Peri-gastrulation embryo model

Abstract

Provided herein are methods, compositions, kits and systems for analysis of the events and constituents of early embryogenesis. In particular, provided herein are reagents and methodologies for generation and use of peri-gastrulation trilaminar embryonic disc (PTED) embryoids.

Description

[0001] PERI-GASTRULATION EMBRYO MODEL

[0002] SEQUENCE LISTING

[0003] The text of the computer readable sequence listing filed herewith, titled “42844- 601_SEQUENCE_LISTING”, created February 12, 2025, having a file size of 5,428 bytes, is hereby incorporated by reference in its entirety.

[0004] FIELD

[0005] Provided herein are methods, compositions, kits and systems for analysis of the events and constituents of early embryogenesis. In particular, provided herein are reagents and methodologies for generation and use of peri-gastrulation trilaminar embryonic disc (PTED) embryo models (or embryoids).

[0006] BACKGROUND

[0007] Despite its importance in human development, gastrulation is difficult to analyze due to technical challenges and limited availability of embryonic tissues of interest. Human stem cell-based embryo models including those that recapitulate aspects of pre- and peri- gastrulation human development are useful experimental technologies to study human embryogenesis. However, existing 3-dimensional human embryo models remain difficult to recapitulate the development of the trilaminar embryonic disc structure flanked by the dorsal amnion and ventral secondary yolk sac, a hallmark of human gastrulation. Accordingly, what is needed is a stem cell-based embryo model that allows scientists to recapitulate the trilaminar embryonic disc structure and thus to study the self-organizing properties of peri- gastrulation human development, and to test the complex cellular events and constituents involved in early human embryogenesis.

[0008] SUMMARY

[0009] Provided herein are methods, compositions, kits and systems for analysis of the events and constituents of early embryogenesis. In particular, provided herein are reagents and methodologies for generation and use of peri-gastrulation trilaminar embryonic disc (PTED) embryoids. Exemplary, non-limiting compositions, systems, kits and methods are described below. In some embodiments, the present invention provides a human embryoid system derived from primed human pluripotent stem cells (hPSCs) that recapitulate diverse aspects of human peri-gastrulation embryonic development including, in some embodiments, formation of trilaminar embryonic layers flanked by the dorsal amnion and ventral secondary yolk sac. In some embodiments, generation of the human peri-gastrulation trilaminar embryonic disc (PTED) embryoid supports analysis, for example immunocytochemistry and transcriptomic analysis, to identify cell lineages in PTED embryoids. In some embodiments, PTED embryoids support analysis of lipid metabolism in extraembryonic endoderm differentiation. In some embodiments, PTED embryoids support analysis of hematopoiesis and blood cell generation.

[0010] In some embodiments, the present invention provides a method of generating a peri- gastrulation trilaminar embryonic disc (PTED) embryoid, comprising: providing one or more pluripotent stem cells (PSCs); treating the one or more PSCs with bone morphogenic protein 4 (BMP4); and applying the one or more PSCs treated with said BMP4 to a micropattemed adhesive surface to generate said PTED embryoid. In some embodiments, the one or more PSCs comprises a mammalian PSC and the PTED embryoid is a mammalian PTED embryoid. In some embodiments, the mammalian PSC is a human PSC (hPSC) and PTED embryoid is a human PTED embryoid. In some embodiments, the hPSC comprises one or more human embryonic stem cells (hESCs). In some embodiments, the hESCs are Hl and / or H9 hESCs.

[0011] In some embodiments the hPSC comprises one or more human induced pluripotent stem cells (hiPSCs). In some embodiments, the method comprises treating one or more hPSCs with Activin A. In some embodiments, the micropatterned adhesive surface comprises a morphogenetic field and / or a geometric boundary confinement. In some embodiments, said micropatterned adhesive surface comprises one or more adhesive islands comprising a basement membrane extract comprising laminin, collagen IV, entactin, and heparin sulfate proteoglycans on one or more glass coverslips, for example, Geltrex™, on one or more glass coverslips. In some embodiments, the PTED embryoid comprises a trilaminar embryonic disc. In some embodiments, the trilaminar embryonic disc comprises a trilaminar embryonic layer. In some embodiments, the trilaminar embryonic layer is between a dorsal amnion and a ventral secondary yolk sac. In some embodiments, the PTED embryoid comprises a secondary yolk sac-like structure. In some embodiments, the method comprises incubating the PSC cells in mTeSRl medium. In some embodiments, the method comprises incubating the PSC cells for at least 48 hours. In some embodiments, the present invention provides a method of testing pre- gastrulation and peri-gastrulation, early human embryogenesis, infertility, human primordial germ cell development, primary hematopoiesis, blood cell generation or toxicity and / or teratogenicity screening, comprising: applying an effective amount of an inhibitory or stimulatory compound to a PTED embryoid of the present invention.

[0012] In some embodiments, the present invention provides a method comprising detecting and / or monitoring at least one property of at least one PTED embryoid wherein the at least one property comprises at least one of: embryoid formation; endoderm differentiation; mesoderm differentiation; primordial germ cell differentiation; amnion formation; secondary yolk sac formation; single-cell dissociation; one or more cell-cell interactions; embryoid branching trajectory; and fluorescence emission; wherein the detecting and / or monitoring comprises one or more of nucleic acid sequencing, RNA sequencing, tissue sectioning, immunohistochemistry, optical detection, light intensity detection, optical imaging, microscopy, photography and videography.

[0013] In some embodiments, the present invention provides a method of endoderm differentiation of a PTED embryoid, comprising: detaching one or more PTED embryoids from one or more coverslips; transferring the one or more PTED embryoids to a multi-well plate; supplementing a medium with an extracellular matrix; incubating the one or more PTED embryoids in the medium daily for 10 days; and testing for the presence of endoderm differentiation of said PTED embryoid. In some embodiments, the medium is Essential 6 (E6) medium. In some embodiments, the extracellular matrix comprises a basement membrane extract comprising laminin, collagen IV, entactin, and heparin sulfate proteoglycans, for example, Geltrex™.

[0014] In some embodiments, a PTED embryoid of the present invention is generated by any method provided above. In some embodiments, the present invention provides use of a PTED embryoid generated by any method provided above. In some embodiments, the present invention provides a PTED embryoid generated in vitro comprising a trilaminar embryonic disc and a secondary yolk sac-like structure.

[0015] DESCRIPTION OF THE FIGURES

[0016] Figure 1 shows a human pluripotent stem cell (hPSC)-derived peri-gastrulation trilaminar embryonic disc (PTED) embryoid with amnion and yolk sac-like structures. Figure 1 A shows H&E staining of a human embryo (left) at Carnegie stage (CS) 7 (https: / / www.ehd.org / virtual-human-embryo / ) and corresponding schematic of its transverse view (right). Scale bar, 100 gm. Figure IB shows a cartoon of Day (D) 8 PTED embryoids (left) and confocal micrographs showing D8 PTED embryoids for multiple lineage markers as indicated (right). Different colored regions in the cartoon mark distinct cellular compartments as indicated. Micrographs were filtered by binary masks made by DAPI signals for visualization. Scale bar, 200 gm. Figure 1C shows micrographs showing zoom-in views of D8 PTED embryoids for different lineage markers as indicated. DAPI counterstains cell nuclei. The regions are highlighted by boxes shown in the schematic in IB, with cyan box for NNE, yellow box for endoderm in the yolk sac-like structure, and magenta box for hematopoiesis site. Further views are included for regions highlighted by white boxes as indicated. White arrowheads indicate ISL1+TFAP2A+cells (cyan box), FOXA2+HNF4A+SOX17+cells (yellow box), and VE-cadherin+CD34+cells (magenta box), respectively. Scale bars, 50 pm. Figure ID shows confocal micrographs of E22 (CS8) Cy monkey embryos stained for different lineage markers as indicated. DAPI counterstains cell nuclei. Zoom-in views are for boxed regions as indicated. Scale bars, 200 pm. EPI, epiblast; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; HEP, hematopoietic endothelial progenitor; PGC, primordial germ cell.

[0017] Figure 2 shows a histocytochemistry analysis of human and monkey embryos. Figure 2A shows schematics of transverse views of human embryo development (top) and corresponding H&E staining of human embryonic tissues (bottom) at Carnegie stage (CS) 5c, 6, and 7 as indicated (https: / / www.ehd.org / virtual-human-embryo / ). Scale bars, 100 gm. Figure 2B shows a schematic sagittal view of E22 (CS8) Cy monkey embryo, with different cell lineages and their markers indicated. Figures 2C-G show confocal micrographs of E22 (CS8) Cy monkey embryos stained for different lineage markers as indicated, showing marker expression for amnion and extraembryonic endoderm (2C), embryonic ectoderm and gastrulating cells (2D), amnion (2E), primordial germ cells (2F), and hematopoietic endothelial progenitors (2G). Zoom-in views are for boxed regions as indicated. White arrowheads in F mark TFAP2C+BLIMP1+SOX17+cells. Scale bars, 200 gm.

[0018] Figure 3 shows development of peri-gastrulation trilaminar embryonic disc (PTED) embryoids between Day 0 and Day 4. Figure 3 A shows a schematic protocol for generating PTED embryoids. Rho-associated kinase (ROCK) inhibitor Y-27632 (Y-2732, Y-27). Figure 3B shows a phase-contrast image showing a regular array of DO PTED embryoids. The inset shows a zoom-in view of a single PTED embryoid. Scale bar, 1,000 gm. Figure 3C shows cartoons (top) and bright-field images (bottom) showing PTED embryoid development from Day 0 to Day 4. Different colored regions in cartoons mark distinct cellular compartments as indicated. Scale bar, 200 pm. Figure 3D shows a box plot showing PTED embryoid heights from Day 0 to Day 4. Box: 25% - 75%, bar-in-box: median, rectangle-in-box: mean, and whiskers: 5% and 95%. Figures 3E-I show confocal micrographs showing orthogonal views from x-y, x-z, and y-z planes of PTED embryoids on Day 0 (3E), Day 1 (3F), Day 2 (3G), Day 3 (3H), and Day 4 (31). PTED embryoids were stained for TFAP2A, NANOG, and FOXA2 or OCT4, SOX2, and Bra, as indicated. Different x-y views of PTED embryoids are provided at different z-focal planes as indicated. The x-z and y-z views are expanded threefold along z-axis for visualization. Zoom-in views in G mark “gastrulation-like” nodes formed by Bra+ gastrulating-like cells. Plots show normalized maximum z-proj ection intensities of indicated lineage markers along colony radius of PTED embryoids on different culture days. Scale bars, 200 pm. EPI, epiblast; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; PGC, primordial germ cell.

[0019] Figure 4 shows a characterization of D5 PTED embryoids. Figure 4A shows a phasecontrast image showing a regular array of D5 PTED embryoids (left) and bright-field image showing a single D5 PTED embryoid (right). White arrowhead marks an embryoid detached from underlying glass coverslip. Scale bars, 1,000 pm (left) and 200 pm (right). Figure 4B shows a cartoon showing D5 PTED embryoid structure, with different colored regions marking distinct cellular compartments as indicated. Figures 4C-D show confocal micrographs showing D5 PTED embryoids stained for different lineage markers as indicated. Zoom-in views are for boxed regions as indicated. White arrowheads in D mark ISL1+TFAP2A+FOXA2‘ cells. Scale bars, 200 pm. EPI, epiblast; EN, endoderm; NNE, non- neural ectoderm; NE, neural ectoderm; PGC, primordial germ cell.

[0020] Figure 5 shows a characterization of D8 PTED embryoids. Figure 5 A shows a bright- field image of a single D8 PTED embryoid (left) and cartoon showing its structure (right). Different colored regions in the cartoon mark different cellular compartments as indicated. AM, amnio. YS, yolk sac. Scale bar, 200 pm. Figure 5B shows a photo showing D8 PTED embryoids collected in tissue culture plate. Scale bar, 1 mm. Figure 5C (left) shows a box plot of length of PTED embryoids on Day 5 and Day 8. Box: 25% - 75%, bar-in-box: median, rectangle-in-box: mean, and whiskers: 5% and 95% (middle). Stacked bar plots show projected areas of different compartments of PTED embryoids on Day 5 and Day 8 as indicated. Error bars indicate standard deviations. AM, amnion. YS, yolk sac. Mid, middle part of PTED embryoids, (right) Box plot of efficiency of PTED embryoid formation on Day 5 and Day 8. Box: 25% - 75%, bar-in-box: median, rectangle-in-box: mean, and whiskers: 5% and 95%. Figures 5D-I show confocal micrographs of D8 PTED embryoids stained for different lineage markers as indicated. Zoom-in views of boxed regions are included as indicated. White arrowheads in Figure 5E mark ISL1+GATA3+FOXA2‘ cells (top) or ISL1+TFAP2A+FOXA2‘ cells (bottom). White arrowheads in Figure F mark FOXA2+HNF4A+SOX17+cells. White arrowheads in Figure 51 mark VE-cadherin+CD34+cells. Scale bars, 200 pm. EPI, epiblast; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; HEP, hematopoietic endothelial progenitor; PGC, primordial germ cell.

[0021] Figure 6 shows PTED embryoids with a trilaminar embryonic disc-like structure and primordial germ cell-like cells (PGCLCs). Figure 6A shows a cartoon of D8 PTED embryoids (left) and confocal micrographs showing D8 PTED embryoids for different lineage markers as indicated (right). Different colored regions in the cartoon mark distinct cellular compartments as indicated. Scale bar, 200 pm. Figures 6B-C show micrographs showing lineage marker expression pattern in the trilaminar embryonic disc-like structure, as highlighted by the red box in the schematic in 6A. DAPI counterstains cell nuclei. Yellow arrowheads in 6C mark Bra+NANOG FOXA2‘ cells. Scale bars, 50 pm. Figure 6D shows a confocal micrograph of E22 (CS8) Cy monkey embryo stained for SOX2 and Bra. DAPI counterstains cell nuclei. Scale bar, 200 pm. Figure 6E shows micrographs showing expression of PGC -related lineage markers as indicated in regions highlighted by cyan and yellow boxes in the schematic in 6 A. DAPI counterstains cell nuclei. White arrowheads mark TFAP2C+BLIMP1+SOX17+cells. Scale bars, 20 pm. Figure 6F shows confocal micrographs of E22 (CS8) Cy monkey embryo stained for different lineage markers as indicated. DAPI counterstains cell nuclei. Zoom-in views are for a boxed region as indicated. White arrowheads mark TFAP2C+BLIMP1+SOX17+cells. Scale bar, 200 pm. EPI, epiblast; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; HEP, hematopoietic endothelial progenitor; PGC, primordial germ cell.

[0022] Figures 7A-C show a characterization of trilaminar embryonic disc-like structures in D8

[0023] PTED embryoids. Confocal micrographs of D8 PTED embryoids were stained for different lineage markers as indicated. Zoom-in views of boxed regions are included as indicated. In 7C, red arrowheads mark OCT4+NANOG SOX2+cells, whereas white arrowheads label OCT4+NANOG+SOX2- cells.

[0024] Figure 8 shows development of human primordial germ cell-like cells in PTED embryoids. Figures 8A-D show confocal micrographs showing orthogonal views from x-y, x-z, and y-z planes of PTED embryoids on Day 3 (8 A) and Day 4 (8B). PTED embryoids were stained for TFAP2C, TFAP2A, and S0X17 as indicated. Different x-y views of PTED embryoids are provided at different z-focal planes as indicated. The x-z and y-z views are expanded threefold along the z-axis for visualization. White arrowheads mark TFAP2C+SOX17+human primordial germ cell-like cells (hPGCLCs). Zoom-in views are for boxed regions as indicated. Scale bars, 200 pm. Figure 8C shows confocal micrographs showing D5 PTED embryoids stained for TFAP2C, TFAP2A, and SOX17, as indicated. Zoom-in views are for color-coded boxed regions as indicated. White arrowheads mark TFAP2C+SOX17+hPGCLCs. Scale bar, 200 pm. Figure 8D shows confocal micrographs showing D8 PTED embryoids stained for different PGC markers as indicated. Zoom-in views are for color-coded boxed regions as indicated. White arrowheads mark TFAP2C+SOX17+hPGCLCs. Scale bars, 200 pm.

[0025] Figure 9 shows PTED embryoids generated from NODAL-KO hPSCs. Figure 9A shows a schematic of NODAL-KO D3 PTED embryoids (left). Confocal micrographs show orthogonal views from x-y, x-z, and y-z planes of D3 NODAL-KO PTED embryoids stained for TFAP2A, NANOG, and FOXA2 or OCT4, SOX2, and Bra as indicated (middle). Different x-y views of PTED embryoids are provided at different z-focal planes as indicated. The x-z and y-z views are magnified threefold along z-axis. Scale bars, 200 pm. The plot on the right shows normalized maximum z-proj ection intensities of indicated lineage markers along a colony radius of D3 NODAL-KO PTED embryoids. Figure 9B shows confocal micrographs showing D3 NODAL-KO PTED embryoids stained for TFAP2C, TFAP2A, and SOX17. Different x-y views of PTED embryoids are provided at different z-focal planes as indicated. The x-z and y-z views are expanded threefold along the z-axis for visualization. Zoom-in views are provided for boxed regions as indicated. White arrowheads mark TFAP2C+SOX17+hPGCLCs. Scale bars, 200 pm. Figure 9C shows a schematic of D5 NODAL-KO PTED embryoids (left). Confocal micrographs in the middle show orthogonal views from x-y, x-z, and y-z planes of D5 PTED embryoids stained for TFAP2A, NANOG, and FOXA2 as indicated. Different x-y views of PTED embryoids are provided at different z- focal planes as indicated. The x-z and y-z views are expanded threefold along z-axis for visualization. Scale bars, 200 pm. Plot on the right showing normalized maximum z- projection intensities of indicated lineage markers along colony radius of D5 NODAL-KO PTED embryoids. EPI, epiblast; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; PGC, primordial germ cell.

[0026] Figure 10 shows PTED embryoids generated from Hl hESCs and another hiPSC line. Confocal micrographs show D5 and D8 PTED embryoids generated from Hl hESCs (Figure 10A) and D8 PTED embryoids generated from a hiPSC line (Figure 10B) stained for different lineage markers as indicated. Zoom-in views of boxed regions are included as indicated. White arrowheads mark VE-cadherin+CD34+cells. Scale bars, 200 pm.

[0027] Figure 11 shows the effects of colony size and cell seeding density on PTED embryoid development. Figure 11 A shows arrays of DO PTED embryoids with initial hPSC colony diameters of 400 pm (left) and 800 pm (right). Scale bars, 500 pm. Figure 1 IB shows a cartoon of D3 PTED embryoids generated from initial hPSC colonies with a diameter of 400 pm (left). Different colored regions in the cartoon mark different cellular compartments as indicated. Figure 1 IB shows confocal micrographs showing orthogonal views from x-y, x-z, and y-z planes of D3 PTED embryoids stained for TFAP2A, NANOG, and FOXA2 (right). Different x-y views of PTED embryoids are provided at different z-focal planes as indicated. The x-z and y-z views are expanded threefold along z-axis for visualization. Scale bars, 100 pm. Figure 11C shows plots showing normalized maximum z-projection intensities of indicated lineage markers along colony radius of D3 PTED embryoids generated from hPSC colonies of different sizes as indicated. Figure 1 ID shows a cartoon of D5 PTED embryoids generated from initial hPSC colonies with a diameter of 400 pm (left). Different colored regions in the cartoon mark different cellular compartments as indicated. Confocal micrographs show orthogonal views from x-y, x-z, and y-z planes of D5 PTED embryoids stained for TFAP2A, NANOG, and FOXA2 (right). Different x-y views of PTED embryoids are provided at different z-focal planes as indicated. The x-z and y-z views are expanded threefold along z-axis for visualization. Scale bars, 100 pm. Figure 1 IE shows a schematic of D2 PTED embryoids generated from initial hPSC colonies with a diameter of 800 pm under different cell seeding density conditions as indicated (left). Different colored regions in cartoons mark different cellular compartments as indicated. Confocal micrographs showing orthogonal views from x-y, x-z, and y-z planes of D2 PTED embryoids were stained for TFAP2A, NANOG, and Bra (middle). Different x-y views of PTED embryoids are provided at different z-focal planes as indicated. The x-z and y-z views are expanded threefold along z- axis for visualization. Scale bars, 200 pm. Plots on the right show normalized maximum z- projection intensities of indicated lineage markers along colony radius of D2 PTED embryoids. Figure 1 IF shows a box plot showing the percentage of D2 PTED embryoids with a top center amnion region as a function of initial cell seeding density. Box: 25% - 75%, bar-in-box: median, rectangle-in-box: mean, and whiskers: 5% and 95%. ***, p < 0.001. EPI, epiblast; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm. Figure 12 shows a single-cell transcriptomic analysis of PTED embryoids. Figure 12A shows a unified manifold approximation and projection (UMAP) visualization of integrated data combining scRNA-seq data of Day 2, Day 5, and Day 8 PTED embryoids, color-coded according to either culture times (left) or cell identity annotations (right). Figure 12B shows a dot plot illustrating expression of key marker genes across different cell clusters in PTED embryoids. Dot sizes represent proportions of cells expressing corresponding genes, while dot colors indicate averaged scaled values of log-transformed expression levels. Figure 12C shows a UMAP visualization of integrated data combining scRNA-seq data of Day 8 PTED embryoids and CS7 human embryo (Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021) color-coded according to either sample origins (left) or cell identity annotations (right). Figure 12D shows a heatmap showing correlation coefficients between paired cell clusters from CS7 human embryo and Day 8 PTED embryoids as indicated. Figure 12E shows a UMAP visualization of integrated data combining scRNA-seq data of Day 8 PTED embryoids and E20 (CS8) monkey embryo (Zhai, J. et al. Primate gastrulation and early organogenesis at single-cell resolution. Nature 612, 732-738 (2022) color-coded according to either sample origins (left) or cell identity annotations (right). Figure 12F shows a heatmap showing correlation coefficients between paired cell clusters from E20 monkey embryo and Day 8 PTED embryoids as indicated. EPI, epiblast; PS, primitive streak; NM, nascent mesoderm; EM, emergent mesoderm; AM, advanced mesoderm; ExM, extraembryonic mesoderm; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; HEP, hematopoietic endothelial progenitor; PGC, primordial germ cell; blood, blood cell; AxM, axial mesoderm.

[0028] Figure 13 shows a single-cell transcriptomic analysis of D2, D5, and D8 PTED embryoids, respectively. Figure 13 A shows a UMAP visualization of scRNA-seq data of D2 PTED embryoids, color-coded according to cell identity annotations, n indicates cell number. Figure 13B shows feature plots showing expression patterns of selected lineage markers used for cell identity annotations in UMAP plot of D5 PTED embryoid. Figure 13C shows a UMAP visualization of scRNA-seq data of D8 PTED embryoids, color-coded according to cell identity annotations, n indicates cell number. Figure 13D shows feature plots showing expression patterns of selected lineage markers used for cell identity annotations in UMAP plot of D5 PTED embryoid. Figure 13E shows a UMAP visualization of scRNA-seq data of D8 PTED embryoids, color-coded according to cell identity annotations, n indicates cell number. Figure 13F shows a dot plot showing expression of key marker genes across different cell clusters in D8 PTED embryoids. Dot sizes represent proportions of cells expressing corresponding genes, while dot colors indicate averaged scaled values of log- transformed expression levels. Figure 13G shows feature plots showing expression patterns of selected lineage markers used for cell identity annotations in UMAP plot of D8 PTED embryoid. EPI, epiblast; PS, primitive streak; NM, nascent mesoderm; EM, emergent mesoderm; AM, advanced mesoderm; ExM, extraembryonic mesoderm; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; HEP, hematopoietic endothelial progenitor; PGC, primordial germ cell.

[0029] Figure 14 shows a single-cell transcriptome analysis of integrated datasest of D2, D5, and D8 PTED embryoids. Figure 14A shows a UMAP visualization of integrated scRNA-seq data of D2, D5, and D8 PTED embryoids separated by culture day from integrated UMAP plot in Figure 12A. Data are color-coded according to cell identity annotations, n indicates cell numbers. Figure 14B shows a stacked bar plot showing cellular compositions in D2, D5, and D8 PTED embryoids as indicated. Figure 14C shows a heatmap showing expression levels of differentially expressed genes identified from integrated scRNA-seq data of D2, D5, and D8 PTED embryoids. Color bars above the heatmap indicate cell identity and culture day as indicated. Figure 14D shows feature plots showing expression patterns of selected lineage markers used for cell identity annotations in integrated UMAP plot in Fig. 12A. Figure 14E shows a subclustering analysis of scRNA-seq data of NNE cluster separated from integrated data of D2, D5, and D8 PTED embryoids, showing 2 subclusters annotated as Amnion and EmNNE (embryonic non-neural ectoderm). UMAP plots are color-coded according to culture time (left) or cell subcluster identity annotations (right), n indicates cell number. Figure 14F shows a dot plot showing expression of key marker genes in EmNNE and Amnion subclusters. Dot sizes represent proportions of cells expressing corresponding genes. Dot colors indicate averaged scaled values of log-transformed expression levels. Figure 14G shows feature plots showing expression patterns of selected lineage markers in UMAP plots of EmNNE and Amnion subclusters.

[0030] Figure 15 shows CellChat, developmental trajectory and gene regulatory analyses of integrated scRNA-seq data of D2, D5, and D8 PTED embryoids. Figure 15 A shows circle plots depicting incoming and outgoing signaling pathways across different cell clusters in integrated scRNA-seq data of D2, D5, and D8 PTED embryoids. Figure 15B shows a heatmap showing incoming and outgoing signaling pathways across different cell clusters in integrated scRNA-seq data of D2, D5, and D8 PTED embryoids. Selected signaling patterns are highlighted as indicated. Figure 15C shows developmental branches of cell differentiation based on pseudotime analysis of integrated scRNA-seq data of D2, D5, and D8 PTED embryoids. Figure 15D shows a gene regulatory network (GRN) analysis of different cell clusters in integrated scRNA-seq data of D2, D5, and D8 PTED embryoids. EPI, epiblast; PS, primitive streak; NM, nascent mesoderm; EM, emergent mesoderm; AM, advanced mesoderm; ExM, extraembryonic mesoderm; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; HEP, hematopoietic endothelial progenitor; PGC, primordial germ cell.

[0031] Figure 16 shows transcriptomic comparisons between D8 PTED embryoids and CS7 human gastrula and between D8 PTED embryoids and E20 Cy monkey embryo. Figure 16A shows a UMAP showing scRNA-seq data of D8 PTED embryoids (left) and CS7 human gastrula (right) separated from integrated data of D8 PTED embryoids and CS7 human gastrula. Data are color-coded according to cell identity annotations, n indicates cell numbers. Figure 16B shows a UMAP of scRNA-seq data of endodermal cells separated from integrated scRNA-seq data of D8 PTED embryoids and CS7 human embryo, showing two subclusters annotated as embryonic endoderm (EEN) and extraembryonic endoderm (ExEN). Data are color-coded according to cell origins (left) or cell identity annotations (right) as indicated, n indicates cell number. Figure 16C shows a dot plot showing expression of key marker genes in EEN and ExEN subclusters, which are separated from integrated scRNA-seq data of D8 PTED embryoids and CS7 human embryo. Dot sizes represent proportions of cells expressing corresponding genes. Dot colors indicate averaged scaled values of log-transformed expression levels. Figure 16D shows feature plots showing expression patterns of selected lineage markers in EEN and ExEN subclusters which are separated from integrated scRNA- seq data of D8 PTED embryoids and CS7 human embryo. Figure 16E shows a pathway enrichment analysis of an ExEN subcluster identified from integrated scRNA-seq data of D8 PTED embryoids and CS7 human embryo. Figure 16F shows a UMAP of scRNA-seq data of D8 PTED embryoids (left) and E20 Cy monkey embryo (right), separated of integrated scRNA-seq data of D8 PTED embryoids and E20 Cy monkey embryo. Data are color-coded according to cell identity annotations, n indicates cell numbers. Figure 16G shows a UMAP of scRNA-seq data of endodermal cells isolated from integrated scRNA-seq data of D8 PTED embryoids and E20 Cy monkey embryo, showing 3 subclusters annotated as EEN, ExENl, and ExEN2. Data are color-coded according to cell origins (left) or cell identity annotations (right) as indicated, n indicates cell numbers. Figure 16H shows a dot plot showing expression levels of key marker genes in EEN, ExENl, and ExEN2 subclusters which are separated from integrated scRNA-seq data of D8 PTED embryoids and E20 Cy monkey embryo. Dot sizes represent proportions of cells expressing corresponding genes. Dot colors indicate averaged scaled values of log-transformed expression levels. Figure 161 shows feature plots showing expression patterns of selected lineage markers in EEN, ExENl, and ExEN2 subclusters, which are separated from integrated scRNA-seq data of D8 PTED embryoids and E20 Cy monkey embryo. Figure 16J shows a pathway enrichment analysis of ExEN subcluster identified from integrated scRNA-seq data of D8 PTED embryoids and E20 Cy monkey embryo. EPI, epiblast; PS, primitive streak; NM, nascent mesoderm; EM, emergent mesoderm; AM, advanced mesoderm; ExM, extraembryonic mesoderm; EN, endoderm; NNE, non-neural ectoderm; NE, neural ectoderm; HEP, hematopoietic endothelial progenitor; PGC, primordial germ cell; blood, blood cell; AxM, axial mesoderm; EEN, embryonic endoderm; ExEN, extraembryonic endoderm.

[0032] Figure 17 shows extraembryonic endoderm and blood cells development in PTED embryoids with promoted endoderm differentiation. Figure 17A shows a schematic showing the generation of PTED embryoids with promoted endoderm differentiation. ExENLC, extraembryonic endoderm-like cell; EENLC, embryonic endoderm-like cell. Figure 17B shows confocal micrographs showing Day 3 PTED embryoids stained for F0XA2, HNF4A, and SOX17 (left) and Day 6 PTED embryoids stained for VE-Cadherin, CD34, and F0XA2 (right), respectively. Zoom-in views are for boxed regions as indicated. White dashed line marks the boundary between FOXA2+SOX17HIGHHNF4A EENLCS and FOXA2+SOX17LOWHNF4A+EXENLCS. Scale bars, 200 pm. Figure 17C shows confocal micrographs showing Day 6 PTED embryoids stained for VE-Cadherin, CD34, and FOXA2, with cultures supplemented with different drugs as indicated from Day 3 to Day 6. Scale bars, 200 pm. Figure 17D shows a schematic (left) and brightfield micrographs (right) showing blood cell generation in Day 14 PTED embryoids. Blood cells exhibiting round morphologies are marked by white arrowheads. Scale bars, 100 pm. Figure 17E shows phase (Ph) and immunofluorescence micrographs showing blood cell generation in Day 14 PTED embryoids stained for PU. l and CD34 (top and middle) and RUNX1 and CD36 (bottom). DAPI counterstains cell nuclei. Zoom-in views showed for boxed regions as indicated. White arrowheads mark multi-lobed nuclei of PU.1+cells (top), PU.1+CD34+cells (middle), and CD36+RUNX1+cells (bottom), respectively. Scale bars, 50 pm.

[0033] Figure 18 shows extraembryonic endoderm development in PTED embryoids with enhanced endoderm differentiation. Figure 18A shows schematics on the left showing PTED embryoids with enhanced endoderm differentiation cultured under different conditions as indicated (top: default condition; middle: lipid-rich BSA condition; bottom: lipid-free BSA condition). ExENLC, extraembryonic endoderm-like cell; EENLC, embryonic endoderm-like cell. Confocal images on the right show PTED embryoids cultured under each condition stained for F0XA2, HNF4A, and SOX17 on Day 3. Zoom-in views are for boxed regions as indicated. White dashed line marks the boundary between FOXA2+SOX17HIGHHNF4A' EENLCs and FOXA2+SOX17LOWHNF4A+EXENLCS. Scale bars, 200 pm. Figure 18B shows a UMAP of scRNA-seq data of D3 PTED embryoids cultured under default condition. Data are color-coded according to cell identity annotations, n indicates cell number. Figure 18C shows a dot plot showing expression of key marker genes in different cell clusters identified in D3 PTED embryoids cultured under default enhanced endoderm differentiation condition. Dot sizes represent proportions of cells expressing corresponding genes. Dot colors indicate averaged scaled values of log-transformed expression levels. Figure 18D shows confocal micrographs showing endoderm differentiation under different 2D culture conditions as indicated with hPSCs seeded as either single cells (left) or cell clusters (right). Cells were stained for FOXA2, HNF4A, and SOX17 on Day 3. Scale bars, 100 pm. Figure 18E shows oil red O staining of hPSCs on Day 3 cultured with default basal medium (E6) as indicated. Scale bar, 20 pm. EPI, epiblast; PS, primitive streak; NM, nascent mesoderm; EM, emergent mesoderm; AM, advanced mesoderm; NNE, non-neural ectoderm; PGC, primordial germ cell; EEN, embryonic endoderm; ExEN, extraembryonic endoderm.

[0034] Figure 19 shows hematopoiesis in PTED embryoids with enhanced endoderm differentiation. Figure 19A shows a schematic (top left) showing continuous culture of PTED embryoids under default condition. Confocal micrographs showing PTED embryoids stained for different lineage markers on Day 6 (top right) and Day 14 (bottom) as indicated. Zoom-in views are for boxed regions as indicated. White arrowheads mark PU.1+CD34+FOXA2‘ cells, CD45+Ibal+RUNXF cells, or CDl lb+RUNXF cells. Yellow arrowheads mark CD45+Ibal+RUNX1+cells. Scale bars, 200 pm (top right and first group of images for Day 14) or 50 pm (images for Day 14 except the first group). Figure 19B shows a schematic (top) showing continuous culture of PTED embryoids under lipid-rich BSA culture condition. Confocal micrographs showing PTED embryoids stained for different lineage markers on Day 6 (bottom left) and Day 14 (bottom right) as indicated. Zoom-in views are for boxed regions as indicated. Scale bars, 200 pm (bottom left) or 50 pm (bottom right).

[0035] Figure 20 shows hematopoiesis and blood cell development in PTED embryoids. Figure 20A shows schematics showing progressive hematopoiesis and blood cell development in PTED embryoids with enhanced endoderm differentiation. Figure 20B shows a 3D reconstruction micrograph showing D6 PTED embryoids stained for indicated lineage markers. DAPI counterstains cell nuclei. Scale bars, 50 pm. Figure 20C shows a 3D reconstruction micrograph showing D9 PTED embryoids stained for indicated lineage makers. DAPI counterstains cell nuclei. Zoom-in views are for boxed regions as indicated. Scale bars, 50 pm.

[0036] Figure 20D shows confocal micrographs (top) and 3D reconstruction images (bottom) showing D9 PTED embryoids stained for indicated lineage markers. DAPI counterstains cell nuclei. White arrowheads mark hematopoietic endothelial or blood progenitors. Scale bars, 50 pm.

[0037] Figure 20E shows confocal micrographs (left) and 3D reconstruction images (middle and right) of D14 PTED embryoids stained for indicated lineage markers. Zoom-in views are provided for boxed regions as indicated. White arrowheads mark membrane buddings of CD42b+megakaryoid-like cells. DAPI counterstains cell nuclei. Scale bars, 50 pm. Figure 20F shows a UMAP visualization of scRNA-seq data of D14 PTED embryoids, color-coded according to cell identity annotations, n indicates cell number. Figure 20G shows a dot plot showing expression of key marker genes across different cell clusters in D14 PTED embryoids. Dot sizes represent proportions of cells expressing corresponding genes, while dot colors indicate averaged scaled values of log-transformed expression levels. Figure 20H shows bright-field images showing colonies generated from individual cells in D14 PTED embryoids from colony forming unit (CFU) experiments. Scale bars, 200 pm. Figure 201 shows a stacked bar plot showing cell colony numbers per 10,000 cells from D14 PTED embryoids in CFU experiments. Error bars indicate standard deviations. Figure 20J shows flow cytometry scatterplots showing CD34 and CD43 expression in CD235ab+erythroid populations on different culture days of PTED embryoids (left), and the percentages of CD34+CD43+, CD34+CD43‘, and CD34 CD43+erythroid progenitors in CD235ab+erythroid populations as a function of culture days (right).

[0038] Figure 21 shows development of PTED embryoids with enhanced endoderm differentiation. Figure 21A shows a schematic showing development of PTED embryoids with enhanced endoderm differentiation. Y27, Y-2732. ExEndo, extraembryonic endoderm. EmEndo, embryonic endoderm. Figure 2 IB shows confocal micrographs showing D3 PTED embryoids stained for indicated endodermal lineage markers. Zoom-in views are provided for boxed regions as indicated. White dashed line marks the boundary between FOXA2+SOX17HIGHHNF4A’ EmEndo-like cells and FOXA2+SOX17LOWHNF4A+ExEndo- like cells. DAPI counterstains cell nuclei. Scale bar, 200 pm. Figure 21C shows a Uniform Manifold Approximation and Projection (UMAP) algorithm applied to RNA sequencing data of D3 PTED embryoids with enhanced endoderm differentiation. Data are color-coded according to cell identity annotations, n indicates cell number. Figure 2 ID shows a dot plot showing expression of key marker genes across different cell clusters identified in D3 PTED embryoids. Dot sizes correspond to proportions of cells expressing corresponding genes, while dot colors indicate averaged scaled values of log-transformed expression levels. Figure 2 IE shows feature plots showing expression patterns of selected hematopoiesis-related markers in UMAP plots. Figure 21G shows schematics (left) and confocal micrographs (right) showing endoderm differentiation under 2D culture conditions, with hPSCs seeded as either single cells (top) or cell clusters (bottom). Cells were stained for indicated endodermal lineage markers on D3. DAPI counterstains cell nuclei. Scale bars, 100 pm.

[0039] Figure 22 shows hematopoiesis and blood cell development in PTED embryoids, with enhanced endoderm differentiation. Figure 22 A shows schematics showing progressive hematopoiesis and blood cell development in PTED embryoids with enhanced endoderm differentiation. Figure 22B shows: confocal micrographs of D6 PTED embryoids stained for indicated lineage markers (left). Zoom-in views are provided for boxed regions as indicated. Scale bar, 200 pm; confocal micrographs showing PTED embryoids treated with indicated VEGF / KIT inhibitors on D6 stained for different lineage markers (middle). Scale bars, 200 pm; and confocal micrographs showing orthogonal views from x- , x-z and y-z planes of D6 PTED embryoids, stained for indicated lineage markers. Scale bars, 50 pm. DAPI counterstains cell nuclei (right). Figure 22C shows confocal micrographs showing orthogonal views from x- , x-z and y-z planes and 3D reconstruction micrographs of D9 PTED embryoids, stained for indicated lineage markers. DAPI counterstains cell nuclei. Zoom-in views are provided for boxed regions as indicated. Scale bars, 50 pm. Figure 22D shows confocal micrographs showing orthogonal views from x- , x-z or y-z planes and 3D reconstruction images of D14 PTED embryoids, stained for indicated lineage markers. DAPI counterstains cell nuclei.

[0040] Figure 23 shows blood cell development in PTED embryoids. Figure 23 A shows confocal micrographs showing D5 PTED embryoids stained for indicated lineage markers. White arrowhead marks CD34+CD42b+cells. DAPI counterstains cell nuclei. Scale bar, 50 pm. Figure 23B shows confocal micrographs showing D6 PTED embryoids stained for indicated lineage markers. White arrowheads mark CD235ab+CD42b+cells, brown arrowhead marks CD235ab+CD34+cells, and blue arrowhead marks CD34+CD235ab'CD42b' cells. DAPI counterstains cell nuclei. Scale bars, 50 pm. Figure 23C shows confocal micrographs showing D7 PTED embryoids stained for indicated lineage markers. DAPI counterstains cell nuclei. Scale bar, 50 pm. Figure 23D shows confocal micrographs showing D9 PTED embryoids stained for indicated lineage markers. Zoom-in views are provided for boxed regions as indicated. White arrowheads mark CD34+CD42b+cells, brown arrowheads label CD235ab+CD34+cells, and blue arrowheads mark CD34+CD235ab'CD42b' cells. DAPI counterstains cell nuclei. Scale bars, 50 pm. Figure 23E shows phase-contrast and confocal micrographs showing D14 PTED embryoids stained for indicated lineage markers. Zoom-in views are provided for boxed regions as indicated. White arrowheads mark CD34+PU.F CD42b' cells, brown arrowheads label CD34+blood progenitors that are also CD235ab+, CD42b+, or PU.1+, and blue arrowheads mark blood cells that have detached from CD34+hematopoietic endothelial progenitors. DAPI counterstains cell nuclei. Scale bars, 200 pm (for the first row of images) or 50 pm.

[0041] Figure 24 shows blood cell, mesodermal and endodermal lineage developments in PTED embryoids. Figure 24A shows a schematic showing erythropoiesis and megakaryopoiesis from CD235ab+erythroid-like cells and CD42b+megakaryoid-like cells, respectively. Figure 24B shows phase-contrast and confocal micrographs showing erythroid- and megakaryoid-like cells in D14 PTED embryoids stained for indicated lineage markers. DAPI counterstains cell nuclei. Scale bars, 200 pm. Figure 24C shows phase-contrast, confocal, and 3D reconstruction images showing CD235ab+erythroid- and CD42b+megakaryoid-like cells in D14 PTED embryoids. Zoom-in views are provided for boxed regions as indicated. White arrowheads mark RUNXl+CD235ab+or RUNXl+CD42b+cells, while brown arrowheads mark RUNXFCD235ab+or RUNXFCD42b+cells. Scale bars, 50 pm. Figure 24D shows bright-field micrographs showing spontaneous beating cardiac tissues in PTED embryoids on different culture days. White arrowheads mark spontaneous beating sites. Scale bars, 100 pm. Figure 24E shows confocal micrographs and 3D reconstruction image showing D14 PTED embryoids stained for different lineage markers associated with myocardium tissues. Scale bars, 20 pm.

[0042] Figure 24F shows confocal micrographs showing D7 and D14 PTED embryoids stained for indicated extraembryonic endoderm markers. Scale bars, 20 pm. Figure 25 shows single-cell transcriptomic and flow cytometry analysis of hematopoiesis in PTED embryoids. Figure 25A shows a subclustering analysis of scRNA- seq data of hematopoiesis-related cell clusters (Endothelium, MEP, Erythroid 1, Erythroid 2, Megakaryoid 1, Megakaryoid 2, and Myeloid / Lymphoid) separated from D14 PTED embryoids with enhanced endoderm differentiation. UMAP plot is color-coded according to cell subcluster identity annotations, n indicates cell number. Figure 25B shows plots showing expression patterns of selected lineage markers in UMAP plots of hematopoiesis-related cell clusters. Figure 25C shows a diffusion map of cells in hematopoiesis-related cell clusters. Data are color-coded according to cell identity annotations (top) or pseudotime (bottom) as indicated. Figure 25D shows a flow cytometric analysis of CD 15+CD31+neutrophil-like cells, CD 14+monocyte- or macrophage-like cells, CD117+lymphoid-like progenitors, and CD19+B cell progenitor-like cells in the CD43+CD45+population of D9 PTED embryoid cultures; CD235ab+erythroid-like and CD42b+megakaryoid-like populations in D12 PTED cultures; CD33+myeloid-like cells, CD235 ab+erythrocyte progenitors-like cells, and CD42b+megakaryocyte progenitors-like cells in the CD43+population of D12 PTED cultures; CD56+CD49d+natural killer-like cells and CD3+T cell progenitor-like cells in the CD45+CD14 CD19‘ populations of D18 PTED cultures. Figure 25E shows a flow cytometric analysis of CD34 and CD43 expression on cells in the entire PTED culture (top) or in the CD235ab+erythroid-like population (bottom) on different culture days as indicated (left), and plots showing percentages of CD34+CD43+, CD34+CD43‘, and CD34 CD43+cells among the entire PTED cell population (top) or in the CD235ab+erythroid-like population (bottom) on different culture days (right).

[0043] Figure 26 shows a single-cell transcriptomic analysis of endodermal and mesodermal clusters in PTED embryoids with enhanced endoderm differentiation. Figure 26A shows a ubclustering analysis of scRNA-seq data of ExEndo cluster separated from D14 PTED embryoids with enhanced endoderm differentiation. The UMAP plot is color-coded according to cell subcluster identity annotations. ExEndo, extraembryonic endoderm, n indicates cell number. Figure 26B shows feature plots showing expression patterns of selected endodermal lineage markers in UMAP plots of endodermal clusters. Figure 26C shows plots showing expression patterns of selected lineage markers, which are highly expressed in ExEndo or hepatocytes, or associated with extracellular matrix (ECM) or different hematopoietic lineages, as indicated. Figure 26D shows a subclustering analysis of scRNA-seq data of mesodermal clusters (Meso 1 and Meso 2) separated from D14 PTED embryoids with enhanced endoderm differentiation. The UMAP plot is color-coded according to cell subcluster identity annotations, n indicates cell number. Figure 26E shows a dot plot showing expression of key marker genes in different mesodermal subclusters. Dot sizes correspond to proportions of cells expressing corresponding genes, while dot colors indicate averaged scaled values of log-transformed expression levels.

[0044] Figure 26F shjows plots showing expression patterns of selected mesodermal lineage markers in UMAP plots of mesodermal clusters.

[0045] Figure 27A shows an analysis of cell lineage interactions and signaling pathways using scRNA-seq data of D14 PTED embryoids with enhanced endoderm differentiation. Figure 27A shows a UMAP visualization of scRNA-seq data of D14 PTED embryoids, with endodermal and mesodermal subcluster information incorporated. The UMAP plot is color- coded according to cell cluster identity annotations, n indicates cell number. Figure 27B shows circle plots depicting incoming and outgoing signaling pathways across different cell clusters in scRNA-seq data of D14 PTED embryoids. Figure 27C shows a heatmap showing incoming and outgoing signaling pathways across different cell clusters in scRNA-seq data of D14 PTED embryoids.

[0046] DEFINITIONS

[0047] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0048] In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.” The term “one or more,” as used herein, refers to a number higher than one. For example, the term “one or more” encompasses any of the following: two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, fifty or more, 100 or more, or an even greater number.

[0049] The term “one or more but less than a higher number,” “two or more but less than a higher number,” “three or more but less than a higher number,” “four or more but less than a higher number,” “five or more but less than a higher number,” “six or more but less than a higher number,” “seven or more but less than a higher number,” “eight or more but less than a higher number,” “nine or more but less than a higher number,” “ten or more but less than a higher number,” “eleven or more but less than a higher number,” “twelve or more but less than a higher number,” “thirteen or more but less than a higher number,” “fourteen or more but less than a higher number,” or “fifteen or more but less than a higher number” is not limited to a higher number. For example, the higher number can be 10,000, 1,000, 100, 50, etc. For example, the higher number can be approximately 50 (e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2).

[0050] As used herein, the term a “nucleic acid” or “nucleic acid molecule” generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. “Nucleic acids” include, without limitation, single- and double-stranded nucleic acids. As used herein, the term “nucleic acid” also includes DNA as described above that contains one or more modified bases. Thus, DNA with a backbone modified for stability or for other reasons is a “nucleic acid.” The term “nucleic acid” as it is used herein embraces such chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, including for example, simple and complex cells.

[0051] The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or “nucleic acid” refer to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides for DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides for RNA are uracil, adenine, cytosine, and guanine.

[0052] The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or of a polypeptide or its precursor. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained. The term “portion” when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, “a nucleotide comprising at least a portion of a “gene” may comprise fragments of the gene or the entire gene.

[0053] The term “gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends, e.g., for a distance of about 1 kb on either end, such that the gene corresponds to the length of the full- length mRNA (e.g., comprising coding, regulatory, structural and other sequences). The sequences that are located 5' of the coding region and that are present on the mRNA are referred to as 5' non-translated or untranslated sequences. The sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' nontranslated or 3' untranslated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0054] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' ends of the sequences that are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage, and polyadenylation.

[0055] The term “primer” refers to an oligonucleotide, whether occurring naturally as, e.g., a nucleic acid fragment from a restriction digest, or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid template strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer, and the use of the method.

[0056] The term “probe” refers to an oligonucleotide (e.g., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly, or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification, and isolation of particular nucleic acid sequences (e.g., a “capture probe”). It is contemplated that any probe used in the embodiments of the present disclosure may, in some embodiments, be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the various embodiments of the present disclosure be limited to any particular detection system or label.

[0057] The term “target,” as used herein refers to a nucleic acid sought to be sorted out from other nucleic acids, e.g., by probe binding, amplification, isolation, capture, etc. For example, when used in reference to the polymerase chain reaction, “target” refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction, while when used in an assay in which target DNA is not amplified.

[0058] Accordingly, as used herein, “non-targef ’, e.g., as it is used to describe a nucleic acid such as a DNA, refers to nucleic acid that may be present in a reaction, but that is not the subject of detection or characterization by the reaction. In some embodiments, non-target nucleic acid may refer to nucleic acid present in a sample that does not, e.g., contain a target sequence, while in some embodiments, non-target may refer to exogenous nucleic acid, i.e., nucleic acid that does not originate from a sample containing or suspected of containing a target nucleic acid, and that is added to a reaction, e.g., to normalize the activity of an enzyme (e.g., polymerase) to reduce variability in the performance of the enzyme in the reaction.

[0059] As used herein, the terms “patient” or “subject” refer to organisms to be subject to various tests described herein. The term “subject” includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Further with respect to diagnostic methods, a preferred subject is a vertebrate subject. A preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal. A preferred mammal is most preferably a human. As used herein, the term “subject” includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As such, the present disclosure provides for the diagnosis of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and / or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; pinnipeds; and horses. Thus, also provided is the diagnosis and treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including racehorses), and the like.

[0060] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a sub-portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR’s) regulated under the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a sub-portion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0061] As used herein, the term “information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term “information related to a subject” refers to facts or data pertaining to a subject (e.g., a human, plant, or animal). The term “genomic information” refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, percentage methylation, allele frequencies, RNA expression levels, protein expression, phenotypes correlating to genotypes, etc.

[0062] As used herein the term “stem cell” (“SC”) refers to cells that can self-renew and differentiate into multiple lineages. A stem cell is a developmentally pluripotent or multipotent cell. A stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor ("transit") cell, which then proliferates into the tissue's mature, fully formed cells. Stem cells may be derived, for example, from embryonic sources ("embryonic stem cells") or derived from adult sources. For example, U.S. Pat. No. 5,843,780 to Thompson describes the production of stem cell lines from human embryos. PCT publications WO 00 / 52145 and WO 01 / 00650 describe the use of cells from adult humans in a nuclear transfer procedure to produce stem cell lines.

[0063] Examples of adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and bone marrow stromal cells. These stem cells have demonstrated the ability to differentiate into a variety of cell types including adipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromal cells, and thymic stroma (mesenchymal stem cells); hepatocytes, vascular cells, and muscle cells (hematopoietic stem cells); myocytes, hepatocytes, and glial cells (bone marrow stromal cells) and, indeed, cells from all three germ layers (adult neural stem cells).

[0064] As used herein, the term “totipotent cell” refers to a cell that is able to form a complete embryo (e.g., a blastocyst).

[0065] As used herein, the term “pluripotent cell” or “pluripotent stem cell” refers to a cell that has complete differentiation versatility, e.g., the capacity to grow into any of the mammalian body's approximately 260 cell types. A pluripotent cell can be self-renewing, and can remain dormant or quiescent within a tissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell), a pluripotent cell, even a pluripotent embryonic stem cell, cannot usually form a new blastocyst.

[0066] As used herein, the term “induced pluripotent stem cells” (“iPSCs”) refers to a stem cell induced from a somatic cell, e.g., a differentiated somatic cell, and that has a higher potency than said somatic cell. iPS cells are capable of self-renewal and differentiation into mature cells.

[0067] As used herein, the term “multipotent cell” refers to a cell that has the capacity to grow into a subset of the mammalian body's approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all of the cell types. As used herein, the term “progenitor cell” refers to a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue.

[0068] As used herein, the term "embryonic stem cell" (“ES cell” or ESC”) refers to a pluripotent cell that is derived from the inner cell mass of a blastocyst (e.g., a 4- to 5-day-old human embryo), and has the ability to yield many or all of the cell types present in a mature animal.

[0069] As used herein the term “feeder cells” refers to cells used as a growth support in some tissue culture systems. Feeder cells may be embryonic striatum cells or stromal cells.

[0070] As used herein, the term “chemically defined media” refers to culture media of known or essentially-known chemical composition, both quantitatively and qualitatively. Chemically defined media is free of all animal products, including serum or serum-derived components (e.g., albumin).

[0071] As used herein, the term “serum-free media” refers to culture media that is devoid of serum, but not necessarily of other undefined components.

[0072] DETAILED DESCRIPTION

[0073] Provided herein are methods, compositions, kits and systems for analysis of the events and constituents of early embryogenesis. In particular, provided herein are reagents and methodologies for generation and use of peri-gastrulation trilaminar embryonic disc (PTED) embryoids. Exemplary, non-limiting methods, compositions, kits and systems are described below.

[0074] Gastrulation is among the most crucial milestones in embryonic development (Arnold, S. J. & Robertson, E. J. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat. Rev. Mol. Cell Biol. 10, 91-103 (2009), Solnica- Krezel, L. Gastrulation: From Embryonic Pattern to Form Preface. Gastrulation: From Embryonic Pattern to Form 136, Xvii-Xxiv (2020). O'Rahilly, R. & Muller, F. Developmental Stages in Human Embryos: Revised and New Measurements. Cells Tissues Organs 192, 73-84 (2010)). Gastrulation leads to development of the basic body plan and heralds organogenesis in the embryo. Through the gastrulation process, a homogeneous population of pluripotent epiblast cells in the bilaminar disc of human embryo self-organizes and forms the trilaminar embryonic disc consisting of embryonic ectoderm, mesoderm, and endoderm along the dorsal (D)-ventral (V) axis that is flanked by dorsal amnion and ventral secondary yolk sac structures (O'Rahilly, R. & Muller, F. Developmental Stages in Human Embryos: Revised and New Measurements. Cells Tissues Organs 192, 73-84 (2010) (Figures 1 A and 2A). Despite its importance in human development, direct study of human gastrulation remains challenging due to technical issues associated with intrauterine development after implantation and availability of precursor cells and tissues. Understanding of peri-gastrulation human development remains incomplete. The current knowledge of peri- gastrulation human development is derived from studies of animal models such as the mouse (Brennan, J. et al. Nodal signaling in the epiblast patterns the early mouse embryo. Nature 411, 965-969 (2001), Rivera-Perez, J. A. & Magnuson, T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3. Dev. Biol. 288, 363-371 (2005), Ben-Haim, N. et al. The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4. Dev. Cell 11, 313-323 (2006), Peng, G. D. et al. Spatial Transcriptome for the Molecular Annotation of Lineage Fates and Cell Identity in Mid-gastrula Mouse Embryo. Dev. Cell 36, 681-697 (2016), Scialdone, A. et al. Resolving early mesoderm diversification through single-cell expression profiling. Nature 535, 289-293 (2016)), or sparse data from human (Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021), or non-human primate monkey (Bergmann, S. et al. Spatial profiling of early primate gastrulation in utero. Nature 609, 136-143 (2022), Cui, G. et al. Spatial molecular anatomy of germ layers in the gastrulating cynomolgus monkey embryo. Cell Rep. 40, 111285 (2022), Zhai, J. et al. Primate gastrulation and early organogenesis at single-cell resolution. Nature 612, 732-738 (2022)) gastrula samples based on histocytochemistry or transcriptomic analysis.

[0075] Stem cell-derived human embryo models (“embryoids”) provide experimental tools to promote fundamental understanding of human development and advance reproductive and regenerative medicine (Shahbazi, M. N., Siggia, E. D. & Zemicka-Goetz, M. Selforganization of stem cells into embryos: A window on early mammalian development. Science 364, 948-951 (2019), Fu, J., Warmflash, A. & Lutolf, M. P. Stem-cell-based embryo models for fundamental research and translation. Nat. Mater. 20, 132-144 (2021), Rossant, J. & Tam, P. P. L.

[0076] Opportunities and challenges with stem cell-based embryo models. Stem Cell Rep. 16, 1031- 1038 (2021)). Human embryoids recapitulate important aspects of human pre- and peri- gastrulation development (Ai, Z. et al. Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids. Cell Res. 33, 661-678 (2023), Karvas, R. M. et al. 3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages. Cell Stem Cell 30, 1148-1165 el 147 (2023), Liu, L. et al. Modeling post-implantation stages of human development into early organogenesis with stem-cell- derived peri-gastruloids. Cell 186, 3776-3792 (2023), Oldak, B. et al. Complete human day 14 post-implantation embryo models from naive ES cells. Nature (2023), Pedroza, M. et al. Self-patterning of human stem cells into post-implantation lineages. Nature (2023), Weatherbee, B. A. T. et al. Pluripotent stem cell-derived model of the post-implantation human embryo. Nature (2023)), including embryonic disk and bi laminar disk formation, anterior-posterior symmetry breaking of the epiblast, and primitive streak development. However, it’s not certain if the current embryoid systems are capable of modeling the development and organization of the human trilaminar embryonic disc structure, a hallmark of gastrulation in vertebrate species (Arnold, S. J. & Robertson, E. J. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat. Rev. Mol. Cell Biol. 10, 91-103 (2009), Solnica-Krezel, L. Gastrulation: From Embryonic Pattern to Form Preface. Gastrulation: From Embryonic Pattern to Form 136, Xvii-Xxiv (2020)).

[0077] In some embodiments, the present invention provides methods, compositions, kits and systems comprising a human peri-gastrulation development model generated from primed human pluripotent stem cells (hPSCs) termed a peri-gastrulation trilaminar embryonic disc embryoid (PTED). Development of a PTED through spontaneous cell differentiation and organization bypasses blastocyst- or implantation-like developmental stages and recapitulates aspects of peri-gastrulation human embryonic development including formation of trilaminar embryonic layers flanked by dorsal amnion and ventral secondary yolk sac, and primary hematopoiesis and blood cell generation in the secondary yolk sac. Development of a PTED does not generate a structure mimicking the primitive streak, a transient structure associated with mammalian gastrulation (Rivera-Perez, J. A. & Magnuson, T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3. Dev. Biol. 288, 363-371 (2005). Bergmann, S. et al. Spatial profiling of early primate gastrulation in utero. Nature 609, 136-143 (2022)). Accordingly, the PTED bypass certain gastrulati on-related cellular events but generate key outcomes of the gastrulation with a recognizable tissue structure containing organized cellular lineages with spatially defined identities in an emerging coordinate system (Peng, G. D. et al. Spatial Transcriptome for the Molecular Annotation of Lineage Fates and Cell Identity in Mid-gastrula Mouse Embryo. Dev. Cell 36, 681-697 (2016), Cui, G. et al. Spatial molecular anatomy of germ layers in the gastrulating cynomolgus monkey embryo. Cell Rep. 40, 111285 (2022), Liu, L. et al. Modeling postimplantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids. Cell 186, 3776-3792 (2023)). In some embodiments, generation of PTEDs comprises confining primed hPSCs in micropatterned adhesive surfaces and treating these primed hPSCs with bone morphogenic protein 4 (BMP4) as an alternative to other human embryoids for modeling human pre- and peri-gastrulation development (Ai, Z. et al. Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids. Cell Res. 33, 661-678 (2023), Karvas, R. M. et al. 3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages. Cell Stem Cell 30, 1148-1165 el 147 (2023), Liu, L. et al. Modeling post-implantation stages of human development into early organogenesis with stem-cell- derived peri-gastruloids. Cell 186, 3776-3792 (2023), Oldak, B. et al. Complete human day 14 post-implantation embryo models from naive ES cells. Nature (2023), Pedroza, M. et al. Self-patterning of human stem cells into post-implantation lineages. Nature (2023), Weatherbee, B. A. T. et al. Pluripotent stem cell-derived model of the post-implantation human embryo. Nature (2023)). BMP4 treatment initiates differentiation of primed hPSCs in PTED embryoids with differentiation of germ layer lineages and extraembryonic amnion cells and their self-organization, and formation of PTED embryoids thereby. Geometric boundary confinement endowed by micropattemed adhesive surfaces provides an effective morphogenetic field to promote cellular interaction and organization during PTED embryoid development.

[0078] Generation of a secondary yolk sac-like structure in PTED embryoids is of note. In human embryos, formation of the secondary yolk sac occurs during gastrulation with participation of hypoblast-derived lineages (Ross, C. & Boroviak, T. E. Origin and function of the yolk sac in primate embryogenesis. Nat. Commun. 11, 3760 (2020) that are not present in PTED embryoids. The cellular dynamics and lineage origins underlying the secondary yolk sac development in vivo are incompletely resolved. Embryonic mesoderm and extraembryonic mesoderm (ExM) cells in PTED embryoids may share common progenies. The origin of yolk sac endoderm-like cells and / or extraembryonic endoderm-like cells (ExENLCs) in PTED embryoids are unresolved. It is possible that ExENLCs and other endodermal cells share common progenies in gastrulating cells that appear early in PTED embryoid development. In some embodiments, the methods, compositions, kits and systems of the present invention identify the spatial distribution and organization of embryonic and extraembryonic mesoderm and endoderm cells in PTED embryoids. In some embodiments, PTED embryoids comprise extraembryonic endoderm and extraembryonic mesoderm interactions in the secondary yolk sac in primary hematopoiesis. By recapitulating both embryonic and extraembryonic development during human gastrulation, including the formation and organization of embryonic germ layers and dorsal amnion and ventral secondary yolk sac (and associated primary hematopoiesis), PTED embryoids of the present invention model the self-organizing properties of peri-gastrulation human development and complex cellular events involved in early human embryogenesis. In some embodiments, human embryoid systems derived from human pluripotent stem cells (hPSCs) replicate diverse features of peri-gastrulation human embryonic development including formation of trilaminar embryonic layers flanked by dorsal amnion and ventral secondary yolk sac.

[0079] In some embodiments of the present invention, methods, compositions, kits, and systems comprising peri-gastrulation trilaminar embryonic disc (PTED) embryoids support screening compounds and exposures for safety and efficacy. In some embodiments, the compound is a drug. In some embodiments, the compound is a toxin and / or pollutant. In some embodiments, toxicity of the compound varies depending on the genetic and epigenetic composition of the PTED. In some embodiments, a PTED is modified to increase or decrease susceptibility by, for example, site-directed mutagenesis, CRISPR and the like. In some embodiments, the exposure is to ambient factors in the environment including, for example, heat, cold, humidity, light, radiation, magnetic field, and the like.

[0080] In some embodiments of the present invention, methods, compositions, kits, and systems comprising peri-gastrulation trilaminar embryonic disc (PTED) embryoids support generation of differentiated tissues and organs of use, for example, in clinical and research applications. In some embodiments, the clinical application is transplantation of tissue including, for example, blood tissue, amnion tissue, liver tissue, kidney tissue, central nervous system tissue, skin, and the like. In some embodiments, the clinical application is infertility. In some embodiments, the research application is investigation of ill-health and / or aging. In some embodiments, the clinical application is replacement of organs damaged by ill-health and / or aging with transplantation of tissues derived from PTED embryoids. In some embodiments, the research application comprises investigation of bulk, single cell, single nucleus and spatial cells and tissues comprising diverse technologies, platforms, analytic pipelines, statistical algorithms and integrative data systems. In some embodiments, the diverse technologies, platforms, analytic pipelines, statistical algorithms and integrative data systems comprise genomic data, epigenomic data, methylomic data, transcriptomic data, proteomic data, metabolomic data, biochemical data, imaging data, histologic data, and cell and tissue function data,

[0081] In some embodiments, methods, compositions, kits, and systems of the present invention, comprise generation PTED embryoids from non-human mammals. In some embodiments, the non-human mammals are non-human primates, agricultural mammals including, for example, bovine mammals, porcine mammals, ovine mammals, caprine mammals, equine mammals and the like, companion mammals including, for example, canine mammals, feline mammals and the like, and non-domestic mammals. In some embodiments, the non-human mammal is a rat or a mouse. In some embodiments, the events and constituents of early embryogenesis are compared between mammalian species and genera using peri-gastrulation trilaminar embryonic disc (PTED) embryoids of the present invention.

[0082] In some embodiments, the present invention provides methods, compositions, kits, and systems comprising peri-gastrulation trilaminar embryonic disc (PTED) embryoids that support generation of differentiated tissues and organs as a yolk sac hematopoiesis and blood cell formation technology for research and therapy. Compared to conventional blood cell generation methods (for example Mikkola, H. K. A. Yolk sac steps up to the plate. J Exp Med 219, e20212315 (2022), Tamaoki, N. et al. Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells. Cell Rep Methods 3, 100460 (2023), Sugimura, R. et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438 (2017), Motazedian, A. et al. Multipotent ragl+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids. Nat. Cell Biol. 22, 60-73 (2020), Zheng, H. Q. et al. Generating hematopoietic cells from human pluripotent stem cells: Approaches, progress and challenges. Cell Regen 12, 31 (2023), Atkins, M. H. et al. Modeling human yolk sac hematopoiesis with pluripotent stem cells. J Exp Med 219, e20211924 (2021), Lim, W. F. et al. Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells. Stem Cell Res Ther 4, 71 (2013), PTED embryoids of the present invention need not comprise supplementation of exogenous hematopoiesis-related soluble factors in keeping with an autonomous and self-organizing hematopoietic process in the yolk sac through intricate interactions between the extraembryonic lineages comprising the definitive yolk sac structure. In some embodiments the present invention provides mature erythrocytes and megakaryocytes, the presence of myeloid and lymphoid cells, and the transition from CD34+to CD43+progenitors. In some embodiments, the present invention provides methods, compositions, kits, and systems for disease modeling in, for example, conditions that affect early embryo development. In some embodiments, the present invention provides methods, compositions, kits, and systems for drug and environmental exposure screening in, for example, settings that model drug and environmental exposures in embryos. In some embodiments, the present invention provides methods, compositions, kits, and systems for generation of blood cells including, for example, erythrocytes and megakaryocytes of use in research and therapy.

[0083] In some embodiments, the methods, compositions, systems and kits of the present invention provide a modified yolk sac model with enhanced myeloid and lymphoid generation, including generation of monocytes and macrophages. Myeloid cells, including granulocytes and lymphoid cells, are enhanced during transient definitive wave hematopoiesis and definitive wave hematopoiesis. Modification of culture conditions including altering the culture environment (for example, use of 4% Geltrex and / or providing a liquid-air interface) indicate that both myeloid and lymphoid generation may be enhanced. In some embodiments, the modified model of the present invention provides a platform for testing early immune cell generation with clinical applications in immune cell generation.

[0084] In some embodiments, the methods, compositions, systems and kits of the present invention provide co-culture of yolk sac endoderm and mesoderm for induction of hematopoiesis. The yolk sac comprises a yolk sac endoderm layer enveloped by yolk sac mesoderm layer with primary hematopoiesis occurring within the mesoderm layer. Primary hematopoiesis is induced through interactions between the yolk sac endoderm and yolk sac mesoderm. In vitro, yolk sac mesoderm may be generated and isolated by differentiating naive or primed human pluripotent stem cells (hPSCs), while yolk sac endoderm may be generated using a yolk sac model of the present invention. In some embodiments of the present invention, purified yolk sac mesoderm and endoderm cells are co-cultured to form layered structures to initiate the primary hematopoiesis process. In some embodiments, the present invention provides models that support lineage tracing and perturbation assays, with different lineages labeled and tracked during development to detect differentiation trajectories of use, for example, to test gene function loss and gain and drug perturbations on the developing embryo.

[0085] In some embodiments, the present invention provides methods, compositions, systems and kits that recapitulate a structured trilaminar embryonic disc comprising, for example, primary hematopoiesis within the yolk sac compartment. In some embodiments, the present invention provides a transgene-free PTED model derived from wild-type, primed hPSCs that accurately corresponds to peri-gastrulation human development, including formation of trilaminar embryonic layers between the dorsal amnion and ventral yolk sac. Conventional methods for blood cell generation depend on induction of vascularization and blood generation from mesodermal cells through the addition of diverse and multiple soluble factors. In some embodiments, the PTED models of the present invention do not depend on supplementation of exogenous hematopoiesis-related soluble factors. For example, after initial induction of endoderm and mesoderm for 3 days, tissue of the present invention may be cultured in a basal medium without soluble factors during the subsequent hematopoiesis process, and without gene modification of hPSCs and overexpression of a gene (for example, GA TA6). In some embodiments, the methods, compositions, kits and systems of the present invention provide efficient blood cell generation with enhanced cell number and maturation rate of generated cells.

[0086] EXPERIMENTAL EXAMPLES

[0087] EXAMPLE 1 - Development of peri-gastrulation trilaminar embryonic disc embryoid

[0088] Markers of different cell lineages based on immunohistochemistry analysis of an in vivo, peri-gastrulation Cynomolgus (Cy) monkey embryo on embryonic day 22 (E22, or Carnegie stage 8, or CS8) were validated (Figure 2B-G). Non-neural ectoderm (NNE, including amnion) upregulates expression of TFAP2A, ISL1, and GATA3 (Yang, R. et al. Amnion signals are essential for mesoderm formation in primates. Nat. Commun. 12, 5126 (2021)), embryonic ectoderm retains SOX2 expression, gastrulating cells exhibit marked expression of BRACHYURY (or Bra), extraembryonic endoderm upregulates expression of SOX17 and HNF4A (Mackinlay, K. M. L. et al. An in vitro stem cell model of human epiblast and yolk sac interaction. Elife 10, e63930 (2021), Chen, L. et al. The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac. Nat. Commun. 12, 2886 (2021)), primordial germ cells (PGCs) express TFAP2C, BLIMP 1, and SOX17, and hematopoietic endothelial progenitors (HEPs) in the extraembryonic mesoderm compartment surrounding the secondary yolk sac show upregulated expression of VE-Cadherin and CD34 (Ross, C. & Boroviak, T. E. Origin and function of the yolk sac in primate embryogenesis. Nat. Commun. 11, 3760 (2020)) (Figure 2B-G).

[0089] Studies of mammalian gastrulation, based on both mouse embryos (Arnold, S. J. & Robertson, E. J. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat. Rev. Mol. Cell Biol. 10, 91-103 (2009), Brennan, J. et al. Nodal signaling in the epiblast patterns the early mouse embryo. Nature 411, 965-969 (2001), Rivera-Perez, J. A. & Magnuson, T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3. Dev. Biol. 288, 363-371 (2005), Ben-Haim, N. et al. The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4. Dev. Cell 11, 313-323 (2006)) and human peri- gastrulation embryoids (Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847-854 (2014), Chhabra, S., Liu, L., Goh, R., Kong, X. & Warmflash, A. Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids. PLoS Biol. 17 (2019), Muncie, J. M. et al. Mechanical Tension Promotes Formation of Gastrulation-like Nodes and Patterns Mesoderm Specification in Human Embryonic Stem Cells. Dev. Cell 55, 679-694 (2020)) show that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is integral for initiating the gastrulation process. BMP signaling in extraembryonic tissues adjacent to the pre-gastrulation epiblast initiates the gastrulation signaling cascade activating WNT followed by NODAL in the epiblast to drive the process of gastrulation (Rivera-Perez, J. A. & Magnuson, T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3. Dev. Biol. 288, 363-371 (2005), Ben-Haim, N. et al. The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4. Dev. Cell 11, 313-323 (2006)). To develop PTED embryoids, exogeneous BMP4 was used to treat primed hPSCs, which reside in a developmental state similar to post-implantation pluripotent epiblasts (Tesar, P. J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196-199 (2007), O'Leary, T. et al. Tracking the progression of the human inner cell mass during embryonic stem cell derivation. Nat. BiotechnoL 30, 278-282 (2012)) and have been used for modeling peri-gastrulation human development (Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847-854 (2014), Chhabra, S., Liu, L., Goh, R., Kong, X. & Warmflash, A. Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids. PLoS Biol. 17 (2019), Muncie, J. M. et al. Mechanical Tension Promotes Formation of Gastrulation-like Nodes and Patterns Mesoderm Specification in Human Embryonic Stem Cells. Dev. Cell 55, 679-694 (2020), Shao, Y. et al. A pluripotent stem cell-based model for post-implantation human amniotic sac development. Nat. Commun. 8, 208 (2017), Simunovic, M. et al. A 3D model of a human epiblast reveals BMP4-driven symmetry breaking. Nat. Cell Biol. 21, 900-910 (2019), Zheng, Y. et al. Controlled modelling of human epiblast and amnion development using stem cells. Nature 573, 421-425 (2019)). H9 human embryonic stem cells (hESCs) are first seeded onto circular Geltrex adhesive islands with a diameter of 800 m printed onto glass coverslips using microcontact printing (Figures 3 A, 3B). Geometric boundary confinement provides an effective morphogenetic field to support cellular organization during embryoid development (Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847-854 (2014), Chhabra, S., Liu, L., Goh, R., Kong, X. & Warmflash, A. Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids. PLoS Biol. 17 (2019), Muncie, J. M. el al. Mechanical Tension Promotes Formation of Gastrulati on-like Nodes and Patterns Mesoderm Specification in Human Embryonic Stem Cells. Dev. Cell 55, 679-694 (2020), Karzbrun, E. et al. Human neural tube morphogenesis in vitro by geometric constraints. Nature 599, 268- 272 (2021)). With a cell seeding density of 5,000 cells mm’2, approximately 1,800 hESCs become attached to each adhesive island. Cells are cultured in mTeSRl medium for the first 2 days before BMP4 is supplemented into mTeSRl for the following two days to initiate differentiation of hESCs (Figure 3 A). The day on which BMP4 is added into mTeSRl is designated as Day 0 (Figure 3 A). BMP4 stimulations drive hESC colonies to transform from a two-dimensional (2D) monolayer structure to a three-dimensional (3D) multi-layered structure, evident on Day 1 (Figures 3C, 3F). Two regions of TFAP2A+NNE cells emerge in PTED embryoids on Day 1, with one located at the boundary and the other at the top central region of PTED embryoids (Figure 3F). On Day 2, Bra+gastrulating-like cells emerge and form “gastrulation-like” nodes that are embedded inside PTED embryoids at discrete, peripheral regions (Figure 3G). Even with exogeneous BMP4 removed from culture medium from Day 3 onwards hESC colonies continue to develop and self-organize (Figures 3H, 31). On Day 3, FOXA2+endodermal cells are detected with a majority of FOXA2+endodermal cells appearing within a concentric ring region at the bottom surfaces of PTED embryoids (Figure 3H). Bra+gastrulating-like cells also form a concentric ring pattern at peripheral regions of bottom surfaces of PTED embryoids on Day 3 (Figure 3H). From Day 3 onwards TFAP2A+NNE cells accumulate at top central regions of PTED embryoids (Figures 3h, 31).

[0090] On Day 5 large cavities become evident in PTED embryoids, and PTED embryoids start to detach from underlying glass coverslips (Figure 4A). Immunofluorescence staining of paraffin sections of Day 5 (D5) PTED embryoids shows 3 distinct regions along their lengths, with one pole accumulated with TFAP2A+ISL1+amnion cells, the opposite pole containing ISL1+TFAP2A' mesodermal and SOX17+FOXA2+endodermal cells, and regions between occupied by OCT4+and SOX2+epiblast-like cells (Figures 4B-D). ISL1+TFAP2A' mesodermal regions often contain visible cavities (Figure 4D). In areas adjacent to the cavities, smaller openings are may be evident enclosed by a single layer of FOXA2+SOX17+endodermal cells (Figure 4D). HNF4A, a marker of yolk sac endoderm, is upregulated in the FOXA2+SOX17+endodermal cells (Figure 4D) indicating their identity as yolk sac endoderm-like cells (Mackinlay, K. M. L. et al. An in vitro stem cell model of human epiblast and yolk sac interaction. Elife 10, e63930 (2021), Chen, L. et al. The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac. Nat. Commun. 12, 2886 (2021)).

[0091] Between Day 5 and Day 8, a majority of PTED embryoids become detached from glass coverslips and are free-floating in culture medium (Figures 5A, 5B). PTED embryoids grow in size and become more elongated, exhibiting a distinct tissue architecture characterized by the presence of 2 cavities at 2 ends flanking a central region containing densely packed cells (Figure IB, Figures 5A-D). Immunofluorescence staining of paraffin sections of D8 PTED embryoids shows TFAP2A+NNE cells and SOX17+or FOXA2+endodermal cells lining the 2 cavities, respectively, and most of the cells between retaining SOX2 or NANOG expression (Figure 5D). Analysis of additional lineage markers shows that cavities at one pole of D8 PTED embryoids are surrounded by TFAP2A+ISL1+or GATA3+ISL1+amnion cells (Figures 1C, 5E). This pole is designated as the dorsal (D) pole of PTED embryoids, with the opposite pole identified as the ventral (V) pole. Within dorsal amniotic-like cavities, ISL1+TFAP2A' mesodermal cells are often evident (Figures 1C, 5E). Cavities at the ventral pole of D8 PTED embryoids are lined with FOXA2+SOX17+HNF4A+yolk sac endoderm-like cells (Figures 1C, 5E) which are surrounded by mesodermal cells that express varying levels of SNAIL, ISL1, GATA4, and ACTIN2 (Figure 5E). Immunostaining for extracellular matrix (ECM) proteins Laminin and DECORIN (DCN) shows that these mesodermal cells are actively secreting ECM proteins (Figure 5H). Within mesodermal tissues surrounding yolk sac endoderm-like cells there are a small number of cells coexpressing CD34 and VE-Cadherin, markers associated with HEPs (Ross, C. & Boroviak, T. E. Origin and function of the yolk sac in primate embryogenesis. Nat. Commun. 11, 3760 (2020), supporting primary hematopoiesis in ventral yolk sac-like structures (Figures 1C, 51). Formation of ventral yolk sac-like structures in PTED embryoids may comprise spontaneous splitting of mesodermal tissues and their interactions with yolk sac endoderm-like lineages, consistent with other human embryoids that develop yolk sac-like structures (Ai, Z. et al. Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids. Cell Res. 33, 661-678 (2023), Oldak, B. et al. Complete human day 14 postimplantation embryo models from naive ES cells. Nature (2023). Expression of cell lineage markers and the spatial organization of the amniotic cavity and the secondary yolk sac are confirmed using the CS8 Cy monkey embryo (Figure ID).

[0092] Immunofluorescence staining for lineage markers associated with embryonic germ layers (ectoderm: OCT4, NANOG; mesoderm: Bra; endoderm: FOXA2) show a clear layered organization of ectodermal, mesodermal, and endodermal cells along the D-V axis of PTED embryoids in keeping with the formation of a trilaminar embryonic disc-like structure (Figures 6A, 6B, 7A). In some D8 PTED embryoids, Bra+ gastrulating-like cells are localized on one side of the trilaminar embryonic disc-like structure, which is similar to CS8 Cy monkey embryo data (Figures 6A,6B, 7A), indicating the possible establishment of an anterior (A)-posterior (P) axis. Additionally, there are clusters of SOX2+OCT4+NANOG' and SOX2 OCT4+NANOG+epiblast-like or ectodermal cells detectable in dorsal regions of the trilaminar embryonic disc-like structure indicative of neural ectoderm development in embryonic ectoderm-like cells (Figure 7C).

[0093] In primates, primordial germ cells emerge during peri-gastrulation development (Chen, D. et al. Human Primordial Germ Cells Are Specified from Lineage-Primed Progenitors. Cell Rep. 29, 4568-4582 (2019), Sasaki, K. et al. The Germ Cell Fate of Cynomolgus Monkeys Is Specified in the Nascent Amnion. Dev. Cell 39, 169-185 (2016). PTED embryoids also give rise to primordial germ cell-like cells (PGCLCs). Small clusters of TFAP2C+SOX17+PGCLCs are evident in PTED embryoids as early as Day 3 (Figures 8A-C). In D8 PTED embryoids, a proportion of TFAP2C+SOX17+PGCLCs express BLIMP 1 or NANOG indicating a fully committed stage to germline cell development (Figures 6E, 8D) consistent with CS8 Cy monkey embryo data (Figure 6F).

[0094] Embryoids generated from mammalian stem cells are amenable to genetic analysis (Yang, R. et al. Amnion signals are essential for mesoderm formation in primates. Nat. Commun. 12, 5126 (2021). Morgani, S. M., Metzger, J. J., Nichols, J., Siggia, E. D. & Hadjantonakis, A.-K. Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning. Elife 7, e32839 (2018). To investigate the role of NODAL signaling in PTED embryoids, given that NODAL signaling is activated in the epiblast and acts as a key regulator of embryonic mesoderm and endoderm development during mammalian gastrulation (Brennan, J. et al. Nodal signaling in the epiblast patterns the early mouse embryo. Nature 411, 965-969 (2001). Ben-Haim, N. et al. The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4. Dev. Cell 11, 313-323 (2006), a NODA / .-knockout (KO) H9 hESC line (Zheng, Y. et al. Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development. Cell Stem Cell 29, 1402-1419 (2022) was used for to analyze PTED embryoid development. Immunostaining analysis of D3 NODAL- PTED embryoids shows that NODAL -KO hESCs give rise to TFAP2A+NNE cells, Bra+gastrulating cells, and TFAP2C+SOX17+PGCLCs (Figures 9A-C) consistent with findings from previous peri-gastrulation human embryoids generated from NODAL-KO hESCs (Zheng, Y. et al. Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development. Cell Stem Cell 29, 1402-1419 (2022). However, endoderm differentiation is completely inhibited in D3 or D5 NODAL-K PTED embryoids evidenced by an absence of FOXA2+cells (Figures 9A and 9C). There is also an absence of cavities in D5 NODAL- O PTED embryoids (Figure 9C). These data support the role of NODAL signaling in human peri-gastrulation development.

[0095] PTED embryoids may be generated from different hPSC lines, including hESCs (Hl and H9 hESCs) and human induced pluripotent stem cells (hiPSCs) (Figures 10A, 10B). Efficiencies of PTED embryoid formation from different hESC lines are comparable. PTED embryoid formation efficiency from hiPSCs may be less than from hESC lines.

[0096] EXAMPLE 2 - Effects of colony size and cell seeding density in embryoid development

[0097] Initial culture conditions play a role in peri-gastrulation embryoid generation (Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847-854 (2014), Shao, Y. et al. A pluripotent stem cell-based model for post-implantation human amniotic sac development. Nat. Commun. 8, 208 (2017). Moris, N. et al. An in vitro model of early anteroposterior organization during human development. Nature 582, 410-415 (2020).). When H9 hESCs are seeded onto Geltrex adhesive islands with a diameter of 400 pm, TFAP2A+NNE cells envelop the entire cell colony, whereas the number of FOXA2+endodermal cells is reduced (Figures 11 A-C). On Day 5 no cavities are present in cell colonies, suggesting failure to generate PTED embryoid structures (Figure 1 ID).

[0098] Cell seeding density plays a role in PTED generation efficiency. As seeding densities of H9 hESCs decrease from 5,000 cells mm'2to 313 cells mm'2for 800 pm diameter adhesive islands, the efficiency of PTED embryoid formation decreases significantly (Figures HE, 1 IF). With low cell seeding densities, hESC colonies may remain as 2D structures, with TFAP2A+NNE cells developing only at colony borders consistent with findings from other 2D human peri-gastrulation embryoids (Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847-854 (2014), Manfrin, A. et al. Engineered signaling centers for the spatially controlled patterning of human pluripotent stem cells. Nat. Methods 16, 640-648 (2019).

[0099] EXAMPLE 3 - Single-cell transcriptomic analysis

[0100] Single-cell RNA-sequencing (scRNA-seq) was used for transcriptomic analysis of D2, D5 and D8 PTED embryoids. Unbiased clustering of cells from 3 PTED embryoids shows distinct cell populations annotated as EPI (epiblast), PS (primitive streak), NM / EM (nascent or emergent mesoderm), AM (advanced mesoderm), ExM (extraembryonic mesoderm), EN (endoderm), NNE, NE (neural ectoderm), PGC, and HEP based on lineage marker expression patterns (Figures 12A, IB). Cell cluster annotations are based on those used for a CS7 human gastrula (Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021). The NNE cluster shows heightened expression of ISI.l, TFAP2A, GATA3, and GABRP, the EN clusters shows expression of SOX17 and FOXA2, and the PS cluster upregulates ZB Tbut downregulates NANOG and SOX2. Cells with NE identity show upregulated SOX2 but downregulated POU5F1 (gene encoding OCT4) ox NANOG, PGC cluster exhibits prominent expression of POU5F1, NANOG, TFAP2C, and SOX17, and HEP cluster shows upregulated expression of CD34 and CDH5 (gene encoding VE-Cadherin) (Figures 12B, 13). PAX6 is not expressed in the NE cluster indicating that cells in the NE cluster may yet to be fully committed.

[0101] Analyzing cellular compositions of D2, D5, and D8 PTED embryoids, respectively, based on scRNA-seq data, shows that only cells associated with EPI, PS, and NNE identities are present in D2 PTED embryoids, whereas other cell clusters, except NE and HEP, appear in D5 PTED embryoids (Figures 13A-F) NE and HEP clusters emerge in D8 PTED embryoids (Figures 13E-G) consistent with immunostaining data in Figures 1C, 5 and 7). Between Day 2 to Day 8, proportions of mesodermal lineages, including cells in PS, NM, EM, AM, and ExM clusters, increase (Figures 14A-D). Differentially expressed genes are calculated for each cluster (Figure 14C) showing gene expression profiles consistent with immunostaining results in Figures 3-5 and 7). Subclustering analysis was conducted for NNE cluster showing 2 distinct subpopulations annotated as amniotic ectoderm or Amnion and embryonic non-neural ectoderm or EmNNE (Figure 14E). The Amnion subcluster shows upregulated expression of ISI.l, TFAP2A, GABRP, VTCN1, ACTC1, and TGFBI. In contrast, the EmNNE subcluster expresses CLDN10, MCM5, TUBG1, GRHL3, and GATA3 consistent with the gene expression profile of embryonic surface ectoderm (Tyser, R. C. V. et al. Singlecell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021) (Figures 14F, 14G).

[0102] CellChat analysis was conducted to infer cell-cell interactions in PTED embryoids, and showed mesodermal lineages (PS, NM, EM, AM, and ExM clusters) as the main sources of BMP, WNT, non-canonical WNT, and TGF-P signals, and EN cluster as a signaling center of FGF signals (Figure 15A). VEGF and KIT ligands that are critically involved in primary hematopoiesis in the secondary yolk sac (Tamaoki, N. et al. Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells. Cells Rep. Methods 3, 100460 (2023), Ivanovs, A. et al. Human haematopoietic stem cell development: from the embryo to the dish. Development 144, 2323-2337 (2017), are mainly secreted from cells with AM, ExM, and HEP and EN identities, respectively (Figure 15 A). Heatmaps of incoming and outgoing signaling patterns further support mesodermal (AM, ExM, and HEP) and endodermal (EN) lineages as the main sources of diverse developmental signals (Figure 15B). ExM-, HEP-, and EN-associated cells show greater outgoing signals associated with ECM-related pathways (such as COLLAGEN and LAMININ) (Figure 15B) indicating their active ECM secretion states consistent with immunostaining results in Figure 5H. EPL, PS-, and PGC- related cells show heightened outgoing NODAL signal intensities, whereas NMZEM- and AM-associated cells appear as the only cells responding to NODAL signals (Figures 15 A, 15B) consistent with the kety role of NODAL in driving mesoendoderm development in vivo (Brennan, J. et al. Nodal signaling in the epiblast patterns the early mouse embryo. Nature 411, 965-969 (2001). Ben-Haim, N. et al. The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4. Dev. Cell 11, 313-323 (2006), and with present data showing inhibition of endoderm differentiation in NODAL-KO PTED embryoids in Figure 9. Developmental trajectory branches were inferred based on pseudotime analysis of integrated scRNA-seq data of D2, D5, and D8 PTED embryoids (Figure 15C). This analysis indicates that the EPI cluster gives rise to 3 main branches, one towards NE development, another towards PGC development, and the third leading to NNE, EN, HEP, ExM, and AM development (Figure 15C). This developmental trajectory branch analysis indicates that embryonic mesoderm and ExM cells in PTED embryoids share common progenies (Figure 15C). Gene regulatory network (GRN) analysis for each cell cluster identified regulons associated with each cluster (Figure 15D).

[0103] D8 PTED embryoid transcriptomes were compared with scRNA-seq data of the CS7 human gastrula (Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021) or with an E20 monkey embryo (CS8) (Shahbazi, M. N., Siggia, E. D. & Zernicka-Goetz, M. Self-organization of stem cells into embryos: A window on early mammalian development. Science 364, 948-951 (2019), respectively. When D8 PTED embryoid data are integrated with those of CS7 human gastrula or of E20 monkey embryo, respectively, high concordances are evident between PTED embryoid and human or monkey cells (Figures 12C-F, 16). Cell clusters of D8 PTED embryoid overlap with their counterparts from CS7 human gastrula and from E20 monkey embryo, respectively (Figures 12C, 12E, 16A, 16F). There are differences as well. The D8 PTED embryoid comprises a NE population that is not present in CS7 human gastrula or in E20 monkey embryo (Figures (Figures 12C, 12E, 16A, 16F). Axial mesoderm (AxM) and blood cell populations in human gastrula are not present in D8 PTED embryoids (Figures 12C, 16A). Blood cell populations evident in E20 monkey embryos is absent in D8 PTED embryoids (Figures 12E, 16F). The lack of blood cell population in D8 PTED embryoid may arise from the absence of hypoblast-derived cells in PTED embryoids. Additional correlation analysis between annotated cell clusters from D8 PTED embryoids and human / monkey embryos further supports transcriptome similarities between corresponding cell clusters from PTED embryoids and human / monkey embryos, respectively, except for EM and EN clusters (Figure 12D, 12F).

[0104] Immunostaining data support the presence of FOXA2+SOX17+HNF4A+yolk sac endoderm-like cells in D8 PTED embryoids (Figures 1C, 5F). Accordingly, subclustering analysis was performed for EN clusters identified from integrated data of D8 PTED embryoid and CS7 human gastrula, or of D8 PTED embryoid and E20 monkey embryo (Figures 16B, 16G). Two subclusters annotate as EEN (embryonic endoderm) characterized by heightened expression of FOXA2 and SOX17, and ExEN (extraembryonic endoderm), marked by upregulated expression of HNF4A and APOA / B / E (Figures 16C, 16D, 16H, 161). ExEN subclusters contain cells both from PTED embryoids as well as from human or monkey embryos (Figures 16B, 16G). Pathway enrichment analysis based on expressed genes in ExEN cells indicates activation of signaling pathways related to metabolic absorption and digestion (Figures 16E, 16J) consistent with a primary function of the yolk sac for nutrient absorption.

[0105] EXAMPLE 4 - Hematopoiesis in PTED embryoids

[0106] Evidence for primary hematopoiesis during early human development is incomplete (Ross, C. & Boroviak, T. E. Origin and function of the yolk sac in primate embryogenesis. Nat. Commun. 11, 3760 (2020)., Tamaoki, N. et al. Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells. Cells Rep. Methods 3, 100460 (2023). Ivanovs, A. et al. Human haematopoietic stem cell development: from the embryo to the dish. Development 144, 2323- 2337 (2017). To investigate primary hematopoiesis using PTED embryoids, a protocol was adopted to further promote EN differentiation in PTED embryoids. The protocol comprises 1- day treatment with both BMP4 and Activin A followed by a 2-day treatment with Activin A alone (Figures 17A, 18A). On Day 3, both FOXA2+SOX17HIGHHNF4A’ EEN-like cells (EENLCs) and FOXA2+SOX17LOWHNF4A+ExEN-like cells (ExENLCs) are evident in PTED embryoids (Figures 18B, 18 A). Both EENLCs and ExENLCs form concentric ring patterns in PTED embryoids with EENLCs predominantly observed at colony border regions, and ExENLCs located adjacent to EENLCs but closer to colony center (Figures 17B, 18 A). scRNA-seq analysis was conducted for D3 PTED embryoids developed under the EN differentiation-enhancing condition (Figures 18B, 18C). Unbiased clustering shows cell clusters annotated as EPI, PS / NNE, NM, EM, AM, EEN, ExEN, and PGC, based on lineage marker expression (Figures 18B, 18C). Cell cluster annotations are mainly based on those used for the CS7 human gastrula (Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021). Under the EN differentiation-enhancing condition, PTED embryoids give rise to cell populations consistent with those from PTED embryoids treated only with BMP4, albeit with expeditated differentiation. In addition, endodermal cells in PTED embryoids become clearly resolved into 2 distinct populations of EENLCs and ExENLCs (Figures 18C, 18D). Geometric boundary confinement of PTED embryoids is necessary for ExENLC differentiation. Under the same EN differentiation-enhancing culture condition, hPSCs seeded onto confinement- free, 2D tissue culture plates, either as single cells or as cell clusters, only give rise to a few if any FOXA2+SOX17LOWHNF4A+ExENLCs (Figure 18D).

[0107] In view of heightened metabolic activities in ExENLCs as shown by pathway enrichment analysis in Figures 16E and 16J, the impact of exogenous lipid supplementation on ExENLC development was tested. Differentiation of ExENLCs is inhibited in PTED embryoids as well as in 2D EN differentiation-enhancing cultures when lipid-rich BSA is supplemented between Day 0 to Day 3 (Figures 18 A, 18D). In contrast, supplementation of lipid-free BSA does not exhibit similar inhibitory effect on ExENLC differentiation (Figure 18 A). In hPSCs under 2D cultures with E6 and lipid-rich BSA, lipid droplets are more abundant and larger compared to in hPSCs cultured without lipid-rich BSA or with lipid-free BSA (Figure 18E). These data support the role of active lipid metabolism by hPSCs in ExENLC development during PTED embryoid formation.

[0108] To test hematopoiesis, PTED embryoids were cultured continuously. Exogeneous morphogens were removed from PTED embryoid culture from Day 3 onwards (Figure 19 A). On Day 6, expression of CD34 and VE-cadherin is evident in PTED embryoids, particularly in cells at colony border regions in keeping with active hematopoiesis (Figures 17B, 19A). In contrast, PTED embryoids treated with lipid-rich BSA between Day 0 to Day 3 do not express CD34 and show limited if any VE-cadherin expression on Day 6 (Figure 19B). Because CellChat analysis in Figure 15A shows prominent VEGF and KIT signals during PTED embryoid development and because both are critically involved in hematopoiesis (Tamaoki, N. et al. Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells. Cells Rep. Methods 3, 100460 (2023), Ivanovs, A. et al. Human haematopoietic stem cell development: from the embryo to the dish. Development 144, 2323-2337 (2017), different VEGFR and c- KIT inhibitors (Axitinib, Motesanib, Linifanib, and Cediranib), respectively, were supplemented in PTED embryoid culture between Day 3 to Day 6. Supplementation of these small molecule drugs inhibits hematopoiesis in PTED embryoids (Figure 17C).

[0109] On Day 14, extensive blood cell formation is evident in PTED embryoids, with blood cells exhibiting round morphologies (Figures 17D, 17F, 19A). A proportion of spherical cells express PU.l, RUNX1, or CD36, indicating their blood cell identities and maturation states (Figures 17E, 19A). A proportion of PU.1+cells display multi-lobed nuclei, a characteristic feature of granulocytes of the immune system (Figure 17E, 19A). A proportion of PU.1+cells also retain expression of CD34 and are in close proximity to a network of CD34+cells (Figures 17E, 19A) indicating a lineage relationship between blood cells and endothelial -like cells in PTED embryoids. Microglial markers CD45, CD1 lb, and Ibal are upregulated in a proportion of mesenchymal-like cells indicating microglial development in PTED embryoids (Figure 19A). PTED embryoids treated with lipid-rich BSA between Day 0 to Day 3 do not exhibit signs of hematopoiesis on Day 14 (Figure 19B). EXAMPLE 5 - Hematopoiesis in PTED embryoid yolk sac

[0110] During mammalian development, a first wave of blood cell production, or primary hematopoiesis, begins when vascular and hematopoietic cells differentiate from ExM progenitors in the yolk sac and progressively organize themselves into blood islands. (Ross, C. & Boroviak, T. E. Origin and function of the yolk sac in primate embryogenesis. Nat Commun 11, 3760 (2020), Tamaoki, N. et al. Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells. Cell Rep Methods 3, 100460 (2023)., Ivanovs, A. et al. Human haematopoietic stem cell development: From the embryo to the dish. Development 144, 2323- 2337 (2017).) In experiments conducted in the development of the present invention, hematopoietic sites in the ExM-like compartment surrounding the yolk sac endoderm-like tissue in PTED embryoids were identified. (Figures 20A, 20B.). CellChat analysis further showed signaling activities in the Endo population that promote HEP induction and hematopoiesis. To test primary hematopoiesis through PTED embryoids with enhanced endoderm differentiation, Activin A was supplemented into PTED embryoid culture between Day 0 and Day 3 (Figure 21 A). On Day 3, both FOXA2+SOX17HIGHHNF4 A' EmEndo- and FOXA2+SOX17LOWHNF4A+ExEndo-like cells were evident forming concentric ring patterns at colony borders (Figure 2 IB). scRNA-seq for these D3 PTED embryoids (Figures 21C, 2 ID, 2 IE) showed that unbiased clustering identifies cell clusters annotated as EpiLC, Gast / NNE, NasM, EmgM, AdvM, EmEndo, ExEndo, and PGCLC Annotations of these cell clusters follow those used for the CS7 human gastrula. (Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021). With enhanced endoderm differentiation, PTED embryoids give rise to consistent peri- gastrulation cell lineages with expedited differentiation and segregation of EmEndo- and ExEndo-like cells. D3 PTED embryoids upregulate KDR (gene encoding VEGFR) and GYPA (gene encoding CD235a) (Figure 21E). Few cells in D3 PTED embryoids express HEP markers CDH5 and CD34. (Goh, I. et al. Yolk sac cell atlas reveals multiorgan functions during human early development. Science 381, eadd7564 (2023).

[0111] Geometric boundary confinement supports ExEndo-like cell differentiation in PTED embryoids. Under the same culture condition as for Activin A-treated PTED embryoids, hPSCs seeded onto confinement-free, 2D tissue culture plates, either as single cells or as cell clusters, gave rise to very few FOXA2+SOX17+HNF4A+ExEndo-like cells Figure 21F). Activin A-treated PTED embryoids begin to detach from underlying glass coverslips on Day 3. Once free-floating, it is difficult to analysis hematopoiesis. Accordingly, re-plated PTED embryoids on glass coverslips on Day 3 were kept under basal E6 medium from Day 3 onwards. On Day 6, positive immunostaining for CD34 and VE-cadherin was evident, particularly in cells at colony borders, supporting HEP development (Figures 20A, 22A, 22B)). To test the roles of VEGF and KIT signaling, which are implicated in PTED embryoid development and are critical for hematopoiesis (Sturgeon, C. M. et al. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561 (2014), Axitinib, motesanib, linifanib, or cediranib was supplemented into E6 medium between Day 3 and Day 6. Each inhibitor blocks HEP development in PTED embryoids (Figure 22B).

[0112] Spatial organization and differentiation of blood cell lineages were examined based on immunostaining of PTED embryoids. On Day 6, HEPs and blood progenitors, marked by CD34 or RUNX1, emerged beneath or among FOXA2+endodermal cells (Figures 20B, 22B). By Day 9, tubular structures composed of CD34+HEPs were evident, often with PU.1+blood progenitors and CD235ab+erythroid cells appearing inside or right outside of the tubular structures (Figures 20C, 22C). Decorin (DCN), an ECM molecule, was detected to separate FOXA2+endodermal layers and hematopoietic endothelial tubes (Figures 20C, 22C). The spatial tissue organization was consistent with hematopoietic islands in the definitive yolk sac (Hislop, J. et al. Modeling post-implantation human development to yolk sac blood emergence. Nature 626, 367-376 (2023), Ross, C. & Boroviak, T. E. Origin and function of the yolk sac in primate embryogenesis. Nat Commun 11, 3760 (2020), Goh, I. et al. Yolk sac cell atlas reveals multi organ functions during human early development. Science 381, eadd7564 (2023). Hematopoietic island-like spatial organization in PTED embryoids was maintained over time, with the hematopoietic sites continuously expanding and more blood cells forming (Figure 22D). On Day 14, a proportion of blood cell colonies without adjacent CD34+HEPs wer observed consistent with HEP exhaustion or blood colony formation from re-inhabited blood progenitors (Figure 22D).

[0113] During primary hematopoiesis, blood cells are derived from hemogenic endothelial cells. (Goh, I. et al. Yolk sac cell atlas reveals multiorgan functions during human early development. Science 381, eadd7564 (2023), Canu, G. & Ruhrberg, C. First blood: The endothelial origins of hematopoietic progenitors. Angiogenesis 24, 199-211 (2021), Lange, L., Morgan, M. & Schambach, A. The hemogenic endothelium: A critical source for the generation of psc-derived hematopoietic stem and progenitor cells. Cell Mol Life Sci 78, 4143-4160 (2021), Calvanese, V. et al. Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature 604, 534-540 (2022), Sugimura, R. et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438 (2017), Motazedian, A. et al. Multipotent ragl+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids. Nat. Cell Biol. 22, 60-73 (2020).) To test the lineage relationship between HEPs and blood progenitors on Day 5, CD42b, a marker of megakaryocytes, was observed to express alongside CD34 in HEP clusters (Figure 23 A). On Day 6 and Day 7, CD235ab+CD34+and CD42b+CD34+cells were evident, indicating emergence of erythroid and megakaryocyte progenitors from CD34+HEPs (Figures 23B, 23C). CD235ab+CD42b+common cell progenitors of erythrocytes and megakaryocytes, also referred to as megakaryocyte-erythroid progenitors (MEPs), were observed (Figure 23B). On Day 9 and Day 14, multiple CD235ab+CD34+, CD42b+CD34+and PU.1+CD34+cells were present adjacent to CD34+cells (Figures 20D, 23D, 23E), thereby indicating a sustained HEP-to-blood progenitor transition. By Day 14, many blood progenitors and blood cells were seen free-floating with a round-shaped morphology (Figure 23E).

[0114] To further test erythrocyte and megakaryocyte development and maturation, on Day 14, both CD235ab+erythroid and CD42b+megakaryoid cells exhibited diverse morphologies, indicating that these cells at different developmental stages (Figures 20E, 24A, 24B, 24C). CD235ab+erythroid cells exhibited different sizes with enucleation and cell nuclei polarized to one side of the cells or being squeezed out from the cells to form bleb-like structures (Figures 20E, 24A, 24B, 24C) in addition to also nucleus-free CD235ab+erythroid cells. As well, CD42b+megakaryoid cells exhibited a range of maturation stages, including cells with single nuclei, cells with multi-lobed nuclei, enlarged cells with multi-lobed nuclei, cells showing membrane budding, and nucleus-free platelet-like structures (Figures 20E, 24A, 24B, 24C). The observed processes of erythropoiesis and megakaryopoiesis are consistent with prior observations (Sugimura, R. et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438 (2017), Dzierzak, E. & Philipsen, S. Erythropoiesis: Development and differentiation. Csh PerspectMed3, a011601 (2013), Italiano, J. E. & Hartwig, J. H. Megakaryocyte development and platelet formation. Platelets, 2nd Edition, 23-44 (2007), Machlus, K. R. & Italiano, J. E. The incredible journey: From megakaryocyte development to platelet formation. J Cell Biol 201, 785-796 (2013).

[0115] Additional mesodermal and endodermal lineages were examined (Figures 24D, 24E, 24F). Spontaneous beating of PTED embryoids was observed as early as Day 12, with beating sites increasing over time (Figure 24D). Beating cells are cTnT+NKX2.5+, indicating their cardiomyocyte identity (Figure 24E). (Hofbauer, P. et al. Cardioids reveal selforganizing principles of human cardiogenesis. Cell 184, 3299-3317 (2021), Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021), Schmidt, C. et al. Multi -chamber cardioids unravel human heart development and cardiac defects. Cell 186, 5587-5605 (2023).) Multiple FOXA2+endodermal cells on Day 7 and nearly all on Day 14 showed upregulated expression of HNF4A, AFP, and APOA4, indicating continuous development of the ExEndo-like compartment in PTED embryoids (Figure 24f). (Ross, C. & Boroviak, T. E. Origin and function of the yolk sac in primate embryogenesis. Nat Commun. 3760 (2020), Mackinlay, K. M. L. et al. An in vitro stem cell model of human epiblast and yolk sac interaction. Elife 10, e63930 (2021),

[0116] To further elucidate cell identities in PTED embryoids, scRNA-seq, colony forming unit (CFU) assays, and flow cytometry were performed (Figures 20F, 20G, 20H, 201, 20J, 25, 26, 27. scRNA-seq data from D14 PTED embryoids shows distinct hematopoiesis-related cell clusters annotated as MEP, Erythroid 1 & 2, Megakaryoid 1 & 2, Myeloid & Lymphoid, and Endothelium, each expressing lineage-specific markers, in addition to Mesoderm 1 & 2 and ExEndo clusters (Figures 20F, 20G, 25A, 25B, 26). Certain myeloid and lymphoid subtypes (for example, granulocyte, monocyte / macrophage, B cell, NK cell, and T cell subtypes) were present. (Figures 20G, 25B). A portion of cells in Endothelium cluster express NT5E, denoting their non-hematopoietic endothelium identity (Figures 25B). Hematopoiesis-related cell clusters were isolated for diffusion map analysis with pseudotime calculations. Developmental trajectories of erythroid, megakaryoid, and myeloid & lymphoid lineages originate from endothelium and MEP (Figure 25C).

[0117] CFU analysis showed early erythrocyte progenitors (BFU-E), late erythrocyte progenitors (CFU-E), granulocyte-erythrocyte-megakaryocyte-monocyte common progenitors (CFU-GEMM), granulocyte progenitors (CFU-G), monocyte progenitors (CFU- M), and granulocyte-monocyte common progenitors (CFU-GM) in D14 PTED embryoids (Figures 20H, 201) in keeping with scRNA-seq data. Flow cytometric analysis showed the presence of neutrophil-like cells (CD15+CD31+), lymphoid-like progenitors (CD117+), monocyte- and macrophage-like cells (CD14+), and B cell progenitor-like cells (CD19+) in CD43+CD45+populations of D9 PTED embryoids (Figure 25D). In D12 PTED embryoids, erythroid (CD235ab+) and megakaryoid (CD42b+) populations were detected, together with myeloid progenitor-like cells (CD33+), erythrocyte progenitors (CD235ab+) and megakaryocyte progenitors (CD42b+) within CD43+populations (Figure 25D) thereby confirming distinct blood subtype identities. Natural killer-like cells (CD56+CD49d+) and T cell progenitor-like cells (CD3+) were identified within CD45+CD14 CD19‘ populations on Day 18 (Figure 25D). Analysis of CD34 and CD43 expression showed dynamic changes in the percentages of CD34+CD43+, CD34+CD43‘, and CD34 CD43+cells in PTED embryoids on different days, indicating transitions from CD34+to CD43+progenitors and indicating additional waves of hematopoiesis in PTED embryoids (Figures 20J, 24E). (Zhai, J. et al. Primate gastrulation and early organogenesis at single-cell resolution. Nature 612, 732-738 (2022), Goh, I. et al. Yolk sac cell atlas reveals multiorgan functions during human early development. Science 381, eadd7564 (2023), Canu, G. & Ruhrberg, C. First blood: The endothelial origins of hematopoietic progenitors. Angiogenesis 24, 199-211 (2021).

[0118] Transcriptomic analysis was conducted for the ExEndo and Mesoderm 1 & 2 clusters in D14 PTED embryoids (Figure 26). Two distinct ExEndo subtypes (ExEndo 1 & 2) were observed, both expressing markers characteristic of ExEndo, including HNF4A, AFP, APOA1 / 4 nd APOB (Figures 26A, 26B). ExEndo 2 showed enriched expression of ECM- and hematopoiesis-related genes (Figure 22C). Based on feature plots of expression patterns of genes enriched in ExEndo and hepatocytes, ExEndo-like cells in PTED embryoids showed a closer resemblance to ExEndo lineage (Figure 26C). (Goh, I. et al. Yolk sac cell atlas reveals multiorgan functions during human early development. Science 381, eadd7564 (2023).) Four mesodermal subclusters were identified from subclustering analysis of Mesoderm 1 & 2 clusters: Mesoderm 1, enriched with ECM-related genes; Mesoderm 2, expressing cardiac mesoderm-related genes (Hofbauer, P. et al. Cardioids reveal selforganizing principles of human cardiogenesis. Cell 184, 3299-3317 (2021), Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021), Schmidt, C. et al. Multi -chamber cardioids unravel human heart development and cardiac defects. Cell 186, 5587-5605 (2023); Mesoderm 3, showing mesothelium (Botting, R. A. et al. Multi-organ functions of yolk sac during human early development. bioBxiv, 2022.2008.2003.502475 (2022), ExM-related gene expression and Mesoderm 4 were identified by upregulated expression of hematopoiesis-related genes (Figures 26D, 26F) Endoderm 2 and Mesoderm 4 upregulated certain hematopoiesis-related genes but without apparent expression of HEP markers (Figures 2 IE, 26C, 26E, 26F). (Sturgeon, C. M. et al. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561 (2014), Boting, R. A. et al. Multi-organ functions of yolk sac during human early development. bioRxiv, 2022.2008.2003.502475 (2022).)

[0119] Cell Chat analysis for D14 PTED embr oids showed multiple interactions among different cell types (Figure 27 A). WNT ligands are primarily secreted by endothelial cells and MEPs (Figures 27B, 27C). ncWNT ligands are nearly exclusively secreted by MEPs (Figures 27B, 27C). Both NOTCH and TGF-P pathways showed extensive connectivity between diverse cell types (Figures 27B, 27C). Endogenous expression of hematopoiesisspecific ligands wsas evident including erythropoietin (EPO) and stem cell factor (SCF), supporting PTED embryoids with an intnnsic hematopoietic property (Figures 27B, 27C)

[0120] EXERIMENTAL METHODS

[0121] Cell culture

[0122] Human pluripotent stem cell (hPSC) lines used include Hl human embryonic stem cell (hESC; WA01, WiCell NIH registration number: 0043), H9 hESC (WA09, WiCell; NIH registration number: 0062), and 1196a human induced pluripotent stem cell (hiPSC; from the University of Michigan Pluripotent Stem Cell Core) line. Cells were maintained in a feeder- free culture system using mTeSRl medium (STEMCELL Technologies, cat #85850). Before cell seeding culture plates were coated with 1% lactate dehydrogenase-elevating virus (LDEV)-free and hESC cell-qualified reduced growth factor basement membrane matrix Geltrex (Thermo Fisher Scientific, cat #A1413302). To ensure the quality of hPSCs, a visual examination was performed during each passage to confirm the absence of spontaneously differentiated, mesenchymal-like cells in cell culture. Cells were used before reaching passage 70. An authentication process for the hPSCs was conducted by both the original sources and in-house. Additionally, immunostaining for pluripotency markers and successful differentiation of hPSCs to the 3 definitive germ layers was performed as part of the authentication process. Karyotype analysis was carried out by Cell Line Genetics to confirm karyotypic normality of hPSCs. hPSCs were tested for negative mycoplasma contamination using LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich, cat #MP0035).

[0123] Cynomolgus monkey embryo

[0124] The Cynomolgus (C ) monkies Macaca fascicular is) originates from Southeast Asia and were housed at Xieerxin Biology Resource and maintained at 25 °C with a relative humidity of 40% - 70% on a 12-hour light-dark schedule. The Xieerxin Biology Resource PCT / US25 / 15505 21 April 2025 (21.04.2025)

[0125] REPLACEMENT SHEET

[0126] Client Docket No. 2023-124-02 Attorney Docket No. UM-42844.601 was accredited by the laboratory animal care facility in Beijing. Cy monkeys were given a commercial diet twice a day with tap water ad libitum and fed vegetables and fruits once a day under veterinary supervision. Before performance of experiments, none of the Cy monkeys received clinical or experimental (drug or test) history that would affect physiological aging or increase susceptibility to disease.

[0127] Oocyte collection, intracytoplasmic sperm injection, pre-implantation embryo culture, and transfer of pre-implantation embryos to foster mothers were performed as described by Yamasaki et al (Yamasaki, J. et al. Vitrification and transfer of cynomolgus monkey (Macaca fascicularis) embryos fertilized by intracytoplasmic sperm injection. Theriogenology 76, 33- 38 (2011). At 6-8 years of age, female Cy monkeys were chosen for oocyte collection accomplished by superovulation with a follicle-stimulating hormone (FSH) using an implantable and programmable microfusion device subcutaneously implanted under ultrasound detection. The day on which collected ova were artificially fertilized by sperm injection was designated as embryonic day 0 (EO). Embryos showing blastocoel cavities around E6 - E7 were selected as high-quality embryos and transferred to appropriate recipient female Cy monkeys. Implanted embryos were monitored by ultrasound scanning from E14 onwards to identify successful pregnancies. Pregnant Cy monkeys were anesthetized with ketamine hydrochloride (10 mg kg'1) before uteri were surgically removed at E22 to obtain Cy monkey embryonic tissues.

[0128] Generation of NODAL-KO hPSCs

[0129] To generate NODAL-knockout (KO) hPSCs (Zheng, Y. et al. Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development. Cell Stem Cell 29, 1402-1419 (2022), a 58-bp segment of genomic DNA within NODAL exon 1 was deleted using CRISPR / Cas9 gene editing technology. Two crRNAs were purchased from Thermo Fisher Scientific: NODAL crRNA l (5’-AGGCUCAGCAUGUACGCCAG-3’ (SEQ ID NO: 1)) and NODAL_crRNA_2 (5 -AGACAUCAUCCGCAGCCUAC-3’(SEQ ID NO: 2)). Duplexes of crRNA:tracrRNA were prepared according to standard protocol and introduced into H9 hESCs along with Cas9 enzyme and pCXLE-EGFP expression plasmid (Addgene plasmid # 27082; RRID: Addgene_27082) to support constitutive expression of EGFP. The NEON electroporation system (Thermo Fisher Scientific) was used to introduce components into hESCs. EGFP-expressing single hESCs were isolated by fluorescence- activated cell sorting (FACS Aria Fusion, BD Biosciences) and seeded onto 96-well plates precoated with Matrigel (Thermo Fisher Scientific) under mTeSR Plus medium supplemented with CloneR single-cell culture supplement (STEMCELL Technologies). To confirm desired deletion, genomic DNA was isolated from single cell-derived clones, and PCR was performed using primers designed for amplification of NODAL exon 1 (forward primer: 5’-CTTCCTTCTGCACGCCTGGTGG-3’(SEQ ID NO: 3); reverse primer: 5’- CCAACCCACAGCACTTCCCGAG-3’(SEQ ID NO: 4)). Resulting amplicons were subjected to Sanger sequencing using primer (5’-CTTCCTTCTGCACGCCTGGTGG- 3’(SEQ ID NO: 5)).

[0130] Microcontact printing

[0131] Polydimethylsiloxane (PDMS; Sylgard 184; Dow Coming) stamps containing circular micropattems were fabricated using a microfabricated silicon mold. PDMS prepolymer, with a curing agent to base polymer ratio of 1 :20, was poured onto the mold and baked at 110°C for 1 hour. After thermal curing, PDMS stamps were peeled off the mold before immersion in 1% Geltrex solution (v / v, diluted in DMEM / F12) and incubated at 37°C for 1 hour. Concurrently, glass coverslips (Thermo Fisher Scientific) were treated with ultraviolet ozone using an Ozone cleaner (Jelight) for 7 minutes. After blow drying with nitrogen gas, Geltrex- coated PDMS stamps were placed in conformal contact with ultraviolet ozone-treated coverslips, to transfer Geltrex adhesive patterns onto coverslips.

[0132] Generation of PTED Embryoids

[0133] On Day 2, hPSCs in tissue culture plates were dissociated using Accutase at 37°C for 8 minutes. Cells were then centrifuged and re-suspended in mTeSRl containing 10 pM Y- 27632 (Tocris, cat #1254), at a concentration of 4 x 106cells mL'1. 140 pL of cell solution was dropped onto 12-mm diameter coverslips that were pre-printed with Geltrex adhesive islands resulting in a cell density of about 5,000 cells mm'2. Coverslips holding cell solutions were placed into a 24-well plate and incubated at 37°C for 30 minutes. Coverslips were gently washed with DMEM / F12 to remove unattached cells before 500 pL of mTeSRl containing Y-27632 (10 pM) was added to the well plate. On Day 1, the culture medium was replenished with 500 pL of mTeSRl without Y-27632 On Days 0 and 1, the culture medium was switched to 500 pL of mTeSRl supplemented with BMP4 (50 ng mL'1; R&D Systems, cat #314-BP-050). On Day 2, the culture medium was switched to 500 pL of mTeSRl. Between Days 5 to 8, embryoids that began to detach from the coverslips were collected and transferred to a low-attachment 96-well plate with one embryoid per well. Of the culture medium in the 96-well plates, half was replenished with fresh mTeSRl daily between Days 5 and 8.

[0134] Whole-mount immunocytochemistry

[0135] Between Days 0 to 4, embryoids were collected, cover-slipped and washed in PBS before fixation with 4% paraformaldehyde (PFA) buffered in 1 x PBS at room temperature (RT) for 1 hour. After fixation, embryoids were permeabilized and blocked with a solution containing 0.3% Triton X-100 and 4% donkey serum at 4°C for 24 hours. Embryoids were then incubated with primary antibody solutions at 4°C for an additional 48 hours. After incubations with primary antibodies, embryoids were labeled with DAPI and donkey-raised secondary antibodies at 4°C for 24 hours. Both primary and secondary antibodies were prepared in 4% donkey serum and 0.3% Triton X-100. After immunostainmg, embryoids were washed with PBS and mounted with Fluoromount-G®. Immunofluorescence images were then captured using the Nikon Al si confocal laser scanning microscope (inverted).

[0136] On Day 8, PTED embryoids were washed with PBS before fixation with 4% PFA buffered in 1 x PBS at RT for 1 hour. After fixation, embryoids were treated with a permeabilization solution comprising PBS containing 0.3% Triton X-100, 2% Glycine, and 20% DMSO at RT for 24 hours. The embryoids were placed in a blocking solution of PBS containing 0.3% Triton X-100, 10% DMSO, and 6% donkey serum at room temperature (RT) for 24 hours. Embry oids were then incubated with primary antibody at RT for an additional 48 hours. Following incubation of the embryoids in the primary antibodies, embryoids were labeled with DAPI and donkey-raised secondary antibodies at RT for 48 hours. Both primary antibodies and secondary antibodies were prepared in a supplemented with 0.2% Tween-20, 10 pg / mL Heparin, 5% DMSO, and 3% donkey serum After immunostaining, embryoids were washed and mounted with Easy Index (LifeCanvas Technologies, RI = 1.52). Immunofluorescence images were captured using the ZEISS LIGHT SHEET 7 and processed using Arivis.

[0137] Quantification of embryoid formation efficiency, length, projected area, and height

[0138] Efficiency of embryoid formation was assessed using bright-field imaging. On Day 5, embryoids displayed asymmetric structural features, with one pole larger than the opposite pole and containing a fluid-filled cavity , while the middle part showed an epithelial-like structure. These structures were characterized as successful embryoids. On Day 8, the embryoid structures were evaluated. Those displaying one pole of a greater size and a cavity with a thick wall, the opposite pole with a smaller size with a cavity enclosed by a monolayer of cells, and a middle part with a columnar epithelial-like structure were considered as successful embryoids. The percentage of successful embryoids was calculated by determining the efficiency of embryoid generation. Bright-held images of successful embryoids were recorded to quantify their length and projected area. To measure the charactenstic size features of Day 5 embry oids, gentle application was employed by use of a slow-flowing pipette to detach the embryoids from culture surfaces.

[0139] To measure the height of the Cy monkey embryoids, cells were seeded and embryoids were cultured on a glass-bottom dish, while keeping the other procedures as described above. After immunostaining, a tissue clearing solution was added. The tissue clearing solution comprised 6.3 mL ddH2O, 9.2 mL OptiPrep™ Densify Gradient Medium (MilliporeSigma, cat #D1556), 4 g N-methyl-D-glucamine (MilliporeSigma, cat M2004), and 5g Diatrizoic acid (MilliporeSigma, cat #D9268). Z-stack confocal images were captured, and the heights of embryoids were measured based on the orthogonal views of DAPI stained images.

[0140] Paraffin-embedded sectioning and immunohistochemistry

[0141] Embryoids were collected and fixed in 4% PFA at 4°C overnight. Embryoids were then washed with PBS and mounted in a mixture of 2% low-melting agarose and 2.5% gelatin (McClelland, K. S., Ng, E. T. & Bowles, J. Agarose / gelatin immobilization of tissues or embryo segments for orientated paraffin embedding and sectioning. Differentiation 91, 68- 71 (2016). Mounted embryoids were then soaked in 70% ethanol at RT for at least 24 hours. Subsequently, a tissue processing dehydration run was performed for 4-6 hours. Mounted embryoids were embedded in paraffin. Tissue sections were obtained using a microtome with a thickness of 5pm, and placed on Superfrost Plus Microscope Slides (Thermo Fisher Scientific). Tissue sections were dried at RT for 48 hours.

[0142] To remove paraffin, tissue sections were washed twice with xylene followed by two washes of 100% ethanol, two washes of 95% ethanol, and lastly two washes of water, each for 5 minutes. The slides were then soaked in IX SignalStain® Citrate Unmasking Solution and heated to 90°C for at least 30 minutes before cooling to RT. After permeabilization and blocking with 0.3% Triton X-100 and 4% donkey serum at RT for 1 hour, tissue sections were incubated in primary antibody solutions at RT for 2 hours. Following primary antibody incubations, tissue sections were labeled with DAPI and donkey -raised secondary antibodies at RT for 1 hour. Both primary and secondary antibodies were prepared in 4% donkey serum and 0.3% Triton X-100. After being washed with PBS, the slides were mounted with Fluoromount-G®. Immunofluorescence images were captured using the Nikon Al si confocal laser scanning microscope (inverted).

[0143] Paraffin-embedded sectioning and immunohistochemistry of monkey embryos

[0144] Immediately after Cy monkey embryos were retrieved from their maternal uten on E22, embryos were fixed in 4% PFA at 4°C overnight. Cy monkey embryos were washed 3 times with 1 x PBS for 5 minutes before being dehydrated through graded alcohol (30%, 50%, 75%, 85%, 95%, and 100%) and xylene. Cy monkey embryos were then embedded in paraffin with embryos oriented with their sagittal planes directly facing the dissecting microscope. The embedded Cy monkey embryos were serially sectioned with a microtome at a thickness of 5 pm. The sectioned Cy monkey embryonic tissues were mounted on glass slides.

[0145] Sectioned Cy monkey embryonic tissues were dewaxed and rehydrated through xylene and graded alcohol (100%, 95%, 85%, and 75%). Glass slides were immersed in 0.01 M citric acid buffer solution (C6H8O7.H2O:C6H5Na3O7.2H2O, 1:9, pH 6.0) and heated in a microwave oven at 92 - 98°C for 15 minutes for antigen retrieval. After cooling to RT for 2 hours, glass slides were washed once in lx PBS for 5 minutes, before incubation with 1% Triton X-100 for 30 minutes and blocking with 2% bovine serum albumin (BSA) for 30 minutes at RT. Sectioned Cy monkey embryonic tissues were incubated with primary antibodies diluted in 2% BSA overnight at 4°C and washed 3 times with PBST for 5 minutes. Sectioned Cy monkey embryonic tissues were then incubated with secondary antibodies diluted in 2% BSA and DAPI (1 mg mL'1) for 1 hour. After 3 washes in PBST for 5 minutes, the slides were mounted with anti-fade mounting medium (Gibco), immunofluoresced and recorded using a laser scanning confocal microscope LSM 780 (Carl Zeiss) and LSM 880 (Carl Zeiss) The images were processed by Zen 7.0 (Carl Zeiss).

[0146] Generation of PTED embryoids with enhanced endoderm differentiation

[0147] On Day -1, hPSCs in tissue culture plates were dissociated with Accutase at 37°C for 8 min to obtain single-cell suspension. Cells were then centrifuged and re-suspended in mTeSRl containing 10 pM Y-27632 at a density of 4 x io6cells mL'1. 140 pL of cell solution was dropped onto a 12-mm diameter coverslip pre-coated with Geltrex adhesive islands, resulting in a cell density of about 5,000 cells mm'2. Coverslips holding the cell solution were then placed in a 24-well plate and incubated at 37°C for 30 min. Coverslips were washed multiple times with DMEM / F12 to remove unattached cells. After washing, 500 pL of mTeSRl containing 10 pM Y-27632 was added to replenish medium. On Day 0, culture medium was switched to 500 pL of E6 medium containing 50 ng mL'1BMP4 and 100 ng mL'1Activin A. On both Day 1 and Day 2, culture medium was replenished with 500 pl of E6 containing 100 ng mL'1Activin A. Samples were fixed on Day 3 for immunocytochemistry to assess endoderm differentiation.

[0148] For culture conditions with lipid-rich BSA or lipid-free BSA, either 1% (m / m) lipid- rich BSA (AlbuMAX™, Gibco, cat #11020021) or 1% (m I m) lipid-free BSA (fatty' acid-free Bovine Serum Albumin, Sigma-Aldrich, cat # A8806) was supplemented in the culture medium from Days 0 to 3.

[0149] For endoderm 2D cultures, hPSCs were dissociated on Day -1 using either Accutase or Dispase (STEMCELL Technologies, cat #07923) to obtain single cells or cell clusters, respectively. Singly dissociated hPSCs were re-suspended in mTeSRl containing 10 pM Rho-associated kinase (ROCK) inhibitor Y-27632 (Y-2732), whereas hPSC clusters were suspended in mTeSRl without Y-27632. Singly dissociated hPSCs or hPSC clusters were then seeded into a 24-well plate, with a 12-mm coverslip placed in each well, at a cell density of about 300,000 cells per well. On Day 0, culture medium was switched to E6 medium containing 50 ng mL'1BMP4 and 100 ng mL'1Activin A. On both Day 1 and Day 2, culture medium was replenished with 500 pL of E6 medium containing 100 ng'1Activin A. Samples were fixed on Day 3 for immunocytochemistry to assess endoderm differentiation.

[0150] For prolonged culture of PLED embryoids with enhanced endoderm differentiation, embryoids on Day 3 were detached from coverslips by mechanically removing with pipette tips. Detached cell colonies were then transferred to a new 24-well plate, with each well holding one coverslip E6 supplemented with 1% Geltrex was added. On Day 4, culture medium was switched to E6, and replenished with fresh E6 daily from Day 4 to Day 14.

[0151] For the VEGF and KIT inhibitors assay, PTED embryoids with enhanced endoderm differentiation were generated. E6 containing 50 ng mL-1 BMP4 and 100 ng mL-1 Activin A was replenished from Day 0 to Day 1, and then E6 containing 100 ng mL-1 Activin A was replenished from Day 1 to Day 3. Then VEGF and KIT inhibitors were supplemented from Day 3 to Day 6 in E6. The inhibitors used in the assay werel.O pM Axitinib (Selleckchem, cat #S 1005), 1.0 pM Motesanib (Selleckchem, cat #S 1032), 0.5 pM Linifanib (Selleckchem, cat #S1003), or 0.5 pM Cediranib (Selleckchem, cat #S 1017), individually. The samples were fixed on Day 6 for hematopoiesis inhibition assessment. Lipid staining

[0152] On Day 1 tissue culture plates in hPSCs were dissociated using Dispase at 37°C for 7 minutes. Cells were scraped off and broken into small clusters Cell clusters were seeded into well plates containing coverslips pre-coated by 1% Geltrex under mTeSRl as culture medium. On Day 0, the culture medium was switched to E6 alone, E6 supplemented with 1% lipid-rich BSA, or E6 supplemented with 1% lipid-free BSA. From Days 0 to 3, the culture medium was exchanged daily, maintaining respective culture environments based on experimental conditions. On Day 3, Oil Red 0 staining was conducted using Lipid Staining Kit (Abeam, cat #ab287838). Cells were fixed by 10% Formalin before incubation with 60% isopropanol and Oil Red 0 Working Solution. After thoroughly washing with dFLO, coverslips with cells were mounted using Fluoromount-G®. Bnght-field images were recorded using the Vectra Polaris scanner with a 40* objective lens.

[0153] Quantification of fluorescent intensity

[0154] Quantitative analysis of fluorescent intensity was performed using Fiji ImageJ (Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis Nat. Methods 9, 676-682 (2012). Z-stack confocal images were used to generate a maximum intensity projection. Using the oval selection tool in ImageJ a sample region of interest (ROI) was chosen before employing the "Radial Profile" function to plot quantitative fluorescent intensities along the radial direction. The fluorescent intensities for a specific marker were normalized by dividing by the maximum intensities before plotting.

[0155] Colony forming unit (CFU) assay

[0156] Day 14 PTED embryoids with enhanced endoderm differentiation were trypsinized into single cells and suspended in IMDM medium (Thermo Fisher Scientific, Gibco™, cat # 12440053). The cells were then mixed with MethoCult medium (STEMCELL Technologies, MethoCult™ SF H4636, cat #04636) and seeded into 35 mm non-treated dishes at a density of 10,000 cells per dish, following vendor instructions. The colonies were incubated at 37°C with 5% CO2 for 14 days before evaluation and counting.

[0157] Flow cytometry analysis of PTED embryoids with enhanced endoderm differentiation

[0158] Flow cytometry analysis was performed on PTED embryoids with enhanced endoderm differentiation at Day 5, Day 7, Day 9, Day 12, and Day 18. The embryoids were dissociated into single cells as follows. Day 5 and Day 7 PTED embryoids were incubated with Accutase for 0.5-1 hour at 37°C until completely dissociated into single cells. Day 9, Day 12, and Day 18 PTED embry oids were incubated with a 1:1 mixture of 2.5 mg / mL Liberase TL (Millipore-Sigma, cat #5401020001) in HBSS and Dispase, supplemented with 0. 1 mg / mL DNase I (Millipore-Sigma, cat #11284932001) for 0.5 hour at 37°C. The samples were then collected and centrifuged. The supernatant was aspirated, and the pellet was resuspended in Accutase and incubated for 0.5-1 hour at 37°C until the tissue was dissociated into single cells. During their incubation, the samples were pipetted up and down every 10 min to promote cell dissociation.

[0159] The resulting cell solution was strained through a 70 pm filter, pelleted by centrifugation, and resuspended in Live / Dead staining buffer (eBioscience™ Fixable Viability Dye eFluor™ 780, Invitrogen, cat #65-0865-14, 1:5000 dilution in HBSS). The cells were incubated at RT for 30 minutes, pelleted again, and washed once with Cell Staining Buffer (BioLegend, cat #420201). The cells were then resuspended in an antibody cocktail prepared in Cell Staining Buffer containing Fc Receptor Blocking Solution (BioLegend, cat #422301) and Bnlliant Stain Buffer (Invitrogen, cat #00-4409-42), with a cell concentration of approximately 1 x 106cells per 100 pL. The mixture was incubated at RT for 30 minutes, pelleted by centrifugation, and resuspended in Fixation Buffer (BioLegend, cat #420801). The cells were incubated at RT for another 30 minutes, pelleted, and washed once with Cell Staining Buffer. Finally, the cells were resuspended in Cell Staining Buffer and stored at 4°C, protected from light.

[0160] Leukocytes (BioLegend, cat #426004) and CD34 PBMCs (BioLegend, cat #426901) were prepared according to vendor instructions as positive controls. Unstained cells and cells stained only with live / dead dye were used as negative controls. Flow cytometry was performed using the Cytek Aurora Spectral Analyzer following standard procedures. UltraComp eBeads (Invitrogen, cat #01-2222-42) and the Arc Amine Reactive Kit (Invitrogen, cat # Al 0628) are used for compensation before analyzing cells. Data analysis was conducted using FlowJo 10. 10.0.

[0161] Single-cell dissociation and RNA-sequencing

[0162] On Days 2 and 5, embry oids were washed in PBS 2-3 times and incubated with Accutase for 0.5 - 1 hour at 37°C until the embry oids were dissociated into single cells. On Day 8 embryoids, were washed in PBS and incubated with Try psin for 1 hour at 37°C. During their incubation, the samples were pipetted every 10 minutes to promote cell dissociation. Dissociated single cells were collected and placed into PBS containing 2% BSA before being centrifuged at 200 g for 5 minutes. Resultant cell pellets were re-suspended into single cells in PBS containing 2% BSA. Cell filtration was performed using a40-pm cell strainer on Day 2 and Day 5 embryoids. On Day 8 embryoids cell filtration was performed using a 70-pm cell strainer to obtain single-cell suspensions. Within 1 hour after cell dissociation, cells were loaded into the 10x Genomics Chromium system (10x Genomics). The 10x Genomics v.3 libraries were prepared according to the manufacturer’s instructions. Libraries were then sequenced using paired-end sequencing with a minimum coverage of 20,000 raw reads per cell using Illumina NovaSeq-6000. The scRNA-seq data were aligned and quantified using Cell Ranger Single-Cell Software 719 Suite (v.3.1.0, 10x Genomics) against the Homo sapiens (human) genome assembly GRCh38.pl3 from ENSEMBL.

[0163] Data integration, dimensionality reduction, and clustering

[0164] Analysis of scRNA-seq data and integration of scRNA-seq datasets were performed using Seurat (v.4.3.0) R package (Hao, Y. H. etal. Integrated analysis of multimodal single- cell data. Cell 184, 3573-3587 (2021), Stuart, T et al. Comprehensive Integration of SingleCell Data. Cell 177, 1888-1902 (2019), Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411-420 (2018), Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495-502 (2015). Default settings in Seurat R package were used unless noted otherwise. scRNA-seq data for a single cell batch was first filtered based on the total number of detected genes and percentage of mitochondrial genes. Gene expression was then normalized by the raw count divided by the total count before multiplying by 10,000 and log transformed. The top 2,000 highly variable genes were then identified for each dataset using FindVanabl eFeatures. The cell cycle was then regressed out based on cell cycle scores using CellCycleScoring during data scaling process using ScaleData. PCA analysis (RunPCA) was then performed on filtered data followed by embedding into low dimensional space with Uniform Manifold Approximation and Projection (UMAP; RunUMAP). Cell clusters were identified by a shared nearest neighbor (SNN) modularity optimization-based clustering algorithm achieved using FindClusters.

[0165] For integration of different scRNA-seq datasets count matrices of different datasets were filtered separately for each dataset. On Days 2 and 5 datasets were down-sampled by randomly selecting 1,000 cells from the Day 2 dataset and 3,000 cells from the Day 5 dataset to balance cell numbers in different clusters in integrated datasets. Each integrated dataset was normalized separately, and highly variable features were selected before being integrated using IntegrateData based on 2,000 anchor features. After dataset integration, an integrated scRNA-seq dataset was analyzed following a standard Seurat pipeline. Annotation of cell clusters ws based on the expression of canonical lineage marker genes.

[0166] Dot plots and feature plots were generated using DotPlot and FeaturePlot in Seurat, respectively. Differentially expressed genes (DEGs) for each cluster were identified using Find AllMarkers, with a minimal fold difference of 0.25 in the logarithmic scale between the cluster of interest and all other clusters. Heatmaps were plotted based on top 15 highly differentially expressed genes using plot heatmap in Scillus package.

[0167] Analysis of cell-cell interactions

[0168] R package CellChat v 1.6.1 was used to perform cell-cell communication analysis (Jin, S. Q. et al. Inference and analysis of cell-cell communication using CellChat. Nat. Commun. 12, 1088 (2021). CellChat infers and analyzes intercellular communication networks from scRNA-seq data using network analysis and pattern recognition. A Seurat object including the count matnx and clustering results from the integrated dataset of Days 2, 5 and 8 embryoids, was imported to CellChat. A default human database was used for analysis. Only secreted signaling pathways from Kyoto Encyclopedia of Genes and Genomes (KEGG) were used. Default values were used for all parameters.

[0169] Trajectory branches interference using pseudotime

[0170] Developmental trajectories were inferred for embryoid development in the form of a branching tree using R package URD vl.1.1 (Farrell, J. A. et al. Single-cell reconstruction of developmental trajectories dunng zebrafish embryogenesis. Science 360, eaar3131 (2018), Uzquiano, A. et al. Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex. Cell 185, 3770-3788 (2022). The diffusion map was computed using R package Destiny (Angerer, P. et al. destiny: diffusion maps for large-scale single-cell data in R. Bioinformatics 32, 1241-1243 (2016) by invoking the calcDM function from URD on normalized counts from embryoid datasets (sigma. use = “local”). Root cells were manually assigned. For analysis of the embryoids dataset integrated on Days 2, 5 and 8, EPI clusters from Day 2 embryoids were assigned as root cells. Simulating diffusion from the root to each cell, cells were ordered in pseudotime by floodPseudotime (n = 30, minimum. cells. flooded = 5) and HoodPseudotimeProcess functions. Terminal states of the tree (“tips”) were manually selected based on in vivo knowledge. To detect developmental trajectories from scRNA-seq data, 10,000 biased random walks per tip were simulated using simulateRandomWalksFromTips. Walks were then processed into visitation frequencies using RandomWalksFromTips. Branching trees were established using buildTree. The following parameters were used for building the branching tree in this work: visitthreshold = 0.7, minimum. visits = 10, bins. per.pseudotime. window = 5, cells.per.pseudotime.bin = 50, divergence. method = “preference”, and p thresh = 0.05.

[0171] Gene regulatory network analysis

[0172] Regulatory activity of transcription factors associated with specific cell types was assessed using the R-package SCENIC (Single Cell Regulatory Network Inference and Clustering, v. l.1.2-01) and Python package Arboreto (Aibar, S. et al. SCENIC: single-cell regulatory network inference and clustering. Nat. Methods 14, 1083-1086 (2017). Filtered counts of the integrated Seurat object were used as inputs of SCENIC. GRNBoost2 in Arboreto was used to infer co-expression modules between transcription factors and candidate target genes. Each co-expression module was then analyzed using cis-regulatory motif analyses (RcisTarget). Only modules with significant motif enrichment of the correct up-stream regulator were retained. The human motif collection v9 and the cisTarget databases for hg38 were used in the pipeline (resources.aertslab.org / cistarget / databases / old / homo_sapiens / hgl9 / refseq_r45 / mc9nr / gene_b ased / ). Filtered counts of the integrated Seurat object were used as input of SCENIC. All default parameters were used in SCENIC unless noted otherwise. Normalized intensity of regulons with top AUC were plotted in the heatmap.

[0173] Integration of human embryo data

[0174] The scRNA-seq dataset of the CS7 human gastrula (Tyser, R. C. V. etal. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature 600, 285-289 (2021) was integrated with the Day 8 embryoid scRNA-seq dataset. Because the human gastrula scRNA-seq dataset was generated using Smart-seq2 to compare with Day 8 embryoid scRNA-seq dataset generated using the 10 / Genomics Chromium system, raw counts of each cell in the human gastrula were normalized to exon sizes before being utilized to create the Seurat object (input count = raw count x 1,000 / exon size). Exon size information was obtained from GRCh38.pl3, ENSEMBL. To re-analyze the scRNA-seq dataset of the human gastrula, relevant cell types were processed with the default Seurat pipeline, including data normalization, highly variable feature selection, scaling (including cell cycle regression), and processed by PCA and UMAP. To integrate scRNA-seq data from the human gastrula with those of PTED embryoids of the present invention, scRNA-seq datasets from Day 8 embryoids were fdtered, normalized, scaled (including cell cycle regression) and processed by PCA and UMAP before cluster analysis. Down-sampling of the Day 8 embryoid dataset was performed using Subset function that randomly selects 150 cells from every cluster in the original datasets to form anew Seurat object. This step prevents larger datasets from dominating downstream analysis. Thereafter, the Day 8 embryoid dataset and the human embryo dataset were integrated using IntegrateData function. After integration, the integrated scRNA-seq dataset was analyzed following the standard Seurat pipeline. Annotations of cell clusters were based on expression of canonical lineage marker genes. To calculate a correlation matrix, average expression of the top 2,000 highly variable genes were calculated for each cluster grouped by cell lineage identification and cell origin using AverageExpression function. Pearson’s correlation coefficients were then obtained using cor function.

[0175] Integration of cynomolgus monkey embryo data

[0176] The scRNA-seq dataset of CS8 E20 cynomolgus (Cy) monkey (Macaca fascicularis) embryos (Zhai, J. et al. Primate gastrulation and early organogenesis at single-cell resolution. Nature 612, 732-738 (2022) was integrated with the Day 8 embryoid scRNA-seq dataset. To integrate datasets from different species, gene names from each dataset were matched according to orthologs between human and macaque ENSEMBL genes (http: / / useast.ensembl.org / biomart / martview). Datasets of PTED embryoids and Cy monkey were then processed with the default Seurat pipeline, including data normalization, highly variable feature selection and scaling (including cell cycle regression) and processed by PCA and UMAP before their integration using IntegrateData function. The integrated scRNA-seq dataset was then analyzed following the standard Seurat pipeline. Annotations of cell clusters were based on expression of canonical lineage marker genes. To calculate the correlation matrix, average expression of the top 2,000 highly variable genes for each cluster were grouped by cell lineage identification and cell origin. The calculation was performed using the AverageExpression function. Pearson’s correlation coefficients were then obtained using cor function.

[0177] Sub-clustering analysis Clusters of interest were selected using subset function. Subsequently, the selected dataset underwent processing, including scaling that involved cell cycle regression, PC A, UMAP, and clustering identification, as described in the default Seurat pipeline The annotation of cell clusters was based on the expression of canonical lineage marker genes

[0178] Pathway enrichment analysis

[0179] Pathway enrichment analysis was conducted using clusterProfiler v.4.6.2 R package (Wu, T. et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (Camb) 2, 100141 (2021), Yu, G. C„ Wang, L. G., Han, Y Y. & He, Q Y. clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters. Omics-a Journal of Integrative Biology 16, 284-287 (2012). Positive DEGs were selected for extraembryonic endoderm (ExEN) clusters using Find AllMarkers in Seurat package. Enriched pathways were then calculated based on DEGs using gseKEGG function.

[0180] Statistical analysis

[0181] Statistical analysis was conducted using independent, two-tailed Student’s t-tests in Excel (Microsoft), p < 0.05 was considered statistically significant.

[0182] INCORPORATION BY REFERENCE

[0183] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

Claims

CLAIMSWe claim:

1. A method of generating a peri-gastrulation trilaminar embryonic disc (PTED) embryoid, comprising: a) providing one or more pluripotent stem cells (PSCs); b) treating said one or more PSCs with bone morphogenic protein 4 (BMP4); and c) applying said one or more PSCs treated with said BMP4 to a micropattemed adhesive surface to generate said PTED embryoid.

2. The method of claim 1, wherein said one or more PSCs comprises a mammalian PSC and said PTED embryoid is a mammalian PTED embryoid.

3. The method of claim 2, wherein said mammalian PSC is a human PSC (hPSC) and said PTED embryoid is a human PTED embryoid.

4. The method of claim 3, wherein said hPSC comprises one or more human embryonic stem cells (hESCs).

5. The method of claim 4, wherein said hESCs are Hl and / or H9 hESCs.

6. The method of claim 3, wherein said hPSC comprises one or more human induced pluripotent stem cells (hiPSCs).

7. The method of claim 1, further comprising treating said one or more hPSCs with Activin A.

8. The method of claim 1, wherein said micropatterned adhesive surface comprises a morphogenetic field and / or a geometric boundary confinement.

9. The method of claim 1, wherein said micropatterned adhesive surface comprises one or more adhesive islands comprising a basement membrane extract comprising laminin, collagen IV, entactin, and heparin sulfate proteoglycans on one or more glass coverslips.

10. The method of claim 1, wherein said PTED embryoid comprises a trilaminar embryonic disc.

11. The method of claim 10, wherein said trilaminar embryonic disc comprises a trilaminar embryonic layer.

12. The method of claim 11, wherein said trilaminar embryonic layer is between a dorsal amnion and a ventral secondary yolk sac.

13. The method of claim 1, wherein said PTED embryoid comprises a secondary yolk sac-like structure.

14. The method of claim 1, further comprising incubating said PSC cells in mTeSRl medium.

15. The method of claim 14, wherein said incubating comprises incubating said hPSC cells for at least 48 hours.

16. A method of testing pre-gastrulation and peri-gastrulation, early human embryogenesis, infertility, human primordial germ cell development, primary hematopoiesis, blood cell generation or toxicity screening, comprising: applying an effective amount of an inhibitory or stimulatory compound to said PTED embryoid of claim 1.

17. The method of claim 1, further comprising detecting and / or monitoring at least one property of at least one said PTED embryoid wherein said at least one property comprises at least one of: a) embryoid formation; b) endoderm differentiation; c) mesoderm differentiation; d) primordial germ cell differentiation; e) amnion differentiation; f) amnion cavity formation; g) yolk sac cavity formation;h) primary hematopoiesis and blood cell generation; i) single-cell dissociation; j) one or more cell-cell interactions; k) embryoid branching trajectory; and l) fluorescence emission; and wherein said step of detecting and / or monitoring comprises one or more of nucleic acid sequencing, RNA sequencing, tissue sectioning, immunohistochemistry, optical detection, light intensity detection optical imaging, microscopy, photography and videography.

18. A method of endoderm differentiation of a PTED embryoid, comprising: a) detaching one or more PTED embryoids from one or more coverslips; b) transferring said one or more PTED embryoids to a multi-well plate; c) supplementing a medium with an extracellular matrix; d) incubating said one or more PTED embryoids in said medium daily for 10 days; and e) testing for the presence of endoderm differentiation of said PTED embryoid.

19. The method of claim 18, wherein said medium is Essential 6 (E6) medium.

20. The method of claim 18, wherein said extracellular matrix comprises a basement membrane extract comprising laminin, collagen IV, entactin, and heparin sulfate proteoglycans.

21. A PTED embryoid generated by a method of any of claims of 1-15.

22. Use of a PTED embryoid generated by a method of any of claims 1-15.

23. A PTED embryoid generated in vitro comprising a trilaminar embryonic disc and a secondary yolk sac-like structure.

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