System and methods for generation of human fetal progenitors, tissues, and organs from cultured stem cells

Abstract

The present disclosure generally relates to methods and in vitro system for generation of embryo like structures. The disclosure further provides methods for use of the embryo like structures in regenerative medicine and drug screening.

Description

SYSTEM AND METHODS FOR GENERATION OF HUMAN FETAL PROGENITORS, TISSUES, AND ORGANS FROM CULTURED STEM CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63 / 510,612, entitled, “SYSTEM AND METHODS FOR GENERATION OF HUMAN FETAL PROGENITORS, TISSUES AND ORGANS FROM CULTURED STEM CELLS” filed June 27, 2023, and U.S. Provisional Patent Application No. 63 / 590,107, entitled, “SYSTEM AND METHODS FOR GENERATION OF HUMAN FETAL PROGENITORS, TISSUES, AND ORGANS FROM CULTURED STEM CELLS” filed October 13, 2023. The contents of each of the aforementioned applications are hereby incorporated by reference in its entirety.BACKGROUND

[0002] 1. Field

[0003] The present disclosure relates to methods and in vitro culture system for generation of embryo like structures and method of using the same for cell replacement therapy, tissue regeneration or repair, or in vitro drug screening.

[0004] 2. Background

[0005] The extraordinary capacity for self-organization in developing mammalian embryos orchestrates a series of complex and precisely coordinated morphogenetic processes, ultimately leading to the formation of a fully developed organism. Early embryonic development involves a sequence of cleavage divisions and initial cell fate determinations that facilitate the progression of the developing embryo from a totipotent zygote to a blastocyst, encompassing the foundational tissues of a conceptus: the epiblast and two extraembryonic lineages, the hypoblast (also known as primitive endoderm in mice) and trophoblast. The epiblast gives rise to all fetal tissues, while the hypoblast and trophoblast develop into yolk sac and placenta, respectively. Epiblast, hypoblast, and trophoblast cells have been successfully recapitulated in vitro as distinct embryonic and extraembryonic stem cells, such as embryonic stem cells (ESCs), trophoblast stem cells (TSCs), and extraembryonic endoderm (XEN) stem cells. Through either random or guided in vitro differentiation, extensive previous studies have demonstrated that these stem cells can generate cell derivatives that resemble their in vivo counterparts. For instance, ESCs can differentiate into all three germ layers and germ cells, XEN cells can differentiate into visceral endoderm and parietal endoderm, and TSCs are capable of developing into extravillous trophoblast and syncytiotrophoblast.

[0006] Although mouse ESCs primarily remain lineage-restricted, they can differentiate into extraembryonic cell types under specific conditions. For example, spontaneous conversion from ESCs to XENs has been observed. This evidence supports the idea that early epiblast-derived stem cells possess plasticity toward extraembryonic lineages. Consistent with this concept, mouse extended or expanded pluripotent stem cells (EPSCs), which have a transcriptional profile similar to early (E4.5-5.5) mouse epiblast, have demonstrated bidirectional developmental potency. These mouse EPSCs contribute to both embryonic and extraembryonic lineages in vitro and in vivo. Similarly, human EPSCs have been shown to contribute to both embryonic and extraembryonic tissues when injected into early mouse embryos.

[0007] The derivation of various embryonic and extraembryonic stem cells has facilitated the creation of early embryo models through either self-organization or assembly methods. Most notably, integrated embryo models derived from stem cells, which comprise both embryonic and extraembryonic tissues, have demonstrated significant potential for modeling gastrulation and early organogenesis in mice. Conventional human pluripotent stem cells (PSCs), including hESCs and human induced pluripotent stem cells (hiPSCs), represent primed pluripotency with limited potential for developing extraembryonic lineages. Although well-established non-integrated models of human gastrulation based on primed PSCs, such as 2D and 3D gastruloids, exist, their lack of extraembryonic tissues hinders their capacity to study key morphogenetic events during peri-gastrulation development, which include the formation of bilaminar disc, primitive streak, trilaminar disc and yolk sac, and others, as well as interactions between extraembryonic and embryonic tissues. Additionally, although integrated models of human blastocysts, referred to as blastoids, have been recently developed, they exhibit limited capacity for post-implantation development. Thus, there is an unmet need for developing an integrated model that accurately mirrors key processes during peri-gastrulation development.SUMMARY

[0008] In one aspect, the disclosure encompasses a method of generating a peri- gastruloid having embryo-like structures. In such aspects the method comprises obtaining a three-dimensional culture of pluripotent stem cells (PSCs) in a first culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF- P) activator, a Wingless-related integration site (WNT) activator, and a Mitogen-activated protein kinase kinase (MEK) inhibitor; maintaining the cells in the first culture medium for about 1 to 7 days; changing the culture medium to a second culture medium, wherein the second culture medium comprises a FGF activator, a TGF-p activator, and a MEK inhibitor; andmaintaining the three-dimensional culture of pluripotent stem cells (PSCs) in the second culture medium, to generate the peri-gastruloids having embryo-like structures.

[0009] In further aspects, provided herein is a method of generating embryo-like structures from pluripotent stem cells (PSCs). In such aspects, the method comprises obtaining a three- dimensional culture device, wherein the three-dimensional culture device comprises PSCs and a first culture medium, wherein the first culture medium comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, a WNT activator, and a MEK inhibitor; maintaining the cells in the first culture medium for about 1-7 days; changing the culture medium in the three-dimensional culture device to a second culture medium, wherein the second culture medium comprises a FGF activator, a TGF-p activator, and a MEK inhibitor; maintaining the cells in the second culture medium for about 2-7 days; changing the culture medium in the three-dimensional culture device to a third culture medium, wherein the third culture medium comprises IVC medium supplemented with Matrigel®, Geltrex®, or VitroGel®; and maintaining the cells in the third culture medium for about 1-7 days to generate the embryo-like structures.

[0010] The disclosure further encompasses an in vitro system. In such aspects, the in vitro system comprises pluripotent stem cells (PSCs); a three-dimensional culture device; a first culture medium comprising a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, a WNT activator, and a MEK inhibitor; second culture medium comprising a FGF activator, a TGF-p activator, and a MEK inhibitor; and a third culture medium comprising IVC medium supplemented with Matrigel®, Geltrex®, or VitroGel®; and a means for replacing the first culture medium with the second culture medium and / or the second culture medium with the third culture medium.

[0011] In some aspects, the disclosed PSCs comprise a single cell suspension of PSCs. In some aspects, the PSCs comprise extended pluripotent stem cells (EPSCs). In some aspects, the PSCs comprise human PSCs, primate PSCs, rodent PSCs, porcine PSCs, bovine PSCs, ovine PSCs, or equine PSCs. In some aspects, the three-dimensional culture device is a microwell plate. In some aspects, the three-dimensional culture device is a AggreWell™ plate. In some aspects, about 1-200 PSCs are added to an individual well of a three-dimensional culture device.

[0012] In certain aspects, the disclosed first and second culture medium comprise N2B27 basal medium. In some aspects, the FGF activator is FGF2. In some aspects, the FGF2 activator is present in the culture medium at about 1 ng / ml to about 200 ng / ml. In some aspects, the TGF-p activator is Activin-A. In some aspects, the Activin-A is present in the culture medium at about 1 ng / ml -200 ng / ml. In some aspects, the WNT activator is aGSK3 inhibitor. In some aspects, the GSK3 inhibitor is CHIR99021. In some aspects, the CHIR99021 is present in the culture media at about 0.1 pM to about 5 pM. In some aspects, the MEK inhibitor is PD0325901. In some aspects, the MEK inhibitor is present in the culture medium at about 0.1 pM to about 1 pM of PD0325901.

[0013] In some aspects, the method or system disclosed herein further comprises a fourth medium, wherein the fourth culture medium comprises IVC medium supplemented with Matrigel®, Geltrex®, or VitroGel® and Knock out serum (KSR) and maintaining the cells in the fourth culture medium for about 1-14 days. In some aspects, the Matrigel®, Geltrex®, or VitroGel® is present in an amount of about 1-100% (Vol / Vol). In some aspects, KSR is present in the medium at about 1-100% (vol / vol).

[0014] In some aspects, the peri-gastruloids or the embryo-like structures comprise epiblast-like, hypoblast- 1 ike compartments, or any combination thereof. In some aspects, the peri-gastruloids or the embryo-like structures comprise epiblast-like cells, amnion-like cells, hypoblast- 1 ike cells, yolk sac-like cells, or any combination thereof. In some aspects, the peri-gastruloids or the embryo-like structures comprise yolk sac-like cavity, amniotic-like cavity or any combination thereof. In some aspects, the peri-gastruloids or the embryo-like structures comprise primordial germ cell-like cells. In some aspects, the peri-gastruloids or the embryo-like structures comprise bilaminar disk-like structure, trilaminar disk-like structure, primitive streak like structure, embryonic disk-like structure, primitive groove like structure, or any combination thereof. In some aspects, the peri-gastruloids or the embryolike structures comprise neural ectoderm cells, non-neural ectoderm cells, amniotic ectoderm cells, surface ectoderm cells, endoderm cells, yolk sac endoderm cells, nascent mesoderm, advanced mesoderm cells, lateral plate mesoderm, cardiogenic mesoderm, presomitic mesoderm, or any combination thereof.

[0015] In some aspects, the disclosure further encompasses a peri-gastruloid or embryo-like structure produced by the method or system disclosed herein. In certain aspects, an embryo model comprising a peri-gastruloid or embryo-like structure produced by the disclosed or system is further provided.

[0016] In some aspects, the disclosure comprises a method of cell replacement therapy. In such aspects, the method comprises administering in a subject in need thereof, cells from a peri-gastruloid or embryo-like structure disclosed herein.

[0017] In some aspects, a method of repairing or regenerating a tissue or organ comprising administering in a subject in need thereof, cells from a peri-gastruloid or embryolike structure of disclosed herein, is further provided.

[0018] In certain aspects, an in vitro drug or chemical screening system is provided, the system comprising a peri-gastruloid or embryo-like structure disclosed herein.

[0019] In some aspects, provided herein is a method of high-throughput genetic or chemical screening, comprising testing genetic mutations or chemicals that can cause pregnancy loss or congenital defects using the in vitro drug or chemical screening system disclosed herein.

[0020] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGs. 1A-1 M show differentiation and culture of epiblast-like and hypoblast- 1 ike cells from hEPSCs. FIG. 1A shows representative brightfield and fluorescence merged images of hEPSCs colonies (top panel) and hEPSCs cultured in tHDM medium (bottom panel). Green, SOX2-citrine; Red, SOX17-tdTomato. Scale bars, 100 pm. FIG. 1B shows representative brightfield and fluorescence merged images of hEPSCs long-term cultured in tHDM medium, passage #12. Scale bars, 360 pm (top) and 130 pm (bottom). FIG. 1C shows schematic illustrating the chronological sequence and critical stages of the initial peri- gastruloid derivation protocol that starts with tHDM pre-treatment of attached hEPSCs. Four days prior to seeding cells into an AggreWell™ plate, the hEPSCs were treated with tHDM, defined as day -4. On day 0, the cells were seeded into the AggreWell™ plate. In the following days the cells were cultured in the indicated conditions. FIGs. 1D-1J show representative brightfield, immunofluorescence and fluorescence images of day 4 peri-gastruloids. FIG. 1D display Magenta and white, NANOG; Red, GATA4; Green, HUNll (Human Nuclear Antigen). Blue, DAPI. Scale bars: 200 pm (top) and 100 pm (bottom). FIG. 1E displays Cyan, OTX2; Yellow, E-CAD; Magenta, N-CAD; White, DAPI. Scale bar, 100 pm. FIG. 1F displays White, PODXL; Magenta, GATA4; Blue, DAPI. Yellow dotted box indicates cavitation in the epiblast compartment. Red dotted box indicates cavitation in the hypoblast compartment. Scale bar, 100 pm. FIG. 1G displays White, OTX2; Red, FOXA2; Blue, DAPI. Scale bar, 100 pm. FIG. 1H displays Magenta, CER1 ; Blue, DAPI. Scale bar, 100 pm. FIG. 11 displays Magenta, LEFTY; Green, SOX2; Blue, DAPI. Scale bar, 100 pm. FIG. 1J displays Green, B-CATENIN. Scale bar, 170 pm. FIG. 1K shows representative immunofluorescence images of attachment culture of day 4 peri-gastruloids. Day 4 peri-gastruloids (generated via method shown in Figure SIC) cultured on fibronectin coated dish in DMEM / F12 medium supplemented with 10% FBS for 4 days. Scale bar, 100 pm. Green, GATA6 (top) and SOX2 (bottom); Magenta, GAT A3 (top) and T (bottom); White, OCT4 (top) and ISL1 (bottom); Blue, DAPI. FIG. 1L shows representative immunofluorescence images of a day 9 peri-gastruloid (generated via methodshown in Figure S1C) cultured in basal medium (N2B27) supplemented with 4% Matrigel. Magenta, CDX2; Green, SOX2; Red, T; Blue, DAPI. Scale bar, 100 pm. FIG. 1 M shows Quantification of peri-gastruloids generated from the BTAG hEPSC reporter line. On day 4, efficiency was determined based on the presence of epiblast-like (SOX2+) and hypoblast- 1 ike (PRDM 1+) compartments; On day 6, efficiency was determined based on the presence of epiblast-like (SOX2+), primary yolk sac-like (PRDM1+) structures and amniotic-like cavity (TFAP2C+); On day 8, efficiency was determined based on the presence of primary yolk sac- like cavity (PRDM1+) and amniotic-like tissue (TFAP2C+). The presence of above-mentioned lineages was determined when more than 10 positive cells were observed, ‘n’ denotes biological replicates. Data are shown as mean±SD.

[0022] FIGs 2A-2E show generation of human peri-gastruloids from human EPSCs. FIG. 2A shows a schematic illustrating the chronological sequence and critical stages of the experimental procedure. Microwells in an AggreWell™ 800 plate are depicted as inverted pyramids. tHDM: titrated hypoblast differentiation medium. CHIR99021 was removed on day 2 from tHDM, refer to as tHDM(-Chir). On day 4 medium was changed to IVC1 supplemented with 4% Matrigel. On day 6 medium was changed to IVC2 supplemented with 4% Matrigel and extra glucose. On day 6, selected peri-gastruloids were transferred to low attachment wells. FIG. 2B shows representative brightfield images illustrating the temporal progression of peri- gastruloids in the top panel. A representative scanning electron micrograph (SEM) of a day 8 peri-gastruloid is also shown. A large view of the AggreWell™ plate containing 9 day 5 peri- gastruloids is shown in the bottom panel; and representative images showing large numbers of days 10-11 peri-gastruloids can be generated is shown in middle panel. Scale bars: 100 pm (top panel), 0.5 CM (bottom right). FIG. 2C shows measurements of long diameter of peri- gastruloids over time. FIGs. 2D-2E show efficiency of peri-gastruloids formation on days 4, 6, 8 and 11. On day 4, efficiency was determined based on the presence of epiblast-like (SOX2+) and hypo blast- 1 ike (SOX17+) compartments; on day 6, efficiency was determined based on the presence of epi blast- 1 ike (SOX2+), primary yolk sac-like (SOX17+) structures and amnionlike (CDX2+) cells; on day 8, efficiency was determined based on the presence of gastrulating cells (T+), epiblast-like (SOX2+), and primary yolk sac-like (SOX17+) structures; on day 11 , efficiency was determined based on the presence of neurectoderm cells (PAX6+), epiblastlike (SOX2+), and yolk sac-like (SOX17+) structures. The presence of above-mentioned lineages was determined when more than 10 positive cells were observed, ‘n’ denotes biological replicates. Data are shown as mean±SD.

[0023] FIGs. 3A-3G show epiblast-like and hypoblast-like compartments formation and cavitation. FIG. 3A shows representative merged fluorescence and brightfield images of day 4 aggregates generated from hEPSCRUES2reporter line in the top panels and representativemerged fluorescence and brightfield images of days 1-5 aggregates generated from hEPSCRUES2reporter line in the bottom panel. Green, SOX2-citrine (green); Red, SOX17-tdTomato. Scale bars, 100 pm. FIG. 3B shows representative images of day 4 aggregates stained with NANOG and FOXA2. Scale bar, 100 pm. FIG. 3C shows representative images of day 4 aggregates stained with SOX2 and GATA6. Scale bar, 100 pm. FIG. 3D shows representative image of day 4 aggregates stained with LEFTY and OCT4. Scale bar, 100 pm. FIG. 3E shows representative image of day 5 aggregates stained with Podocalyxin-like protein (PODXL), SOX2, and SOX17, Scale bar, 50 pm. FIG. 3F shows representative images of day 5 aggregates stained with E-CADHERIN (ECAD, also called CDH1), SOX2 and SOX17. MIP, maximum intensity projection. Scale bar, 50 pm. FIG. 3G shows 3D illustration showing different orientations of peri-gastruloids from day 4 onwards in the top panel and a schematic illustrating the emergence of amniotic cavity and primary yolk sac cavity (PYS) in the bottom panel. AC, amniotic cavity; PYS, primary yolk sac cavity; EPI, epiblast. Left, side view; Right, top view.

[0024] FIGs. 4A-4H show amniotic cavity and primary yolk sac formation. FIG. 4A shows a representative brightfield image of a day 6 peri-gastruloid, side view. Scale bar, 100 pm. FIG. 4B shows representative immunofluorescence images of a day 6 peri-gastruloid, side view. Magenta, CDX2; Green, SOX2; Yellow, SOX17; White, DAPI. Inserts: single channels. Scale bar, 100 pm. FIG. 4C shows representative immunofluorescence images of a day 6 peri-gastruloid, top view. Epiblast-like compartment (SOX2, green) is facing upwards, while only the edge of the underlying primary yolk sac-like cavity (SOX17, orange) is visible. Magenta, E-CAD. The optical section (white dotted square) reveals both the epiblast-like compartment and primary yolk sac-like cavity. Scale bar: 50 pm. FIG. 4D shows representative immunofluorescence images of a day 6 peri-gastruloid, side view. Magenta, PDGFRa; Orange, SOX17; White, DAPI. Scale bar, 50 pm. FIG. 4E shows representative immunofluorescence images of a day 6 peri-gastruloid, side view. Cyan, E-CAD; Yellow, OTX2; White, DAPI. Insert (white dotted square): bilaminar disc-like structure. FIG. 4F shows representative immunofluorescence images of a day 6 peri-gastruloid, side view and top view (via optical rotation using Imaris). Green, SOX2; Orange, SOX17; Magenta, T. Cyan dotted circle indicate primitive streak and embryonic disk-like structure. Bottom right showing the optical section indicated by white dotted square in bottom left. FIG. 4G shows representative immunofluorescence images of a day 7 peri-gastruloid, top view. Green, SOX2; Orange, SOX17; Magenta, CDX2; White, DAPI. Insert (yellow dotted square) indicates a primitive streak-like structure. Top right is a schematic illustrating the putative viewing angle for FIG. 4G. Scale bar, 100 pm. AC, amniotic cavity; PYS, primary yolk sac. PS, primitive streak. ED, embryonic disk. MIP, maximum intensity projection. FIG. 4H illustrates supplementation of 4%Matrigel improves the differentiation of amnion-like cells. Amniotic epithelium morphology and CDX2 expression was used to identify amniotic tissue. Unpaired T-test was performed to determine P-Value, n=30.

[0025] FIGs. 5A-5M show cavity formation and gastrulation. FIG. 5A shows SOX2 expression levels decreased asymmetrically among ELCs. Representative images showing a day 4 peri-gastruloid embedded in tHDM (-Chir) and 10% Matrigel and cultured for 3 days. Snapshots were taken on each day. Dotted blue boxes highlight the area for SOX2 intensity quantification. Right, quantification of the SOX2 levels over time. Distance 0 refers to the border furthest from the SOX17 domain. Green, SOX2; Red, SOX17. Scale bars, 100 pm. FIGs. 5B-5D show emergence of amnion-like cells on one side of epiblast (EPI) in day 6 peri- gastruloids. FIG. 5B shows amnion-like cells are induced in the epiblast compartment, top view. White dotted circle indicates the putative amnion-like cells. Green, OCT4; Red, NANOG; Blue, SOX2; Yellow, DAPI. Scale bar, 100 pm. FIG. 5C shows a representative immunofluorescence image of a day 6 peri-gastruloid, top view. Green, SOX2; Magenta, SOX17; White, PODXL. Scale bar, 100 pm. FIG. 5D shows a representative immunofluorescence image of a day 6 peri-gastruloid, top view. Green, SOX2; Blue, SOX17; Magenta, CDX2. Scale bar: 100 pm. FIG. 5E shows a representative immunofluorescence image of a day 6 peri-gastruloid, top view. Green, SOX2; Magenta, SOX17; White, PDGFRa. Scale bar: 100 pm. FIG. 5F shows representative immunofluorescence images of a day 6 peri- gastruloid, top view. Yellow, SOX2; Cyan, SOX17; Magenta, phospho-SMAD1 / 5 / 9 (pSMAD1 / 5 / 9). Scale bar: 100 pm. FIG. 5G shows representative immunofluorescence images of a day 6 peri-gastruloid, top view. Cyan, NANOG; Red, CDX2; Blue DAPI. Dotted orange circle indicates the zoomed-in epiblast-like and amnion-like region. Scale bar, 100 pm. FIG. 5H shows representative immunofluorescence images of a day 6 peri-gastruloid generated from the / VODAL-mCitrine reporter hEPSCs, top view. Epiblast-like compartment is highlighted by yellow dotted circles. Bottom right, a zoomed-in image of CER1-high region indicated by green dotted box in bottom left. FIG. 5I shows quantification of LEFTY1 / 2 and CER1 signal intensities in epiblast-like compartments of day 6 peri-gastruloids. The region of interest (ROI) is indicated by white boxes in bottom images. FIG. 5J shows H&E staining of histological sections of day 6 peri-gastruloids in the left and middle panel, H&E staining of a histological section of a CS6 human embryo (retrieved from the Virtual Human Embryo, section # CS6 34) in the right panel. AC, amniotic cavity; PYS, primary yolk sac cavity. Scale bars, 200 pm. Scale bar, 200 pm. (IC) FIG. 5K shows representative immunofluorescence images of day 8 peri-gastruloids, top view in the left and middle panel and 3D rendering (based on maximal intensity projection) in the right panel. Space coordinates are indicated. Magenta, CDX2 (top) and TFAP2A (bottom); Green, SOX2. Scale bars, 100 pm. FIG. 5L showsrepresentative immunofluorescence images of a day 8 peri-gastruloid. White, F-ACTIN; Magenta, N-CAD; Green, SOX2. Scale bar, 100 m. FIG. 5M shows representative brightfield and immunofluorescence images of a day 8 peri-gastruloid related to FIG. 7C. Green, SOX2; Magenta, T; Yellow, CER1 ; White, DAPI. Scale bar, 100 pm.

[0026] FIGs. 6A-6F show induction of primordial germ cell-like cells (PGCLC) in peri- gastruloids. FIG. 6A shows representative immunofluorescence images of day 6 peri- gastruloids generated from BTAG reporter hEPSCs. Blue dotted circle indicates primary yolk sac (PYS). White dotted circle indicates amniotic cavity (AC). Scale bar, 100 pm. FIG. 6B shows representative immunofluorescence images of a day 6 peri-gastruloid. White dotted box indicates the zoomed-in epiblast-like and amnion-like region. Cyan, NANOG; Red, TFAP2C; Magenta, PRDM1 , Blue, DAPI. Scale bar, 100 pm. FIG. 6C-6D show representative immunofluorescence images of day 6 peri-gastruloids generated from BTAG reporter hEPSCs. Green, TFAP2C; Red, PRDM1 ; Magenta, SOX2; White, DAPI. Scale bar, 100 pm. FIG. 6C shows blue dotted circle indicating primary yolk sac (PYS) and white dotted circle indicating amniotic cavity (AC). FIG. 6E shows representative fluorescence images of a day 5 peri-gastruloid generated from BTAG reporter hEPSCs, 3D rendering (side view) in the top panel with scale bar, 100 pm, orientation angles (theta) of BLIMP 1 (PRDM1) / TFAP2C double positive PGCLCs with respect to the hypoblast plane in the bottom panel, a schematic showing how the angles (theta) between the long diameter of a PGCLC and the hypoblast plane is measured in the right panel. FIG. 6F shows a schematic of PGCLCs migration from the amnion region to the yolk sac region (adapted from K. Sasaki et al., 2016).

[0027] FIGs. 7A-7F show primitive streak formation and gastrulation. FIG. 7A shows representative brightfield (left) and fluorescence (right) images of a day 8 peri-gastruloid, top view. Green, SOX2; Red, SOX17. Scale bar, 100 pm. FIG. 7B shows a schematic illustrating a human embryo undergoing gastrulation. Insert (blue dotted square) shows a cross section of the trilaminar embryonic disk. FIG. 7C shows representative immunofluorescence images of a day 8 peri-gastruloid, top view. Rostral-Caudal axis (also called Anterior-Posterior (A-P) axis) is indicated. High CER1 (yellow) marks rostral (anterior) side, High T (magenta) marks caudal (posterior) side. White dotted circle highlights embryonic disk (ED)-like structure. Primitive streak (PS)-like region is indicated. Middle Z-plane is shown here. Other Z-planes and brightfield images of the same structure are shown in FIG. 5M . FIG. 7D shows representative immunofluorescence images of a day 8 peri-gastruloid, top Z-plane is shown. F-actin indicates the epithelial epiblast (or embryonic disk-like) region and the delaminating gastrulation cells (T+). Red arrowhead indicates the location of gastrulation and primitive streak. Right, 3D-rendering (normal shading) is shown. Scale bar, 100 pm. FIG. 7E shows representative immunofluorescence images showing a trilaminar disk-like structure. Insert(white dotted square) indicate the zoomed-in location. Top right, 3D-rendering (normal shading) of the zoomed-in region. Scale bar, 50 pm. FIG. 7F shows representative immunofluorescence images of an embryonic disk-like and a primitive streak-like structure in a day 8 peri-gastruloid. Scale bar: 100 pm. Right, 3D-rendering (normal shading) of the peri- gastruloid shown. Scale bar: 100 pm.

[0028] FIGs. 8A-8H show early neurulation and organogenesis in advanced peri- gastruloids. FIG. 8A shows a representative brightfield image of a day 9 peri-gastruloid. Top left shows a schematic illustrating the epiblast sandwiched by the above amniotic cavity and an underlying yolk sac. Scale bar: 100 pm. FIG. 8B shows 3D-rendering of a day 9 peri- gastruloid based on the DAPI (white) channel in the top left panel, Z-plane of the structure showing an amniotic-like cavity and yolk sac-like structure in the top right panel and single channels are shown in the bottom panel. Green, SOX2; Orange, SOX17; Magenta, CDX2. Scale car: 100 pm. FIG. 8C shows representative immunofluorescence images of a day 11 peri-gastruloid after tissue clearing. Putative neural plate (NP) / neural groove (NG) (white dotted boxes) and tail bud-like region (yellow dotted boxes) were observed. Scale bar: 100 pm. FIG. 8D shows brightfield and immunofluorescence (top let) images of a cryosection revealing internal architecture of a day 11 peri-gastruloid. Yellow dotted circle indicates SOX2+ (green) region. Top right shows a schematic indicating the sectioning position and orientation. Scale bar: 200 pm. FIG. 8E shows representative immunofluorescence images of a day 11 peri-gastruloid. Green, SOX2; Magenta and white, N-CAD; Blue, DAPI. The epithelial neuroectoderm area, i.e., neural plate (NP) and neural groove (NG), and the delaminating primitive streak (PS) area are indicated. N-CAD+ region is indicated by yellow dotted circle. Scale bar: 100 pm. FIG. 8F shows representative immunofluorescence images of a cryosection of a day 11 peri-gastruloid. White, PAX6; Green, SOX2; Blue, DAPI. Yellow dotted boxes highlight the neural tube / neural groove-like structure. Right panel, zoomed-in images of the highlighted area after optical rotation. Scale bar, 100 pm. FIG. 8G shows H&E staining of a histological section from a CS8 human embryo (retrieved from the Virtual Human Embryo, section # CS8 197) in the top panel, H&E staining of a histological section from a day 11 peri- gastruloid in the bottom left panel. And H&E staining of a histological section from a day 12 peri-gastruloid in the bottom right panel. Scale bars, 200 pm. FIG. 8H shows a schematic of a human embryo at stages CS8-9. Dorsal (top) view. AC, amniotic cavity. EPI, epiblast. YS, yolk sac. MIP, maximum intensity projection. NP / NG, neural plate / neural groove. PS, primitive streak. CLE, caudal epiblast. ExE ect, extraembryonic ectoderm (amniotic ectoderm). Ect, ectoderm. Meso, mesoderm. Endo, endoderm. ExE endo, extraembryonic endoderm.

[0029] FIGs. 9A-9J show early neurulation and organogenesis in advanced peri- gastruloids. FIG. 9A shows representative brightfield (left) and immunofluorescence (right)images of day 10 peri-gastruloids. Green, SOX2; Magenta, HNF4a. Scale bar, 100 pm. FIG. 9B shows Representative brightfield and fluorescence merged images of day 11 peri- gastruloids. Green, SOX2-Citrine; Red, SOX17-Tdtomato. Scale bars, 300 pm. FIG. 9C shows representative immunofluorescence images of cryosections from day 11 peri-gastruloids. White, PAX6. PAX6+ lumens are indicted by yellow asterisks. Scale bar, 1000 pm. FIG. 9D shows representative immunofluorescence of images of cryosections from day 11 peri- gastruloids. PAX6+ lumen is indicted by yellow dotted box. Green, SOX2; White, PAX6; Blue, DAPI. Scale bars, 200 pm (top) and 100 pm (bottom). FIG. 9E shows representative immunofluorescence images of a cryosection from a day 11 peri-gastruloid. Green, SOX2; White, T; Blue, DAPI. Yellow dotted box indicates the TB region. Scale bar: 100 pm. FIG. 9F shows representative immunofluorescence image of a cryosection from a day 11 peri- gastruloid. Green, SOX2; Magenta, ZO-1 ; Cyan, SOX17; Blue, DAPI. Yellow dotted box indicates the NP / NG region, shown in zoomed-in insert. FIG. 9G shows representative immunofluorescence images of a cryosection from a day 11 peri-gastruloid showing the transverse plane of the neural plate region. Section 1 (top) is a position closer to the putative roof plate, with wider inside diameter. Section 2 (bottom) is the position near the putative floor plate, with narrower inside diameter. FIG. 9H shows representative immunofluorescence images of a day 11 peri-gastruloid stained with SOX2 (green) and F-ACTIN (magenta). Yellow box indicates the zoom-in area shown on the right. Blue, DAPI. Scale bars, 100 pm. FIG. 9I shows 3D rendering of a day 11 peri-gastruloid. Yellow dotted box indicates putative neural plate. Green, SOX2; Magenta, PAX6; Blue, DAPI. Scale bar, 100 pm. FIG. 9J shows representative immunofluorescence images of a cryosection from a day 11 peri-gastruloid in the top panel, (Red, SOX17; Green, SOX; White, GATA4), and a schematic illustrating neural plate, gut tube, and yolk sac in the bottom panel. Scale bar, 100 pm. PS, primitive streak; NP, neural plate; NG, neural groove; CLE, caudal epiblast. TB, tail bud. YS, yolk sac.

[0030] FIGs. 10A-10Q show single cell RNA-seq analysis of advanced peri-gastruloids. UMAPs displaying marker genes overrepresented in the corresponding lineages found in peri- gastruloids. The corresponding lineages are indicated. EPI, epiblast; PS, primitive streak; APS, anterior primitive streak; NE, neural ectoderm; NNE, non-neural ectoderm; AME, amniotic ectoderm; Nas. Meso, nascent mesoderm; LPM, lateral plate mesoderm; DE, definitive endoderm; ExE. Endo, extraembryonic endoderm.

[0031] FIGs. 11 A-11 F illustrate single cell RNA-seq analysis of advanced peri-gastruloids.FIG. 11A shows Uniform Manifold Approximation and Projection (UMAP) visualization representing individual cells derived from day 11 peri-gastruloids. 13 distinct colored clusters are identified, corresponding to different cell types: EPI, epiblast; Ect, ectoderm; PS, primitive streak; Nas. Meso, nascent mesoderm; Adv. Meso, advanced mesoderm; DE, definitiveendoderm; ExE endo, extraembryonic endoderm; Intermediate, intermediate cell types; ExE. Meso, extraembryonic mesoderm; SE, surface ectoderm. FIG. 11B shows heatmap displaying the top 25 differentially expressed genes (DEG) for each cluster shown in FIG. 11 A. FIG. 11C shows LIMAP displaying subclusters of ectoderm lineage cells. NE, neural ectoderm; AME, amniotic ectoderm; SE, surface ectoderm. FIG. 11D shows LIMAP displaying subclusters of endoderm lineage cells. DE, definitive endoderm, ExE. endo, Extraembryonic endoderm. FIG. 11E shows LIMAP displaying subclusters of mesoderm lineage cells. Nas. Meso, nascent mesoderm; Gaud meso, caudal mesoderm; PSM, presomitic mesoderm; LPM, lateral plate mesoderm; Cardi, cardiogenic fates; ExE. meso, Extraembryonic mesoderm. FIG. 11 F shows integration of day 11 peri-gastruloids scRNA-seq data with reference scRNA-seq data from natural human and non-human primate embryos-originated cells. Top left, LIMAP showing the mapped cells between peri-gastruloids and human PCW3. Top right, LIMAP showing the mapped cells between peri-gastruloids and ex utero cultured monkey embryos. Bottom left, LIMAP showing the mapped cells between peri-gastruloids and in vivo monkey embryos. Bottom right, LIMAP showing the identified cell types among the integrated cells. Individual cells are color coded for annotation. EPI, epiblast; PS, primitive streak; Ect, ectoderm (including neural ectoderm; neural plate; neural tube; neural crest); Inter. Meso, intermediated mesoderm; Mes, mesenchyme; ExE. meso, extraembryonic mesoderm; NNE, non-neural ectoderm; AME, amniotic ectoderm; SE, surface ectoderm; CTB, cytotrophoblasts; STB, syncytiotrophoblast; EVT extravillous trophoblasts; DE, definitive endoderm; LPM, lateral plate mesoderm; ExE. Endo, extraembryonic endoderm; VE, visceral endoderm; ParE, parietal endoderm; YS. endo, yolk sac endoderm. Cardi, cardiac lineages; Nas. Meso, nascent mesoderm; NMP, neuromesodermal progenitor; EPI, epiblast; PS, primitive streak.

[0032] FIGs. 12A-12D show lineage differentiation in advanced peri-gastruloids. FIG. 12A shows representative immunofluorescence images of day 11 peri-gastruloids. CD34+ cells are highlighted by dotted box. Magenta, CD34 (left) and GATA6 (right); Yellow, SOX17 (left) and FOXA2 (right); Green, CD34 (right). Blue, DAPI. Scale bars, 200 pm. YS, yolk sac. FIG. 12B shows representative brightfield (left) and brightfield and fluorescence merged (right) images showing a day 13 peri-gastruloid generated from HES7-Achilles knock-in reporter hEPSCs. Green, HES7-Achilles. Scale bars, 300 pm. FIG. 12C shows representative brightfield (left), fluorescence (middle) and merged images of a day 13 peri-gastruloid showing rosette-like structures. Green, SOX2-Citrine. Scale bar, 100 pm. FIG. 12D shows a day 13 peri-gastruloid displayed rosette-like structures with a central lumen surrounded by TBX18+ (magenta) cells. The rosette-like structures are highlighted by yellow asterisks. Dotted red box indicates the zoomed-in insert showing one of the rosette-like structures. Blue, DAPI. Scale bar: 100 pm.

[0033] FIGs. 12E-12G show time-resolved single-cell RNA-seq analysis of peri- gastruloids. FIGs. 12E-12F show Uniform Manifold Approximation and Projection (UMAP) visualization representing individual cells derived from days 4-11 peri-gastruloids, respectively. The starting material, extended pluripotent stem cells (EPSCs), labeled as day 0, serves as a control. Hypoblast (Hypo), Endoderm (Endo), Mesoderm (Meso), Epiblast (EPI), Ectoderm (Ect), Primitive streak (PS), and Surface ectoderm (SE). FIG. 12G is a heatmap displaying the tissue-specific marker genes for each identified cell type.

[0034] FIGs. 12H-12I depict single-cell RNA-seq analysis revealing the anterior visceral endoderm specification in peri-gastruloids. FIG. 12H shows Uniform Manifold Approximation and Projection (UMAP) visualization representing individual cells derived from day 4 peri- gastruloids, displaying signatures of human hypoblast (primitive endoderm) cells. FIG. 121 shows UMAP visualization representing individual cells derived from day 4 peri-gastruloids, displaying signatures of human anterior visceral endoderm (AVE) cells.

[0035] FIG. 12J is a heat map of metabolites that are differentially enriched in different stages of peri-gastruloids formation.

[0036] The drawing figures do not limit the present disclosure to the specific aspects disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain aspects of the present disclosure.DETAILED DESCRIPTION

[0037] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0038] The present disclosure is partly based on the discovery of a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, called peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. The inventors have identified that peri-gastruloids can simulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and early neurulation. Further, the inventors observed that advanced human peri-gastruloids show transcriptomic similarities to primary peri-gastrulation cell types found in human and non-human primates. This peri-gastruloid platform allows forfurther exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.I. Terminology

[0039] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred aspects and specific language will be used to describe the same. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs.

[0040] As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0041] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.

[0042] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.

[0043] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0044] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0045] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0046] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1 % to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0047] As used herein, “treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition.

[0048] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and / or degree of onset or severity in the absence of intervention.

[0049] The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0050] As used herein, “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis or therapy is desired, for example, humans, pets, livestock, horses or other animals. As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In one aspect, the subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc. In another aspect, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas. In still another aspect, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another aspect, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In some aspects,the subject can be a human. In other aspects, the subject can be a human in need of repairing or regenerating a tissue or organ.

[0051] As used herein, “effective amount,” “effective dose,” or an “amount effective to”, refers to an amount that is effective in providing at least one desirable biological result.

[0052] As used herein, “FGF activator” refers to a molecule that partially or fully enhances, increases, or stimulates a biological activity of a FGF protein. FGFs are a family of cell signaling proteins that play a role in a wide range of cellular processes. FGF family members comprise FGF proteins (e.g., FGF1 , FGF2, FGB, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11 , FGF12, FGF13, FGF14, FGF15, FGF16, FGF 17, FGF 18, FGF 19, FGF20, FGF21 , FGF22, and FGF23) and FGF receptor (FGFR) proteins (e.g., FGFR1, FGFR2, FGFR3, FGFR4, and FGFRL1). Suitable FGF activators include, but are not limited to, proteins, nucleic acids, small molecules, or any combination thereof. Additionally, FGF activator can be a molecule of any type that enhances the signaling associated with at least one FGF family members (e.g., FGFs or FGFRs) in a cell, for example, either by increasing transcription or translation of a FGF family member, or by increasing FGF activity, or both. In some aspects, the FGF activator may act directly by interacting with a FGF receptor or indirectly by interacting with one or more intracellular components of the FGF signaling pathway. In some aspects, FGF activators as described herein may increase FGF signaling in cells (e.g., reproductive cells or PSCs) by at least 20% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above. The increased FGF signaling in the presence of at least one FGF activator can be determined by conventional methods, e.g., using protein assays such as ELISA or Western blot.

[0053] As used herein, the term “FGF” refers to a FGF polypeptide having the same or similar bioactivity of a wild- type FGF polypeptide. A FGF polypeptide may have an amino acid sequence that is at least 70% or more (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type FGF polypeptide, and is capable of trigger FGF signaling pathway.

[0054] As used herein, “TGF-p activator” refers to a molecule that partially or fully enhances, increases, or stimulates a biological activity of a TGF-p protein. TGF-p is a multifunctional cytokine belonging to the transforming growth factor superfamily that comprises TGF- ps (e.g., TGF-pi , TGF-P2, and TGF-P3), Activins (e.g., Activin-A, Activin-B, and Activin-C), bone morphogenetic proteins (BMPs), and TGF-p receptors (TGFRs) (e.g., type I, type II, and type III receptors). Sequences of genes and proteins in the TGF-p pathway are known in the art, and may be obtained from publicly available databases. Suitable TGF-p activators include, but are not limited to, proteins, nucleic acids, small molecules, or anycombination thereof. Methods for identifying activators of TGF-p may comprise contacting TGF-p with a candidate TGF-p activator and measuring a detectable change in one or more biological activities typically associated with TGF-p. A TGF-p activator can be a molecule of any type that enhances the signaling associated with at least one TGF-p family members (e.g., TGF-p, Activin, BMP, or FGFR) in a cell, for example, either by increasing transcription or translation of a TGF-p family member, or by increasing TGF-p activity, or both. In some aspects, the TGF-p activator may act directly by interacting with a TGF-p receptor or indirectly by interacting with one or more intracellular components of the TGF-p signaling pathway. In some aspects, TGF-p activator as described herein may increase TGF-p signaling in cells (e.g., reproductive cells or PSCs) by at least 20% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above. The increased TGF-p signaling in the presence of at least one TGF- P activator can be determined by conventional methods, e.g., using protein assays such as ELISA or Western blot.

[0055] As used herein, “TGF-p polypeptide” refers to a polypeptide having the same or similar bioactivity of a wild-type TGF-p polypeptide. A TGF-p polypeptide may have an amino acid sequence that is at least 70% or more (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type TGF-p polypeptide, and is capable of trigger TGF-p signaling pathway.

[0056] As used herein, “WNT activator” refers to a molecule that partially or fully enhances, increases, or stimulates a biological activity of a WNT protein. WNTs are a family of secreted proteins important for a wide array of developmental and physiological processes. WNT family members comprise WNT1, WNT2, WNT2b (also called WNT13), WNT3, WNT3a, WNT4, WNT5a, WNT5b, WNT6, WNT7a, WNT7b, WNT8a, WNT8b, WNT9a, WNT9b, WNT10a, WNT10b, WNT11 , and WNT16. Sequences of genes and proteins in the WNT pathway are known in the art and may be obtained from publicly available databases. Suitable WNT activators include, but are not limited to, proteins, nucleic acids, small molecules, or any combination thereof. Methods for identifying activators of WNT may comprise contacting WNT with a candidate WNT activator and measuring a detectable change in one or more biological activities typically associated with WNT. A WNT activator can be a molecule of any type that enhances the signaling associated with at least one WNT family members (e.g., WNT1 , WNT2, WNT3, or WNT3a) in a cell, for example, either by increasing transcription or translation of a WNT family member, or by increasing WNT activity, or both. In some aspects, the WNT activator may act directly by interacting with a WNT receptor or indirectly by interacting with one or more intracellular components of the WNT signaling pathway such as P-catenin, a kinase or phosphatase that acts on p-catenin, a transcription factor that assembles with p-catenin.

[0057] As used herein, “WNT” refers to a WNT polypeptide having the same or similar bioactivity of a wild-type WNT polypeptide. A WNT polypeptide may have an amino acid sequence that is at least 70% or more (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type WNT polypeptide, and is capable of trigger WNT signaling pathway.

[0058] In some aspects, the WNT activator is a glycogen synthase kinase 3 (GSK3) inhibitor. GSK3 is a serine / threonine kinase that has been identified as a regulator of glucose metabolism. As used herein, “GSK3” refers to either or both isoforms of GSK3 (GSK3a and GSK3P). Inhibitors that inhibit either or both of these isoforms are of use. In some aspects, the GSK3 inhibitor specifically inhibits GSK3 and does not substantially inhibit the majority of other mammalian kinases.

[0059] As used herein “MEK inhibitor” is a chemical or drug that inhibits the mitogen- activated protein kinase kinase enzymes MEK1 and / or MEK2. They can be used to inhibit the MAPKZERK pathway. MEK is a kinase that phosphorylates mitogen-activated protein kinase (MAPK). As used herein, “MEK” refers to any isoform of MEK (e.g., MEK1 or MEK2). Inhibitors that inhibit either or both of these isoforms are of use. In some aspects, the MEK inhibitor specifically inhibits MEK and does not substantially inhibit the majority of other mammalian kinases.

[0060] As used herein “peri-gastruloid” encompass embryonic (epiblast) and extraembryonic (hypoblast) tissues.

[0061] As used herein “expression” or “expression level” or “level of expression” refers to amount of a particular analyte (e.g., a biomarker) present in the sample. The amount may be a concentration, number, ratio, proportion, or a percentage of the analyte compared to the control sample or determined using a standard curve. The amount may be an absolute amount or a relative amount.II. Generation of peri-gastruloids or embryo-like structures

[0062] The present disclosure encompasses a method of generating peri-gastruloids having embryo-like structures. In such aspects, the method comprises a three-dimensional culture of pluripotent stem cells (PSCs) in a first culture medium for about 1 to 7 days and changing the culture medium to a second culture medium after about 1 to 7 days.

[0063] In further aspects, the disclosure encompasses generating embryo-like structures from pluripotent stem cells (PSCs). The method comprises obtaining a three-dimensional culture device, wherein the three-dimensional culture device comprises PSCs and a first culture medium, and maintaining the cells in the first culture medium for about 1-7 days. Insome aspects, the method further comprises changing the culture medium in the three- dimensional culture device to a second culture medium, and maintaining the cells in the second culture medium for about 2-7 days. In further aspects, the method comprises changing the culture medium in the three-dimensional culture device to a third culture medium, and maintaining the cells in the third culture medium for about 1-7 days to obtain embryo-like structures. In some aspects, the method further comprises a fourth medium, and maintaining the cells in the fourth culture medium for about 1-14 days.Pluripotent stem cells

[0064] In some aspects, the embryo-like structure or peri-gastruloids having embryo like structure is generated from pluripotent stem cells (PSCs). In some aspects, PSCs may be naive PSC, naive-like PSCs, primed PSCs, formative PSCs, or extended PSCs. In some aspects, the PSCs are naive PSCs. In some aspects, the PSCs are naive-like PSC. In some aspects, the PSCs are primed PSCs. In some aspects, the PSCs are formative PSCs. In some aspects, the PSCs are extended PSCs. In some aspects, PSCs may be conventional human ESCs or induced human PSCs.

[0065] In some aspects, naive PSCs disclosed herein, resemble the embryonic epiblast at an earlier time-point in development, for e.g., cells in vitro resembling the inner cell mass of human embryonic day (E) 6-7 preimplantation blastocysts. Naive pluripotent stem cells can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise naive human pluripotent stem cells (hPSCs).

[0066] In some aspects, naive-like PSCs disclosed herein, are cells that resemble closely the inner cell mass (ICM) and / or closeness of their expression profiles to cells of the ICM. Naive-like pluripotent stem cells can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise naive-like human pluripotent stem cells (hPSCs).

[0067] In some aspects, primed PSCs disclosed herein, display a gene expression signature more similar to the anterior epiblast of a late-gastrula-stage embryo (approximately embryonic day 7, ~E7). Primed PSCs can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise primed human pluripotent stem cells (hPSCs).

[0068] In some aspects, formative PSCs, disclosed herein, comprise epiblast cells poised for gastrulation. Formative PSCs can be established from different embryonic stem cells, pre- / early-gastrula epiblasts and induced PSCs. Similar to pre- / early-gastrula epiblasts, formative PSCs show the transcriptomic features of formative pluripotency, which are distinct from naive ESCs and primed PSCs. In some aspects, formative PSCs share transcriptomic similarities with embryonic day 5-6 (E5-6) epiblasts. Formative PSCs can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise formative human pluripotent stem cells (hPSCs).

[0069] In some aspects, extended (or expanded) PSCs, disclosed herein, can generate both embryonic and extra-embryonic lineages. Extended PSCs can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g. , pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise extended human pluripotent stem cells (hPSCs).

[0070] In some aspects, the PSCs comprise a single cell suspension of PSCs. Single cell suspension of PSCs can be made using any known method in the art. In some aspects, cultured PSCs, can be disassociated into single cells using mechanical dissociation and / or enzyme treatment, for e.g., treatment using Dispase, Collagenase, hyaluronidase, Papain, DNAse-1, Accutase, or TrypLE™.

[0071] In some aspects, PSCs are added or cultured in a three-dimensional culture device. Any suitable three-dimensional culture vessel can be used. In some aspects, three- dimensional culturing methods including methods using scaffold, and scaffold-free methods. In methods using scaffolds, hydrogels may be used as a scaffold. Scaffold-free methods can include low adhesion plate, hanging drop plats, micropatterned surface, spheroid, bioreactor, magnetic levitation, chip, and microfluidic device. In some aspects, the disclosed three- dimensional culture device is a microwell plate. In some aspects, the disclosed three- dimensional culture device is an AggreWell™ plate.

[0072] In further aspects, the three-dimensional device can be pre-treated with an antiadherence solution. Anti-adherence solution as disclosed herein can be any commercially available anti-adherence solution which can prevent attachment of the cells to the device, for e.g., anti-adherence rinsing solution from stem cell technologies (Catalog No. 07010).

[0073] In some aspects, about 1-500 PSCs are added to an individual well of a three- dimensional culture device. In some aspects, about 1 , about 10, about 15, about 20, about 25,about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500 are added to an individual well of a three-dimensional culture device. In some aspects, about 1-200 PSCs are added to an individual well of a three-dimensional culture device. In some aspects, about 30-50 PSCs are added to an individual well of a three-dimensional culture device. In some aspects, not more than 90 PSCs are added to an individual well of a three-dimensional culture device.Media

[0074] In some aspects, a culture medium for generating embryo-like structures or peri- gastruloids from PSCs comprise at least one FGF activator, at least one TGF-p activator, at least one WNT activator, at least one MEK inhibitor, or any combination thereof.

[0075] Activators for any of FGF family members can be used in the media described herein. In some aspects, FGF activators used herein are specific to one FGF family member, e.g., specific to FGF or FGFR. In some aspects, FGF activators are universal to two or more FGF family members, e.g., universal to FGF and FGFR. In some aspects, FGF activators used herein are specific to FGF2. In some aspects, the FGF activator is FGF2.

[0076] Wild-type FGF sequences (e.g., sequences of FGF2) of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human FGF2 is available at NCBI under Accession No. NM_002006.4 and its corresponding amino acid sequence is under Accession No. NP_001997.5. FGF proteins may be isolated from naturally occurring sources (e.g., mammalian cells that naturally produce the protein), produced in eukaryotic or prokaryotic cells using recombinant expression technology, or chemically synthesized. Soluble, biologically active FGF proteins may be prepared in purified form using methods known in the art.

[0077] An effective amount of a FGF activator used in media described herein can be between about 0.1 ng / ml and about 10,000 ng / ml. In some aspects, the effective amount of the FGF activator for the media described herein can be between about 1 ng / ml and about 10,000 ng / ml, between about 10 ng / ml and about 10,000 ng / ml, between about 100 ng / ml and about 10,000 ng / ml, between about 1 ,000 ng / ml and about 10,000 ng / ml, between about5,000 and about 10,000 ng / ml, between about 0.1 ng / ml and about 5,000 ng / ml, between about 1 ng / ml and about 5,000 ng / ml, between about 10 ng / ml and about 5,000 ng / ml, between about 1 ,000 ng / ml and about 5,000 ng / ml, or between about 2,500 ng / ml and about 5,000 ng / ml. In some aspects, FGF activator used in the media described herein can be between about 0.1 ng / ml and about 100 ng / ml. In some aspects, the effective amount of the FGF activator usedin the media described herein can be between about 1 ng / ml and about 200 ng / ml. In some aspects, the effective amount of the FGF activator used in the media described herein is about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 110 ng / ml, about 120 ng / ml, about 130 ng / ml, about 140 ng / ml, about 150 ng / ml, about 160 ng / ml, about 170 ng / ml, about 180 ng / ml, about 190 ng / ml, or about 200 ng / ml. In some aspect, the FGF activator is added to the culture medium at about 10 ng / ml. In some aspects, FGF activator added to the culture medium is FGF2 at about 10 ng / ml.

[0078] Activators for any of TGF-p family member can be used in the media described herein. In some aspects, TGF-p activator used herein are specific to one TGF-p family member, e.g., specific to a TGF-p or an activin (e.g., TGF-P2 or Activin-A). In some aspects, TGF-p activators are universal to two or more TGF- TGF-p family members, e.g., universal to a TGF-p and an Activin (e.g., TGF-p or Activin-A). In some aspects, TGF-p activator used herein is Activin-A.

[0079] Wild-type TGF-p sequences (e.g., sequences of TGF-pi , TGF-P2, or TGF-P3) of various species are available on the world wide web from the NCBI, including, for example, human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human TGF-P2 is available at NCBI under Accession No. NM_001024847.2 and its corresponding amino acid sequence is under Accession No. NP_001020018.1.

[0080] Wild-type activin sequences (e.g., sequences of Activin-A, Activin-B, and Activin- C) of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human Activin- A is available at NCBI under Accession No. NM_002192.3 and its corresponding amino acid sequence is under Accession No. NP 002183.1.

[0081] TGF-p proteins may be isolated from naturally occurring sources (e.g., mammalian cells that naturally produce the protein), produced in eukaryotic or prokaryotic cells using recombinant expression technology, or chemically synthesized. Soluble, biologically active TGF-p proteins may be prepared in purified form using methods known in the art.

[0082] An effective amount of a TGF-p activator used in media described herein can be between about 0.1 ng / ml and about 10,000 ng / ml. In some aspects, the effective amount of the TGF-p activator for the media described herein can be between about 1 ng / ml and about 10,000 ng / ml, between about 10 ng / ml and about 10,000 ng / ml, between about 100 ng / ml and about 10,000 ng / ml, between about 1 ,000 ng / ml and about 10,000 ng / ml, between about5,000ng / ml and about 10,000 ng / ml, between about 0.1 ng / ml and about 5,000 ng / ml, between about 1 and about 5,000 ng / ml, between about 10 ng / ml and about 5,000 ng / ml, between about 1 ,000 ng / ml and about 5,000 ng / ml, or between about 2,500 ng / ml and about 5,000 ng / ml. In some aspects, TGF-p activator used in the media described herein can be between about 0.1 and about 100 ng / ml. In some aspects, the effective amount of the TGF-p activator used in the media described herein can be between about 1 ng / ml and about 200 ng / ml. In some aspects, the effective amount of the TGF-p activator used in the media described herein is about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 110 ng / ml, about 120 ng / ml, about 130 ng / ml, about 140 ng / ml, about 150 ng / ml, about 160 ng / ml, about 170 ng / ml, about 180 ng / ml, about 190 ng / ml, or about 200 ng / ml. In some aspect, the FGF activator is added to the culture medium at about 10 ng / ml. In some aspects, TGF-p activator is added to the culture medium at about 10 ng / ml. In some aspects, the TGF-p activator added to the culture medium is Activin-A at about 10 ng / ml.

[0083] In some aspects, activators for any of WNT family members can be used in the methods described herein. In some aspects, WNT activators used herein are specific to one WNT family member, e.g., specific to WNT1 , WNT2, WNT3, or WNT3a. In some aspects, WNT activators are universal to two or more WNT family members, e.g., universal to WNT3 and WNT3a. In some aspects, WNT activators used herein are specific to WNT3a.

[0084] Wild-type WNT sequences (e.g., sequences of WNT1 , WNT2, WNT3, or WNT3a) of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human WNT3a is available at NCBI under Accession No. NM_033131.3 and its corresponding amino acid sequence is under Accession No. NP_149122.1.

[0085] In some aspects, the WNT activator is a biologically active WNT protein, e.g., WNT3 a protein. In some aspects, the WNT activator is a glycogen synthase kinase 3 (GSK3) inhibitor. In some aspects, the GSK3 inhibitor specifically inhibits GSK3 and does not substantially inhibit the majority of other mammalian kinases. Any GSK3 inhibitor may be used in methods described herein. Exemplary GSK3 inhibitors include, but are not limited to, BIO, AR-A014418, SB 216763, SB-415286, CHIR98014 (CT98014), CHIR98023 (CT98023), CHIR99021 (CT99021), and CHIR99021 trihydrochloride.

[0086] An effective amount of a WNT activator used in media described herein can be between about 0.01 pM and about 10,000 pM. In some aspects, the effective amount of the WNT activator for the media described herein can be between about 0.1 pM and about 10,000 pM, between about 10 and about 10,000 pM, between about 100 pM and about 10,000 pM, between about 1 ,000 pM and about 10,000 pM, between about 5,000 pM and about 10,000 pM, between about 0.1 pM and about 5,000 pM, between about 1 pM and about 5,000 pM, between about 10 pM and about 5,000 pM, between about 1 ,000 pM and about 5,000 pM, or between about 2,500 pM and about 5,000 pM. In some aspects, WNT activator used in the media described herein can be between about 0.01 pM and about 100 pM. In some aspects, the effective amount of the WNT activator used in the media described herein can be between about 0.1 pM and about 5 pM. In some aspects, the effective amount of the WNT activator used in the media described herein is about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 3 pM , about 4 pM , about 5 pM , about 6 pM , about 7 pM , about 8 pM , about 9 pM , about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM. In some aspect, the WNT activator is added to the culture medium at about 1 pM. In some aspects, the WNT activator is added to the culture medium at about 0.3 pM. In some aspects, the WNT activator added to the culture medium is CHIR99021 at about 1 pM. In some aspects, the WNT activator added to the culture medium is CHIR99021 at about 0.3 pM.

[0087] Any MEK inhibitor may be used in media described herein. Exemplary MEK inhibitors include, but are not limited to, GSK1120212, XL518, MEK162, CI-1040, PD0325901 , and TAK-733. In some aspects, the MEK inhibitor should be capable of entering cells in sufficient quantities to inhibit MEK therein. In some aspects, the MEK inhibitor is added to the cell culture medium at a concentration at least equal to the IC50 of the MEK inhibitor. In some aspects, the MEK inhibitor is added to the cell culture medium at a concentration between 0.5 and 50 times the IC50 of the MEK inhibitor.

[0088] An effective amount of a MEK inhibitor used in media described herein can be between about 0.01 pM and about 10,000 pM. In some aspects, the effective amount of the MEK inhibitor for the media described herein can be between about 0. 1 pM and about 10,000 pM, between about 10 pM and about 10,000 pM, between about 100 pM and about 10,000 pM, between about 1 ,000 pM and about 10,000 pM, between about 5,000 pM and about 10,000 pM, between about 0.1 pM and about 5,000 pM, between about 1 pM and about 5,000 pM, between about 10 pM and about 5,000 pM, between about 1 ,000 pM and about 5,000pM, or between about 2,500 pM and about 5,000 pM. In some aspects, MEK inhibitor used in the media described herein can be between about 0.01 pM and about 100 pM. In some aspects, the effective amount of the MEK inhibitor used in the media described herein can be between about 0.1 pM and about 1 pM. In some aspects, the effective amount of the MEK inhibitor used in the media described herein is about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM , about 3 pM , about 4 pM , about 5 pM , about 6 pM , about 7 pM , about 8 pM , about 9 pM , about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM. In some aspects, the MEK inhibitor is added to the culture medium at about 0.3 pM. In some aspects, the MEK inhibitor added to the culture medium is PD0325901 at about 0.3 pM.

[0089] In some aspects, the medium comprises a cell culture medium. In some aspects, the cell culture medium is a basal medium. The basal medium may be any conventional medium used for culturing stem cells. The basal medium may be manually prepared according to conventional methods. Further, the basal medium may be a commercially available medium or a mixture thereof. For example, the basal medium may be selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium; GIBCO), MEM (Minimal Essential Medium; GIBCO), BME (Basal Medium Eagle; GIBCO), RPMI 1640 (GIBCO), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12; GIBCO), DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10; GIBCO), a-MEM (a-Minimal essential Medium; GIBCO), G-MEM (Glasgow's Minimal Essential Medium; GIBCO), IMDM (Isocove's Modified Dulbecco's Medium; GIBCO), Neurobasal medium (GIBCO), and KnockOut DMEM (GIBCO). The basal medium may contain one or more supplements, which includes, but not limited to, KnockOut Serum Replacement (GIBCO), KnockOut SR XenoFree (GIBCO), KnockOut SR XenoFree Growth Factor Cocktail (GIBCO), N2 supplement (GIBCO), B27 supplement (GIBCO).

[0090] In some aspects, the culture medium comprises neurobasal medium. Neurobasal medium is known by those of skill in the art; by way of a non-limiting example, Gibco’s Neurobasal™ medium (GIBCO: 21103049). In some aspects, the neurobasal medium can be used at a concentration between 0.1X to 2X. In some aspects, neurobasal medium is used in the culture medium at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X. In some aspects, the culture medium comprises neurobasal medium at a concentration from about 1 % (vol / vol)to about 100% (vol / vol). In some aspects, about 1% (vol / vol), about 5% (vol / vol), about 10% (vol / vol), about 15% (vol / vol), about 20% (vol / vol), about 25% (vol / vol), about 30% (vol / vol), about 35% (vol / vol), about 40% (vol / vol), 45% (vol / vol), about 50% (vol / vol), about 55% (vol / vol), about 60% (vol / vol), about 65% (vol / vol), about 70% (vol / vol), about 75% (vol / vol), about 80% (vol / vol), about 85% (vol / vol), about 90% (vol / vol), about 95% (vol / vol), or about 99% (vol / vol) of neurobasal medium can be used in the culture medium. In some aspects, the culture medium comprises neurobasal medium at a concentration of at least 40% (vol / vol).

[0091] In some aspects, the culture medium comprises DMEM / F-12 medium. DMEM / F12 medium can be, by way of non-limiting example, Gibco’s DMEM / F12™ medium. In some aspects, the culture medium comprises DMEM / F12 and can be used at a range between 0.1X to 2X. The DMEN / F12 can be, for example, DMEM / F-12 Medium (GIBCO: 11320033). In some aspects, DMEM / F12 medium is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X. In some aspects, the culture medium comprises DM EM / F12 at a concentration from about 1% (vol / vol) to about 100% (vol / vol). In some aspects, about 1 % (vol / vol), about 5% (vol / vol), about 10% (vol / vol), about 15% (vol / vol), about 20% (vol / vol), about 25% (vol / vol), about 30% (vol / vol), about 35% (vol / vol), about 40% (vol / vol), 45% (vol / vol), about 50% (vol / vol), about 55% (vol / vol), about 60% (vol / vol), about 65% (vol / vol), about 70% (vol / vol), about 75% (vol / vol), about 80% (vol / vol), about 85% (vol / vol), about 90% (vol / vol), about 95% (vol / vol), or about 99% (vol / vol) of DMEM / F12 can be used in the culture medium. In some aspects, the culture medium comprises DMEM / F12 at a concentration of at least 40% (vol / vol).

[0092] In some aspects, the media described herein, further comprises at least one additional factor, e.g., N2 supplement (N2), B27 supplement (B27), CEPT (Chroman 1 , Emricasan, Polyamines, and Trans-ISRIB), knockout serum replacement (KSR), Matrigel®, Geltrex®, VitroGel®, Glutamax™, Insulin-Transferrin-Selenium-Ethanolamine (ITS-X), B- estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, heat inactivated Fetal bovine serum (FBS), D-glucose, or any combination thereof.

[0093] In some aspects, the culture medium comprises N2 supplement. The N2 can be, for example, N2 (GIBCO: 17502048). The N2 can be used in the culture medium at a concentration between about 0.1X to about 2X. In some aspects, N2 is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1 ,8X, about 1.9X, or about 2X. In some aspects, the culture medium comprises N2 at a concentration from about 0.1% (vol / vol) to about 5% (vol / vol). In some aspects, about 0.1% (vol / vol), about 0.5% (vol / vol), about 1% (vol / vol), about 1.5% (vol / vol), about 2% (vol / vol),about 2.5% (vol / vol), about 3% (vol / vol), about 3.5% (vol / vol), about 4% (vol / vol), about 4.5% (vol / vol), or about 5% (vol / vol)of N2 can be used in the culture medium. In some aspects, the culture medium comprises N2 at a concentration of at least about 1% (vol / vol).

[0094] In some aspects, the culture medium comprises B27 supplement. The B27 can be, for example, B27 (GIBCO: 17504044). The B27 can be used in the culture medium at a concentration between about 0.1X to about 2X. In some aspects, B27 is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0095] In some aspects, the culture medium comprises B27 at a concentration from about 0.1% (vol / vol) to about 5% (vol / vol). In some aspects, about 0.1% (vol / vol), about 0.5% (vol / vol), about 1% (vol / vol), about 1.5% (vol / vol), about 2% (vol / vol), about 2.5% (vol / vol), about 3% (vol / vol), about 3.5% (vol / vol), about 4% (vol / vol), about 4.5% (vol / vol), or about 5% (vol / vol) of B27 can be used in the culture medium. In some aspects, the culture medium comprises B27 at a concentration of at least about 1% (vol / vol).

[0096] In some aspects, the culture medium comprises a medium supplemented with B27. Non-limiting examples of B27 includes Gibco’s B27™. In some aspects, the culture medium comprises a basal medium supplemented with N2. Non-limiting examples of N2 includes Gibco’s N2™. In some aspects, the culture medium comprises Neurobasal medium supplemented with N2. In some aspects, the culture medium comprises neurobasal medium supplemented with B27. In some aspects, the culture medium comprises a basal medium supplemented with N2 and B27.

[0097] In some aspects, KSR (for e.g, Gibco KnockOut Serum Replacement) used in the described media can be between about 1-100% (vol / vol). In some aspects, the KSR added to the medium for the methods described herein can be about 1% (vol / vol), about 5% (vol / vol), about 10% (vol / vol), about 12% (vol / vol), about 15% (vol / vol), about 17% (vol / vol), about 20% (vol / vol), about 22% (vol / vol), about 25% (vol / vol), about 27% (vol / vol), about 30% (vol / vol), about 35% (vol / vol), about 40% (vol / vol), about 45% (vol / vol), about 50% (vol / vol), about 55% (vol / vol), about 60% (vol / vol), about 65% (vol / vol), about 70% (vol / vol), about 75% (vol / vol), about 80% (vol / vol), about 85% (vol / vol), about 90% (vol / vol), or about 100% (vol / vol). In some aspects, KSR is added to the medium used in the methods described herein at about 30% (vol / vol).

[0098] In some aspects, an extracellular matrix (synthetic, derived from cells, or a combination thereof) can be used in the described medium. In some aspects, Matrigel® (reconstituted basement membrane derived from extracts of Engelbreth-Holm-Swarm mousetumors), Geltrex® (a basement membrane extract), VitroGel® (hydrogel system that closely mimics the natural extracellular matrix (ECM) environment), or similar product can be used in the described media. The Matrigel®, Geltrex®, VitroGel®, or other similar product can be used in an amount between about 1-40% (vol / vol). In some aspects, the Matrigel®, Geltrex®, VitroGel®, or similar product is added to the medium for the methods described herein can be about 1% (vol / vol), about 2% (vol / vol), about 3% (vol / vol), about 4% (vol / vol), about 5% (vol / vol), about 6% (vol / vol), about 7% (vol / vol), about 8% (vol / vol), about 9% (vol / vol), about 10% (vol / vol), about 12% (vol / vol), about 15% (vol / vol), about 17% (vol / vol), about 20% (vol / vol), about 22% (vol / vol), about 25% (vol / vol), about 27% (vol / vol), about 30% (vol / vol), about 35% (vol / vol), or about 40% (vol / vol). In some aspects, Matrigel®, Geltrex®, VitroGel®, or similar product added to the medium used in the methods described herein can be about 4% (vol / vol). In some aspects, Matrigel® is added to the medium used in the methods described herein at about 4% (vol / vol).

[0099] In some aspects, FBS (for e.g., Gibco Fetal Bovine Serum FBS) used in the described media can be between about 1-40% (vol / vol). In some aspects, the FBS added to the medium for the methods described herein can be about 1% (vol / vol), about 5% (vol / vol), about 10% (vol / vol), about 12% (vol / vol), about 15% (vol / vol), about 17% (vol / vol), about 20% (vol / vol), about 22% (vol / vol), about 25% (vol / vol), about 27% (vol / vol), about 30% (vol / vol), about 35% (vol / vol), or about 40% (vol / vol). In some aspects, FBS is added to the medium used in the methods described herein at about 20% (vol / vol).

[0100] In some aspects, one or more of the additional factors CEPT, Glutamax™, ITS-X, or sodium pyruvate, can be used in the described media between about 0.1 ml to about 10 ml. In some aspects, the effective amount of one or more of the additional factors CEPT, Glutamax™, ITS-X, progesterone, or sodium pyruvate used in the media described herein is about 0.1 ml, about 0.2 ml, about 0.3 ml, about 0.4 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1 ml, about 2 ml, about 3 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, or about 10 ml. In some aspects, amount of one or more of the additional factors CEPT, Glutamax™, ITS-X, or sodium pyruvate used in the media described herein is about 1 ml.

[0101] In some aspects, additional factor that can be used in the media described herein is progesterone. In some aspects, progesterone can be used in the described media between about 1-500 ng / ml. In some aspects, progesterone is added to the culture medium at about 1 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 50 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, or about 500 ng / ml. In some aspects, progesterone is added to the culture medium at about 200 ng / ml.

[0102] In some aspects, additional factor used in the described media can be D-glucose. In some aspects, D-glucose can be used in the described media between about 1-100 mg / ml. In some aspects, D-glucose is added to the culture medium at about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 50 ng / ml, or about 100 ng / ml. In some aspects, progesterone is added to the culture medium at about 4 mg / ml.

[0103] In some aspect, the additional factors such as one or more of B-estradiol, or N- acetyl-L-cysteine is added to the culture medium used in the methods described herein at a range between about 1 nm to about 100 pm. In some aspects, the additional factors such as one or more of B-estradiol, or N-acetyl-L-cysteine is added to the culture medium at about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 50 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM , about 500 nM , about 1 pM , about 5 pM , about 15 pM , about 20 pM , about 25 pM , about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, or about 100 pm. In some aspects, B-estradiol can be used in the described media at about 8 nM. In some aspects, N-acetyl-L-cysteine can be used in culture medium of the described methods at about 25 pM.

[0104] In some aspects, the medium disclosed herein comprises a first culture medium. In such aspects, the first culture medium is a neurobasal medium. In some aspects, the first culture medium is a neurobasal medium supplemented with N2 and B27. In further aspects, the first culture medium comprises a FGF activator, a TGF-p activator, a WNT activator, or a MEK inhibitor, or any combination thereof. In some aspects, the first culture medium is a neurobasal medium supplemented with N2, B27, a FGF activator, a TGF-p activator, a WNT activator, a MEK inhibitor, or any combination thereof. In some aspects, the FGF activator used in the first culture medium is FGF2. In some aspects, FGF2 is present in the first culture medium at about 1-200 ng / ml. In some aspects, the TGF-p activator used in the first culture medium is Activin-A. In some aspects, the Activin-A is present in the first culture medium at about 1-200 ng / ml. In some aspects, the WNT activator used in the first culture medium is a GSK3 inhibitor. In some aspects, the GSK3 inhibitor used in the first culture medium is a CHIR99021. In some aspects, CHIR99021 is present in the first culture medium at about 0.1- 5 pM. In some aspects, the MEK inhibitor used in the first culture medium is a PD0325901 . In some aspects, the MEK inhibitor is present in the first culture medium at about 0.1-1 pM of PD0325901. In some aspects, the first culture medium comprises N2B27 basal medium supplemented N2, B27, 10 ng / ml FGF2, 10 ng / ml Activin-A, 1 pM CHIR99021 and 0.3pMPD0325901. In further aspects, the first culture medium may be further supplemented with additional factors disclosed herein. In some aspects, the first culture medium is further supplemented with CEPT. In some aspects, the first culture medium is further supplemented with 1 ml of CEPT.

[0105] In some aspects, the medium disclosed herein comprises a second culture medium. In such aspects, the second culture medium is a neurobasal medium. In some aspects, the second culture medium is a neurobasal medium supplemented with N2 and B27. In further aspects, the second culture medium comprises a FGF activator, a TGF-p activator, a MEK inhibitor, or any combination thereof. In some aspects, the second culture medium is a neurobasal medium supplemented with N2, B27, a FGF activator, a TGF-p activator, a MEK inhibitor, or any combination thereof. In some aspects, the FGF activator used in the second culture medium is FGF2. In some aspects, FGF2 is present in the second culture medium at about 1-200 ng / ml. In some aspects, the TGF-p activator used in the second culture medium is Activin-A. In some aspects, the Activin-A is present in the second culture medium at about 1-200 ng / ml. In some aspects, the MEK inhibitor used in the second culture medium is a PD0325901. In some aspects, the MEK inhibitor is present in the second culture medium at about 0.1-1 pM of PD0325901. In some aspects, the second culture medium comprises N2B27 basal medium supplemented N2, B27, 10 ng / ml FGF2, 10 ng / ml Activin-A, and 0.3 pM PD0325901.

[0106] In some aspects, the medium disclosed herein comprises a third culture medium. In some aspects, the third culture medium comprises In vitro culture medium (IVC). In some aspects, the IVC comprises the basal medium DMEM / F12. In some aspects, the third culture medium comprises IVC supplemented with at least one of Matrigel®, Geltrex®, or VitroGel®. In some aspects, the third culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 1-100% (vol / vol). In some aspects, the third culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 4% (vol / vol). In some aspects, the third culture medium comprises IVC comprising DMEM / F12, Glutamax™, ITS-X, B-estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, FBS, or any combination thereof. In some aspects, the third culture medium comprises IVC comprising DMEM / F12, 4% of Matrigel®, 1 ml of Glutamax™, 1 ml of ITS-X, 8 nM of B-estradiol, 200 ng / ml of progesterone, 25 pM of N-acetyl-L-cysteine, 1 ml sodium pyruvate, 20% FBS, or any combination thereof. In some aspects, the third culture medium comprises IVC comprising DMEM / F12, 4% of Matrigel®, 1 ml of Glutamax™, 1 ml of ITS-X, 8 nM of B-estradiol, 200 ng / ml of progesterone, 25 pM of N-acetyl-L-cysteine, 1 ml sodium pyruvate, and 20% FBS.

[0107] In some aspects, the medium disclosed herein comprises a fourth culture medium. In some aspects, the fourth culture medium comprises IVC. In some aspects, the IVC comprises the basal medium DMEM / F12. In some aspects, the fourth culture medium comprises IVC supplemented with at least one of Matrigel®, Geltrex®, or VitroGel®. In some aspects, the fourth culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 1-100% (vol / vol). In some aspects, the fourth culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 4% (vol / vol). In some aspects, the fourth culture medium comprises IVC comprising DMEM / F12, Glutamax™, ITS-X, B-estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, KSR, or any combination thereof. In some aspects, the fourth culture medium comprises IVC comprising DMEM / F12, 4% of Matrigel®, 1 ml of Glutamax™, 1 ml of ITS-X, 8 nM of B-estradiol, 200 ng / ml of progesterone, 25 pM of N-acetyl-L-cysteine, 1 ml sodium pyruvate, 30% KSR, or any combination thereof. In some aspects, the fourth culture medium comprises IVC comprising DMEM / F12, 4% of Matrigel®, 1 ml of Glutamax™, 1 ml of ITS-X, 8 nM of B-estradiol, 200 ng / ml of progesterone, 25 pM of N-acetyl-L-cysteine, 1 ml sodium pyruvate, and 30% KSR.

[0108] In further aspects, the fourth culture medium may be further supplemented with D- glucose. In some aspects, D-glucose may be supplemented in the fourth medium at about 4 mg / ml.

[0109] In some aspects, the method of generating embryo-like structures or peri- gastruloids from PSCs comprise maintaining PSCs in a first culture medium, in a three- dimensional culture device. In such aspects, the method comprises maintaining PSCs in a first culture medium about 0-10 days. In some aspects, the method comprises maintaining PSCs in a first culture medium for about 0 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In some aspects, the method comprises maintaining PSCs in a first culture medium about 1- 7 days. In some aspects, the method comprises maintaining PSCs in a first culture medium about 1 days. In some aspects, the method comprises maintaining PSCs in a first culture medium about 2 days.

[0110] In further aspects, the method can comprise changing and / or replenishing the first culture medium during when PSCs is maintained in the first culture medium. In some aspects, the method further comprises removing, changing or replenishing the first culture medium. In some aspects, changing or replenishing the first culture medium comprises changing or replenishing with the same first culture medium, with the first culture medium without supplements, or with the first culture medium with additional supplements. For e.g., on 0 days PSCs is maintained in the three-dimensional device with a first culture medium supplementedwith CEPT. In some aspects, on 1 day the first culture medium supplemented with CEPT is removed and fresh first culture medium without CEPT is added to the three-dimensional device.

[0111] In some aspects, the method of generating embryo-like structures or peri- gastruloids from PSCs can further comprise maintaining PSCs in a second culture medium, in a three-dimensional culture device. In such aspects, the method comprises maintaining PSCs in a second culture medium about 0-10 days. In some aspects, the method comprises maintaining PSCs in a second culture medium for about 0 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In some aspects, the method comprises maintaining PSCs in a second culture medium about 2-7 days. In some aspects, the method comprises maintaining PSCs in a second culture medium about 1 day. In some aspects, the method comprises maintaining PSCs in a second culture medium about 2 days.

[0112] In some aspects, the method can further comprise removing, changing and / or replenishing the second culture medium during when PSCs is maintained in the second culture medium. In some aspects, changing or replenishing the second culture medium comprises changing or replenishing with the same second culture medium, with a second culture medium without supplements, or with a second culture medium with additional supplements.

[0113] In some aspects, the method of generating embryo-like structures or peri- gastruloids from PSCs can further comprise maintaining PSCs in a third culture medium, in a three-dimensional culture device. In such aspects, the method comprises maintaining PSCs in a third culture medium about 0-10 days. In some aspects, the method comprises maintaining PSCs in a third culture medium for about 0 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In some aspects, the method comprises maintaining PSCs in a third culture medium about 1-7 days. In some aspects, the method comprises maintaining PSCs in a third culture medium about 1 day. In some aspects, the method comprises maintaining PSCs in a third culture medium about 2 days.

[0114] In some aspects, the method can further comprise removing, changing and / or replenishing the third culture medium during when PSCs is maintained in the third culture medium. In some aspects, changing or replenishing the third culture medium comprises changing or replenishing with the same third culture medium, with a third culture medium without supplements, or with a third culture medium with additional supplements.

[0115] In some aspects, the method of generating embryo-like structures or peri- gastruloids from PSCs can further comprise maintaining PSCs in a fourth culture medium, ina three-dimensional culture device. In such aspects, the method comprises maintaining PSCs in a fourth culture medium about 0-20 days. In some aspects, the method comprises maintaining PSCs in a fourth culture medium for about 0 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days. In some aspects, the method comprises maintaining PSCs in a fourth culture medium about 1-14 days. In some aspects, the method comprises maintaining PSCs in a fourth culture medium about 2 days. In some aspects, the method comprises maintaining PSCs in a fourth culture medium about 4 days.

[0116] In some aspects, the method can further comprise removing, changing and / or replenishing the fourth culture medium during when PSCs is maintained in the fourth culture medium. In some aspects, changing or replenishing the fourth culture medium comprises changing or replenishing with the same fourth culture medium, with a fourth culture medium without supplements, or with a fourth culture medium with additional supplements.

[0117] In some aspects, the method comprises means for replacing the culture medium, for e.g., replacing first culture medium with the second culture medium, the second culture medium with the third culture medium, and / or the third culture medium with the fourth culture medium. Means for replacing the culture medium can be any method or device known in the art, including manually using a pipette, or automated systems such as Portable Culture Medium Replacement System (BMT fluid control solutions).

[0118] In some aspects, the method further comprises picking, and / or removing peri- gastruloids or the embryo-like structures. Means for picking, and / or removing peri-gastruloids or the embryo-like structures can include any method or device known in the art, including mouth pipette, microtweezers, micropipettes, Eppendorf CellTram, and by applying mild suction.

[0119] In some aspects, a method of generating embryo-like structures or peri-gastruloids from PSCs comprises obtaining a three-dimensional culture device, wherein the three- dimensional culture device comprises PSCs and a first culture medium, wherein the first culture medium comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, a WNT activator, and a MEK inhibitor and maintaining the cells in the first culture medium for about 1-7 days. In further aspects, the method comprises changing the culture medium in the three-dimensional culture device to a second culture medium, wherein the second culture medium comprises a FGF activator, a TGF-p activator, and a MEK inhibitor and maintaining the cells in the second culture medium for about 2-7 days. In further aspects, the method comprises changing the culture medium in the three-dimensional culture device to a third culture medium, wherein the third culture medium comprises IVC medium supplemented with Matrigel®, Geltrex®, or VitroGel® and maintaining the cells in the third culture medium for about 1-7 days to obtain embryo-like structures. In some aspects, the method further comprises changing the culture medium in the three- dimensional device to a fourth medium, wherein the fourth culture medium comprises IVC medium supplemented with Matrigel®, Geltrex®, or VitroGel® and Knock out serum (KSR) and maintaining the cells in the fourth culture medium for about 1-14 days. In further aspects, the fourth culture medium can be further supplemented with D-glucose, and the cells can be maintained for about 3-14 days.

[0120] In some aspects, methods of generating embryo-like structures or peri-gastruloids provided herein, the PSCs is cultured for an appropriate period of time sufficient to form the embryo-like structures or peri-gastruloids. In some aspects, the culturing is conducted for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more as needed. In some aspects, the culturing is conducted for about 1-5 days, about 2-6 days, about 3-7 days, about 4-8 days, about 5-9 days, about 6-10 days, or about 2-14 days.

[0121] In some aspects, embryo-like structures or peri-gastruloids are generated from a mammalian PSCs. In some aspects, the mammalian PSCs is a PSC from a human, a mouse, a rat, a rabbit, a cat, a dog, a guinea pig, a hamster, a horse, a cow, a sheep, a pig, a goat, an elephant, a rhinoceros, an orangutan, a gorilla, a bonobo, a chimpanzee, a monkey, a panda, a tiger, a whale, a dolphin, a sea lion, a narwhal, a beluga, a fox, a wolf, a pronghorn, a kangaroo, a sloth, a koala, a hippopotamus, a bear, or a leopard.

[0122] In some aspects, the method further comprises evaluation of PSCs, or structures formed by PSCs using microscopy at one or more stages including during maintenance or during media changes. Non-limiting examples of microscopy techniques for use in evaluation can comprise bright field microscope or epifluorescence microscope. In some aspects, cells or structures can be evaluated using qualitative or quantitative assessment of expression of lineage specific biomarkers. In such aspects, the expression of lineage specific biomarkers in the cells or structures can be compared to expression of such biomarkers in a previously established embryo or peri-gastruloid structure, or an average of expression of the biomarkers generally known in embryos or peri-gastruloid. Further, the expression of the biomarkers and structures can vary with the stage of the embryo or peri-gastruloids. In some aspects, the expression of biomarkers, for e.g., SOX2, SOX17, PRDM1 , TFAP2C, CDX2, T, PAX6, or any combination thereof, is evaluated at different timepoints at and after culture of PSCs and generation of embryo-like structures or peri-gastruloids. Any methods of qualitative or quantitative assessment of expression of lineage specific biomarkers known in the art can beused herein, non-limiting examples of which can include PCR, RT-PCR, immunostaining, and sequencing.

[0123] In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise epiblast-like, hypoblast-like compartments, or any combination thereof. In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise epiblast-like compartments. In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise hypoblast- 1 ike compartments. In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise epiblast-like compartments and hypoblast-like compartments. In some aspects, epiblast-like compartments can be assessed using the presence of at least 1-10 cells that are for e.g., SOX2+. In further aspects, cells of the epiblast-like compartments express SOX2, NANOG, OCT4, OTX2, or any combination thereof. In further aspects, cells of the epiblast-like compartments express SOX2, NANOG, OCT4, and OTX2. In some aspects, hypoblast- 1 ike compartments can be assessed using presence of at least 1-10 cells that are for e.g., SOX17+. In further aspects, cells of the hypoblast-like compartment express SOX17, FOXA2, GATA6, GATA4, LEFTY, CER1 , OTX2, N-CAD, PRDM1 , or any combination thereof. In further aspects, cells of the hypoblast-like compartment express SOX17, FOXA2, GATA6, GATA4, LEFTY, CER1 , OTX2, N-CAD, and PRDM1.

[0124] In further aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise an apical membrane. In some aspects, the apical membrane can be assessed using the presence of apical marker, for e.g., PODXL.

[0125] In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise epiblast-like cells, amnion-like cells, hypoblast- 1 ike cells, yolk sac-like cells, or any combination thereof. In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise epiblastlike cells. In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise amnion-like cells. In some aspects, the peri- gastruloids or the embryo-like structures generated using the methods disclosed herein comprise hypoblast- 1 ike cells. In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise yolk sac-like cells. In some aspects, the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise epiblast-like cells, amnion-like cells, hypoblast-like cells, and yolk sac-like cells. In some aspects, epiblast-like cells express OCT4, SOX2, NANOG. In some aspects, yolk sac-like cells express GATA6, GATA4, SOX17. In some aspects, the hypoblast-like cells express GATA6, GATA4, S0X17. In some aspects, amnion-like cells express GATA3, OCT4, ISL1 , CDX2, TFAP2A, TFAP2C, and / or T (also called Brachyury).

[0126] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise yolk sac-like cavity, amniotic-like cavity or any combination thereof. In some aspects, peri-gastruloids or the embryo-like structures generated using the disclosed methods comprise yolk sac-like cavity. In some aspects, peri- gastruloids or the embryo-like structures generated using the disclosed methods comprise amniotic-like cavity. In some aspects, peri-gastruloids or the embryo-like structures generated using the disclosed methods comprise yolk sac-like cavity, and amniotic-like cavity. In some aspects, yolk sac-like cavity comprise PDGFRa+ cells and / or PODXL+ cells. In some aspects, cells of the amniotic-like cavity comprise columnar cells. In some aspects, cells of the amniotic- like cavity comprise SOX2, OCT4, and / or NANOG.

[0127] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise T+ cells marking the site of gastrulation.

[0128] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise primordial germ cell-like cells (PGCLCs). In some aspects, PGCLCs express NANOG, TFAP2C, PRDM1 , or any combination thereof. In some aspects, PGCLCs express NANOG, TFAP2C, and PRDM1. In some aspects, PGCLCs express TFAP2C, and PRDM 1. In some aspects, PGCLCs are PRDM1+, OCT4+, TFAP2C+, SOX2- or any combination thereof. In some aspects, PGCLCs are PRDM1+, OCT4+, TFAP2C+ and SOX2-. In some aspects, PGCLCs express SOX17, PRDM1 (BLIMP1), NANOS3, or any combination thereof. In some aspects, PGCLCs express SOX17, PRDM 1 (BLIMP1), and NANOS3.

[0129] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise anterior- posted or (A-P) axis. In some aspects, peri- gastruloids or the embryo-like structures generated using the methods described herein comprise gradients of CER1 and LEFTY expression along a putative A-P axis, respectively, and an opposite gradient of NODAL expression. In some aspects, A-P axis express T at one end of the embryonic disk and / or epiblast.

[0130] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise bilaminar disk-like structure, trilaminar disk-like structure, primitive streak like structure, embryonic disk-like structure, primitive groove like structure, or any combination thereof. In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise bilaminar disk-like structure. In some aspects, peri-gastruloids or the embryo-like structures generated using themethods described herein comprise trilaminar disk-like structure. In some aspects, peri- gastruloids or the embryo-like structures generated using the methods described herein comprise primitive streak like structure. In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise embryonic disk-like structure. In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise primitive groove like structure. In some aspects, peri- gastruloids or the embryo-like structures generated using the methods described herein comprise bilaminar disk-like structure, trilaminar disk-like structure, primitive streak like structure, embryonic disk-like structure, and primitive groove like structure. In some aspects, cells of the primitive streak like structure express TDGF1, CDH1, FGF8, or any combination thereof. In some aspects, bilaminar disk-like structure, trilaminar disk-like structure, primitive streak like structure, embryonic disk-like structure, and / or primitive groove like structure can be examined or assessed using microscopic techniques available in the art, non-limiting examples include bright field microscope or epifluorescence microscope. In some aspects, cells of the primitive streak like structure express TDGF1, CDH1, CDH2, and FGF8.

[0131] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise neural ectoderm cells, non-neural ectoderm cells, amniotic ectoderm cells, surface ectoderm cells, endoderm cells, yolk sac endoderm cells, nascent mesoderm, advanced mesoderm cells, lateral plate mesoderm, cardiogenic mesoderm, presomitic mesoderm, or any combination thereof. In some aspects, peri- gastruloids or the embryo-like structures generated using the methods described herein comprise neural ectoderm cells, non-neural ectoderm cells, amniotic ectoderm cells, endoderm cells, yolk sac endoderm cells, nascent mesoderm, advanced mesoderm cells, lateral plate mesoderm, cardiogenic mesoderm, and presomitic mesoderm.

[0132] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise neural ectoderm cells. In some aspects, neural ectoderm cells express SOX2, PAX6, or any combination thereof. In some aspects, neural ectoderm cells express SOX2, CDH2 and PAX6. In some aspects, neural ectoderm cells express SOX2, SOX3, OTX2, PAX6, or any combination thereof. In some aspects, neural ectoderm cells express SOX2, SOX3, OTX2, and PAX6.

[0133] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise non-neural ectoderm cells. In some aspects, non- neural ectoderm cells express TFAP2A, TFAP2C, GATA3, or any combination thereof. In some aspects, non-neural ectoderm cells express TFAP2A, TFAP2C, and GATA3.

[0134] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise amniotic ectoderm cells. In some aspects, amniotic ectoderm cells express TFAP2A, TFAP2C, ISL1 , CDX2, GABPR, or any combination thereof. In some aspects, amniotic ectoderm cells express TFAP2A, TFAP2C, ISL1 , CDX2, and GABPR.

[0135] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise surface ectoderm cells. In some aspects, surface ectoderm cells express LHX2, BMP7, EYA1 , RAX, SIX3, PAX6, or any combination thereof. In some aspects, surface ectoderm cells express LHX2, BMP7, EYA1 , RAX, SIX3, and PAX6.

[0136] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise endoderm cells. In some aspects, endoderm cells express EOMES, SOX17, FOXA2, TDGF1 , or any combination thereof. In some aspects, endoderm cells express EOMES, SOX17, FOXA2, and TDGF1.

[0137] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise yolk sac endoderm cells. In some aspects, yolk sac endoderm cells express TTR, AFP, APOA1 , APOA2, APOC1 , APOE, or any combination thereof. In some aspects, yolk sac endoderm cells express TTR, AFP, APOA1 , APOA2, APOC1 , and APOE.

[0138] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise nascent mesoderm. In some aspects, nascent mesoderm express FGF8, MIXL1 , EOMES, WNT3, MESP1 , MESP2, or any combination thereof. In some aspects, nascent mesoderm express FGF8, MIXL1 , EOMES, WNT3, MESP1 , and MESP2.

[0139] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise advanced mesoderm cells. In some aspects, advanced mesoderm cells express TMEM88, COL3A1 , ACTC1 , or any combination thereof. In some aspects, advanced mesoderm cells express TMEM88, COL3A1 , and ACTC1.

[0140] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise lateral plate mesoderm. In some aspects, lateral plate mesoderm express HAND1 , MEIS1 , GATA4, PITX1 , PITX2, BMP4, or any combination thereof. In some aspects, lateral plate mesoderm express HAND1 , MEIS1 , GATA4, PITX1 , PITX2, and BMP4.

[0141] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise cardiogenic mesoderm. In some aspects, cardiogenicmesoderm express cTnT, MYL7, KDR, IGFBP3, or any combination thereof. In some aspects, cardiogenic mesoderm express cTnT, MYL7, KDR, and IGFBP3.

[0142] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise pre-somitic mesoderm. In some aspects, pre-somitic mesoderm express TBX6, HES7, MLLT3, TCF15, or any combination thereof. In some aspects, presomitic mesoderm express TBX6, HES7, MLLT3, and TCF15.

[0143] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise hematopoietic progenitors. In some aspects, hematopoietic progenitor cells express CD34.

[0144] In some aspects, peri-gastruloids or the embryo-like structures generated using the methods described herein comprise rosette-like structures. In some aspects, the rosettelike structure comprise TBX18+ cells.III. In vitro system

[0145] Aspects of the present disclosure further provides an in vitro system. In such aspects, the system comprises PSCs, a three-dimensional culture device, a first culture medium, second culture medium, and a third culture medium. In some aspects, the in vitro system further comprises a fourth culture medium. In some aspects, the in vitro system disclosed herein can be used for generation of peri-gastruloids or the embryo-like structures.

[0146] In some aspects of the in vitro system, PSCs are mammalian PSCs. In some aspects, the mammalian PSCs is a PSC from a human, a mouse, a rat, a rabbit, a cat, a dog, a guinea pig, a hamster, a horse, a cow, a sheep, a pig, a goat, an elephant, a rhinoceros, an orangutan, a gorilla, a bonobo, a chimpanzee, a monkey, a panda, a tiger, a whale, a dolphin, a sea lion, a narwhal, a beluga, a fox, a wolf, a pronghorn, a kangaroo, a sloth, a koala, a hippopotamus, a bear, or a leopard.

[0147] In some aspects, PSCs in the in vitro system may be naive PSCs, PSC, naive-like PSCs, primed PSCs, formative PSCs, or extended PSCs. In some aspects, the PSCs is naive PSCs. In some aspects, the PSCs is naive-like PSCs. In some aspects, the PSCs is primed PSCs. In some aspects, the PSCs is formative PSCs. In some aspects, the PSCs is extended PSCs. In some aspects, PSCs may be conventional human ESCs or induced human PSCs.

[0148] In some aspects of the in vitro system, naive PSCs disclosed herein, resemble the embryonic epiblast at an earlier time-point in development, for e.g., cells in vitro resembling the inner cell mass of human embryonic day (E) 6-7 preimplantation blastocysts. Naive pluripotent stem cells can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep),caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise naive human pluripotent stem cells (hPSCs).

[0149] In some aspects of the in vitro system, naive-like PSCs disclosed herein, are cells that resemble closely the inner cell mass (ICM) and / or closeness of their expression profiles to cells of the ICM. Naive-like pluripotent stem cells can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise naive-like human pluripotent stem cells (hPSCs).

[0150] In some aspects of the in vitro system, primed PSCs disclosed herein, display a gene expression signature more similar to the anterior epiblast of a late-gastrula-stage embryo (approximately embryonic day 7, ~E7). Primed PSCs can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise primed human pluripotent stem cells (hPSCs).

[0151] In some aspects of the in vitro system, formative PSCs, disclosed herein, comprise epiblast cells poised for gastrulation. Formative PSCs can be established from different embryonic stem cells, pre- / early-gastrula epiblasts and induced PSCs. Similar to pre- / early- gastrula epiblasts, formative PSCs show the transcriptom ic features of formative pluripotency, which are distinct from naive ESCs and primed PSCs. In some aspects, formative PSCs share transcriptom ic similarities with embryonic day 5-6 (E5-6) epiblasts. Formative PSCs can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise formative human pluripotent stem cells (hPSCs).

[0152] In some aspects of the in vitro system, extended (or expanded) PSCs, disclosed herein, can generate both embryonic and extra-embryonic lineages. Extended PSCs can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ovine (e.g., sheep), caprine (e.g., goat), porcine (e.g., pig), bovine (e.g., cow), an ungulate (e.g., horse) PSCs. In some aspects, the PSCs comprise extended human pluripotent stem cells (hPSCs).

[0153] In some aspects, the PSCs of the in vitro system comprise a single cell suspension of PSCs. Single cell suspension of PSCs can be made using any known method in the art. In some aspects, cultured PSCs, can be disassociated into single cells using mechanicaldissociation and / or enzyme treatment, for e.g., treatment using Dispase, Collagenase, hyaluronidase, Papain, DNAse-l, Accutase, or TrypLE™.

[0154] In some aspects, the three-dimensional culture device of the in vitro system is any suitable three-dimensional culture vessel for generation of peri-gastruloids or the embryo-like structures. In some aspects, three-dimensional culturing methods including methods using scaffold, and scaffold-free methods. In methods using scaffolds, hydrogels may be used as a scaffold. Scaffold-free methods can include low adhesion plate, hanging drop plats, micropatterned surface, spheroid, bioreactor, magnetic levitation, chip, and microfluidic device. In some aspects, the disclosed three-dimensional culture device is a microwell plate. In some aspects, the disclosed three-dimensional culture device is an AggreWell™ plate.

[0155] In some aspects, the three-dimensional device of the disclosed in vitro system can be pre-treated with an anti-adherence solution. Anti-adherence solution as disclosed herein can be any commercially available anti-adherence solution which can prevent attachment of the cells to the device, for e.g., anti-adherence rinsing solution from stem cell technologies (Catalog No. 07010).

[0156] In some aspects, the in vitro system disclosed herein comprises a first culture medium. In such aspects, the first culture medium is a neurobasal medium. In some aspects, the first culture medium is a neurobasal medium supplemented with N2 and B27. In further aspects, the first culture medium comprises a FGF activator, a TGF-p activator, a WNT activator, or a M EK inhibitor, or any combination thereof. In some aspects, the first culture medium is a neurobasal medium supplemented with N2, B27, a FGF activator, a TGF-p activator, a WNT activator, a MEK inhibitor, or any combination thereof. In some aspects, the FGF activator used in the first culture medium is FGF2. In some aspects, FGF2 is present in the first culture medium at about 1-200 ng / ml. In some aspects, the TGF-p activator used in the first culture medium is Activin-A. In some aspects, the Activin-A is present in the first culture medium at about 1-200 ng / ml. In some aspects, the WNT activator used in the first culture medium is a GSK3 inhibitor. In some aspects, the GSK3 inhibitor used in the first culture medium is a CHIR99021. In some aspects, CHIR99021 is present in the first culture medium at about 0.1-5 pM. In some aspects, the MEK inhibitor used in the first culture medium is a PD0325901. In some aspects, the MEK inhibitor is present in the first culture medium at about 0.1-1 pM of PD0325901. In some aspects, the first culture medium comprises N2B27 basal medium supplemented N2, B27, 10 ng / ml FGF2, 10 ng / ml Activin-A, 1 pM CHIR99021 and 0.3pM PD0325901. In further aspects, the first culture medium may be further supplemented with additional factors disclosed herein. In some aspects, the first culture medium is further supplemented with CEPT. In some aspects, the first culture medium is further supplemented with 1 ml of CEPT.

[0157] In some aspects, the in vitro system disclosed herein comprises a second culture medium. In such aspects, the second culture medium is a neurobasal medium. In some aspects, the second culture medium is a neurobasal medium supplemented with N2 and B27. In further aspects, the second culture medium comprises a FGF activator, a TGF-p activator, or a MEK inhibitor, or any combination thereof. In some aspects, the second culture medium is a neurobasal medium supplemented with N2, B27, a FGF activator, a TGF-p activator, a MEK inhibitor, or any combination thereof. In some aspects, the FGF activator used in the second culture medium is FGF2. In some aspects, FGF2 is present in the second culture medium at about 1-200 ng / ml. In some aspects, the TGF-p activator used in the second culture medium is Activin-A. In some aspects, the Activin-A is present in the second culture medium at about 1-200 ng / ml. In some aspects, the MEK inhibitor used in the second culture medium is a PD0325901. In some aspects, the MEK inhibitor is present in the second culture medium at about 0.1-1 pM of PD0325901. In some aspects, the second culture medium comprises N2B27 basal medium supplemented N2, B27, 10 ng / ml FGF2, 10 ng / ml Activin-A, and 0.3pM PD0325901.

[0158] In some aspects, the in vitro system disclosed herein comprises a third culture medium. In some aspects, the third culture medium comprises In vitro culture medium (IVC). In some aspects, the IVC comprises the basal medium DMEM / F12. In some aspects, the third culture medium comprises IVC supplemented with at least one of Matrigel®, Geltrex®, or VitroGel®. In some aspects, the third culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 1-100% (vol / vol). In some aspects, the third culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 4% (vol / vol). In some aspects, the third culture medium comprises IVC comprising DMEM / F12, Glutamax™, ITS-X, B-estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, FBS, or any combination thereof. In some aspects, the third culture medium comprises IVC comprising DMEM / F12, 4% of Matrigel®, 1 ml of Glutamax™, 1 ml of ITS-X, 8 nM of B-estradiol, 200 ng / ml of progesterone, 25 pM of N-acetyl-L-cysteine, 1 ml sodium pyruvate, 20% FBS, or any combination thereof. In some aspects, the third culture medium comprises IVC comprising DMEM / F12, 4% of Matrigel®, 1 ml of Glutamax™, 1 ml of ITS-X, 8 nM of B-estradiol, 200 ng / ml of progesterone, 25 pM of N-acetyl-L-cysteine, 1 ml sodium pyruvate, and 20% FBS.

[0159] In some aspects, the in vitro system disclosed herein comprises a fourth culture medium. In some aspects, the fourth culture medium comprises IVC. In some aspects, the IVC comprises the basal medium DMEM / F12. In some aspects, the fourth culture medium comprises IVC supplemented with at least one of Matrigel®, Geltrex®, or VitroGel®. In some aspects, the fourth culture medium comprises IVC supplemented with Matrigel®, Geltrex®, orVitroGel® at ari amount of about 1-100% (vol / vol). In some aspects, the fourth culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 4% (vol / vol). In some aspects, the fourth culture medium comprises IVC comprising DMEM / F12, Glutamax™, ITS-X, B-estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, KSR, or any combination thereof. In some aspects, the fourth culture medium comprises IVC comprising DMEM / F12, 4% of Matrigel®, 1 ml of Glutamax™, 1 ml of ITS-X, 8 nM of B-estradiol, 200 ng / ml of progesterone, 25 pM of N-acetyl-L-cysteine, 1 ml sodium pyruvate, 30% KSR, or any combination thereof. In some aspects, the fourth culture medium comprises IVC comprising DMEM / F12, 4% of Matrigel®, 1 ml of Glutamax™, 1 ml of ITS-X, 8 nM of B-estradiol, 200 ng / ml of progesterone, 25 pM of N-acetyl-L-cysteine, 1 ml sodium pyruvate, and 30% KSR.

[0160] In further aspects of the in vitro system, the fourth culture medium may be supplemented with D-glucose. In some aspects, D-glucose may be supplemented in the fourth medium at about 4 mg / ml.

[0161] In some aspects, the in vitro system further comprises means for replacing the culture medium, for e.g., replacing first culture medium with the second culture medium, the second culture medium with the third culture medium, and / or the third culture medium with the fourth culture medium. Means for replacing the culture medium can be any method or device known in the art, including manually using a pipette, or automated systems such as Portable Culture Medium Replacement System (BMT fluid control solutions).

[0162] In some aspects, the in vitro system further comprises means for picking, and / or removing peri-gastruloids or the embryo-like structures. Means for picking, and / or removing peri-gastruloids or the embryo-like structures can include any method or device known in the art, including mouth pipette, microtweezers, micropipettes, Eppendorf CellTram, and by applying mild suction.IV. Applications

[0163] The present disclosure further provides peri-gastruloid or embryo-like structure produced by the methods or system disclosed herein. In further aspects, the disclosure encompasses an embryo model comprising a peri-gastruloid or embryo-like structure produced by the method or system described herein. Such embryo models can be used for studying imprinting, reprogramming, rejuvenation, and other biochemical, metabolic, and physiological phenomena associated with embryogenesis and development. In further aspects, embryo models can be used for studying effect of mutations in embryogenesis and development.

[0164] The peri-gastruloid, embryo-like structure, or the embryo model generated using methods and system disclosed herein are further contemplated for a variety of uses including drug screening, reproductive medicine, and other research uses and methods.

[0165] In some aspects, the methods and / or in vitro culture systems involving peri- gastruloid, embryo-like structure, or the embryo model as described herein may be used to generate and isolate cells, for therapeutic transplant. Cells contemplated herein non-limiting examples of which comprise epiblast-like cells, amnion-like cells, hypoblast-like cells, yolk sac-like cells, primordial germ cell-like cells, neural ectoderm cells, non-neural ectoderm cells, amniotic ectoderm cells, endoderm cells, yolk sac endoderm cells, nascent mesoderm, advanced mesoderm cells, lateral plate mesoderm, cardiogenic mesoderm, presomitic mesoderm, or any combination thereof. In some aspects, the disclosed peri-gastruloid, embryo-like structure, embryo model, and / or cells generated can be used for cell replacement therapy. The embryos, stem cells, and the differentiated cells and tissues that are obtained or generated from these for therapeutic transplant according to the present disclosure, are produced and isolated using methods disclosed herein, and methods known in the art.

[0166] In such aspects, further provided is a method of cell replacement therapy. In some aspects, the method comprises administering in a subject in need thereof, cells from a peri- gastruloid or embryo-like structure. The disclosure further encompasses a method of repairing or regenerating a tissue or organ, the method comprising administering in a subject in need thereof, cells from a peri-gastruloid or embryo-like structure.

[0167] In some aspects, the cells are able when introduced into a subject, e.g., a human or animal, to infiltrate and proliferate at a desired target site, e.g., heart, brain, liver, bone marrow, kidney or other organ that requires cell therapy. For example, it is contemplated that progenitors cells will infiltrate into a subject and will rejuvenate the immune system of the individual by migrating to the immune system, i.e., blood and bone marrow. Alternatively, in the case of CNS progenitor cells should preferentially migrate to the brain, e.g., that of a Parkinson's, Alzheimer's, ALS, or a patient suffering from age-related senility.

[0168] Cells of a particular lineage may be selected by known methods. Cells which have commenced becoming committed to desired cell lineages contained in embryos may be identified, for e.g., by assaying for the expression of biomarkers characteristic of a particular cell lineage, e.g., hepatocyte markers in situations wherein cell therapy for treating the liver is warranted or pancreatic markers where the subject has a disorder involving the pancreas, e.g., type I or type II diabetes.

[0169] Therapeutic applications wherein cells described herein are useful for cell therapy includes transplantation, cancer, autoimmune diseases of all kinds, proliferative disorders,inflammatory disorders, neurological disorders, age-related disorders, allergic, disorders, immune disorders, viral infections, burn, trauma, other conditions involving tissue injury, and other conditions wherein replacement cells are desirable. Non-limiting examples of conditions include lupus, diabetes, myasthenia gravis, rheumatoid arthritis, ALS, Parkinson's disease, Alzheimer's disease, Huntington's disease, paralysis, multiple sclerosis, thyroiditis, AIDS, psoriasis, psoriatic arthritis, pancreatitis, hematologic malignancies, non-specific cell damage associated with radiotherapy or chemotherapy, cardiac injuries, e.g., associated with heart attack, Sjogren's syndrome, and many others.

[0170] Cells may be administered by any known methods. Typically, the cells can be administered parietally, e.g., via intravenous injection. The cells may be in solution, for e.g., buffered saline. The number of cells administered will be an amount effective to treat the particular condition. It may be beneficial also for the cells to express a marker, e.g., green fluorescent protein (GFP), while allowing for the detection of sites) and number of cells which have become stably engrafted in the subject. The use of GFP and variants thereof to detect specific cells is well known in the art. In some aspects, the method may further comprise repeatedly administering the cells, for e.g., in the case of chronic diseases such as autoimmune disorders or cancer.

[0171] In some aspects, a peri-gastruloid or embryo-like structure provided herein can further used for determining a drug toxicity. Therefore, the disclosure encompasses an in vitro drug or chemical screening system comprising a peri-gastruloid, embryo-like structure, or cells of the peri-gastruloid, or embryo-like structure. In further aspects, a peri-gastruloid, embryolike structure, or cells of the peri-gastruloid, or embryo-like structure comprising genetic variants, can be used to assess how genetic variants affect the response to drugs.

[0172] In such aspects, methods and / or in vitro culture systems comprising a peri- gastruloid, embryo-like structure, or cells as described herein may be used to determine whether a candidate molecule (e.g., a compound, or a drug) is capable of altering the generation of peri-gastruloid, embryo-like structure, or cells of the peri-gastruloid, or embryolike structure in the in vitro culture system. For example, the candidate molecule can be added into the in vitro culture system as described herein. After being cultured under suitable conditions for a suitable period, the generation of the peri-gastruloid, embryo-like structure, or cells in the culture system can be compared with a control culture system that does not contain the candidate molecule. If the peri-gastruloid, embryo-like structure, or cells in the presence of the candidate molecule is altered as compared to that in the absence of the candidate molecule, it indicates that the candidate molecule may affect embryo development.

[0173] Further disclosed herein is a high throughput platform comprising a peri-gastruloid, embryo-like structure, or cells of the peri-gastruloid, or embryo-like structure described herein. The peri-gastruloid, embryo-like structure, or cells of the peri-gastruloid, or embryo-like structure described herein can be provide a model for studying the effects of genetic variants or chemicals on early embryogenesis. Similarly, peri-gastruloid, embryo-like structure, or cells of the peri-gastruloid, or embryo-like structure model offers a platform for studying the effects of mutations on early embryogenesis. In such aspects, provided herein is a method of high- throughput genetic or chemical screening, comprising testing genetic mutations or chemicals that can cause pregnancy loss or congenital defects using the in vitro drug or chemical screening system.

[0174] In some aspects, high throughput platform comprising a peri-gastruloid, embryolike structure, or cells of the peri-gastruloid, or embryo-like structure, can be treated with a candidate molecule, or could comprise a mutation. After being cultured under suitable conditions for a suitable period, the generation of the peri-gastruloid, embryo-like structure, or cells in the culture system can be compared with a control culture system that does not contain the candidate molecule, or the mutation. If the peri-gastruloid, embryo-like structure, or cells in the presence of the candidate molecule, or mutation is altered as compared to that in the absence of the candidate molecule or the mutation, it indicates that the candidate molecule or the mutation may affect embryo development.

[0175] The peri-gastruloid, embryo-like structure, or cells of the peri-gastruloid, or embryolike structure generated using methods described herein can be genetically altered by any known means. Genetically modified cells and tissues can be obtained by performing nuclear transfer with a genetically modified nuclear PSCs to produce nuclear transfer embryo made up of genetically modified cells. Alternatively, cells and tissues can be genetically modified after they are isolated from peri-gastruloid, or embryo-like structure. In some aspects, it may be desirable for the cells to express or not express a target DNA sequence. This may be accomplished by genetically modifying the genome of the PSCs used to generate the peri- gastruloid, or embryo-like structure. In some aspects, multiple gene modifications or gene knockout may be accomplished by repeated nuclear transfer procedures wherein the genome of PSCs is modified.

[0176] In further aspects, methods and / or in vitro culture systems described herein may be used to study the behavior of stem cells (e.g., the discovery of novel biological pathways or processes involved in pluripotency of stem cells).V. Kits

[0177] The present disclosure provides kits for use in generating the peri-gastruloid, or embryo-like structure described herein. Such kits can include one or more containers comprising one or more activators described herein, e.g., a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, and a WNT activator, and a M EK inhibitor and optionally, a population of PSCs. Kits described herein may further comprise a cell culture medium suitable for culturing a population of PSCs.

[0178] The disclosed kits can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of culturing a population of cells in a medium comprising a FGF pathway activator, a TGF-p pathway activator, a WNT pathway activator, and a MEK inhibitor, as described herein. In other aspects, the instructions may comprise a description of methods for culturing the PSCs and generating the peri-gastruloid, or embryo-like structure described herein.

[0179] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.

[0180] The instructions relating to the use of the activator and inhibitor described herein generally include information as to dosage, and dosing schedule for the intended production of the disclosed peri-gastruloid, or embryo-like structure. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0181] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. A kit may have a sterile access port (e.g., the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[0182] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some aspects, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0183] Having described several aspects, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used withoutdeparting from the spirit of the present disclosure. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present disclosure. Accordingly, this description should not be taken as limiting the scope of the present disclosure.

[0184] Those skilled in the art will appreciate that the presently disclosed aspects teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.EXAMPLES

[0185] The following examples are included to demonstrate aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Materials and MethodsTable 1 : Reagents and resources used in studiesExperimental model

[0186] Human pluripotent stem cell experiments were performed at the UT Southwestern Medical Center and followed the 2021 Guidelines for Stem Cell Research and Clinical Translation released by the International Society for Stem Cell Research (ISSCR). The UT Southwestern Stem Cell Oversight Committee (SCRO) conducted a comprehensive review and approval of the protocol (registration no. 29) in accordance with the requisites stipulated by the 2021 ISSCR guidelines. The duration of the culture for these embryo models was carefully calibrated to the minimal necessary time period for the examination of gastrulation and the incipient stages of organogenesis in human development. A significant point to emphasize is that these embryo models are designed to be incomplete due to their lack of trophoblast cells and are thus not capable of supporting full human development.Stem cell lines and culture conditions

[0187] Human pluripotent stem cells including primed pluripotent stem cells, formative pluripotent stem cells and extended pluripotent stem cells were used in this study. All cell lines used in this study were cultured at 37 °C in a 5% CO2 humidified incubator with daily media changes. Cells were periodically examined for mycoplasma contamination via PCR. Cell lines were authenticated by genomic PCR, immunostaining, and in vitro differentiation tests. The following stem cell lines were used in this study: RUES2-GLR (human embryonic stem cell line), which exhibited a high efficiency in generation of peri-gastruloids and was the most commonly used cell line; Nodal-mCitrine reporter cell line (ESI017 background), was used to visualize the Nodal ligand gradient in the epiblast compartment of day 6 peri-gastruloids; H9 (WA09, human ESC) was used for examination of pluripotency markers in the epiblast compartment; PRDM 1 (Blimp 1)-2A-tdTomato(BT), TFAP2C-2A-EGFP(AG) dual reporter hiPSC line was used to evaluate peri-gastruloids generation efficiency and monitor PGCLC induction. HES7- Achilles reporter hiPSC line (NCMR1 background) was used to reproduce peri-gastruloids and monitor potential presomitic mesoderm induction. B-catenin-GFP reporter hiPSC line (Allen Institute) was used to monitor cavity formation on day 4-5 and WNT signaling on day 8. Human naive pluripotent stem cell line WIBR3 and human formative pluripotent stem cells were used for testing HDM induction of hypoblast-like cells.

[0188] Human primed pluripotent stem cells including conventional human ESCs and iPSCs were cultured on feeder-free Matrigel coated culture plates, in mTeSR Plus medium. To perform routine passaging, Dispase was used to digest the colonies without dissociation of the colonies into single cells. Cells were incubated in Dispase for 5-6 mins before removal of Dispase and then addition of 1 ml fresh mTeSR Plus medium. The colonies were then lifted from culture plate with cell lifters, and pipetted less than 10 times to break the big colonies into small clumps. 1 / 10 - 1 / 20 cells were transferred to a new well for maintenance. Accutase was used for making single cell suspensions. 10 pM ROCK inhibitor was supplemented in media on the day of single cell dissociation.

[0189] Human extended pluripotent stem cells (hEPSCs) were maintained in a condition established previously. Specifically, cells were maintained in LCDM: N2B27 medium supplied with 10 ng / ml recombinant Human LIF, 1 pM CHIR 99021 , 2pM (S)-(+)-Dimethindene maleate, 2pM Minocycline hydrochloride, 0.5 pM IWR-endo-1 , and 2pM ROCK inhibitor Y-27632, on irradiated MEF (6.9 x104 / cm2) plates pre-coated with 0.1% gelatin. Approximately 0.3-0.5 pM PD0325901 (PD03) was supplemented to LCDM to maintain pluripotency by inhibiting spontaneous differentiation. The cells were cultured at 37 °C in a 5% CO2 humidified incubator, with daily medium change. For routine passaging, cells were dissociated into single cells using TrypLE Express and transferred to a new MEF plate at a 1 :10 ratio every 3-4 days. Conversion of primed human ESCs and iPSCs to hEPSCs

[0190] Approximately 1 x105human ESCs or iPSCs were seeded on MEF feeders in mTeSR Plus supplemented with 10 pM ROCK inhibitor Y-27632. 1-2 days later the medium was changed to LCDM. Dome-shaped hEPSC colonies appeared within a few passages. Dome-shaped colonies were picked and pooled for following experiments. For some hEPSC lines, 0.3-0.5 pM PD0325901 was supplemented in LCDM to maintain dome-shaped colony. To promote the conversion efficiency, 3 days following the initial cell seeding, a daily medium change was performed with Chemical Resetting Medium: N2B27 basal medium supplemented with 1 pM PD0325901 , 10 ng / m1 recombinant human LIF and 1 mM valproic acid (VPA). hESCs converted hEPSCs, with or without chemical resetting step was not directly compared, albeit no observable difference was observed.Differentiation on attachment culture

[0191] To differentiate hEPSCs into epiblast- and hypoblast-like cells, hEPSCs were plated on MEF coated plates in LCDM. After 24h the medium was changed to tHDM: N2B27 basal medium supplemented with 10 ng / ml FGF2, 10 ng / ml Activin A, 1 pM CHIR99021) and 0.3 pM PD0325901 . Fresh tHDM medium was changed every day, for 3 days. On day 4, cell fates were evaluated via fluorescent reporter or fluorescent immunostaining to investigate theco-culture of the epiblast and hypoblast-like cells. The differentiated cells were passaged as a mixture.Establishment and optimization of peri-gastruloids protocol

[0192] To ensure high efficiency peri-gastruloids formation, stable extended pluripotent stem cells (EPSCs) of low passage are preferred. Careful quality control of the starting EPSC culture is crucial for high efficiency of peri-gastruloids formation.

[0193] To establish the peri-gastruloid formation protocol, hEPSCs were first treated with tHDM on regular 6-well plates for 4 days and then the differentiated cells were seeded in AggreWell™ plates. Specifically, on day 0, approximately 1x105hEPSCs were seeded in one well of MEF coated 6-well plate, in tHDM. Medium was changed every day. On day 4, cells were dissociated into single cells by incubation with TrypLE at 37 °C for 3-5 minutes. The single cells were resuspended in LCDM supplemented with CEPT (50 nM Chroman 1 , 5 pM Emricasan, IxPolyamines, and 0.7 pM Trans-ISRIB), as a mixture of ELCs and HLCs. Cells were counted using a hemocytometer and then seeded into an AggreWell™ plate at a density of 45 cells per microwell, for instance, 5.4x104per well of AggreWell™ 400 (24-well format) or 1.35x104per well of AggreWell™ 800 (24-well format). The next steps were identical to method version 2.0 described in the following paragraph.

[0194] For peri-gastruloid method version 2.0, on day 0, hEPSCs were dissociated into single cells by incubation with T rypLE at 37 °C for 5 minutes. After 5 minutes, cells were taken off TrypLE and mixed with LCDM at 1 :1 ratio. The cells were spun down, and the pellet was resuspended in LCDM supplemented with CEPT. While preparing the single cell suspension, a 0.1% gelatin coated 6-well plate was prepared for the removal of MEF feeder cells. After resuspension, the single cell solution was transferred to a gelatin- coated well and incubated at 37 °C for 1 hour before being moved to another gelatin- coated well for an additional hour. The complete removal of feeders is critical, it was found that residual feeder cells lowered the Peri-gastruloid formation efficiency significantly. After removal of feeders, the single cell suspension was filtered through a 40 pm cell strainer and then counted using a hemocytometer and then seeded into an AggreWell 800 plate at a density of 90 cells per microwell, which in total resulted in 2.7x104cells per well. Before seeding cells into the AggreWell™, 1 ml of anti-adherence solution was added to each well, and the plate was centrifuged at 1500 Relative Centrifugal Force (RCF) for 5 minutes to remove air bubbles and ensure coating of the bottom of the well. The plate was then left at room temperature for 15- 20 minutes before removal of anti-adherence solution, after which all wells were washed with 2 ml N2B27. To seed the cells into pre-treated AggreWell 800 wells, the concentration of cell suspension was adjusted to 2.7x104cells / ml tHDM, meanwhile the wash medium wasremoved from the AggreWell™ and 1 ml tHDM was added back to each well. 1 ml tHDM cell suspension was then added into each well in a dropwise manner. Then the AggreWell™ plate was spun at 120RCF for 3 minutes. After centrifugation, cells in the individual microwell were counted with a brightfield microscope. It is important to make sure that most of the microwells evenly received no less than about 30-50 cells (of note, variation might be introduced during operation such as hand counting and pipetting. Although 90 cells per microwell was the calculated target, the actual cell number was usually less than 90 due to handling loss and other uncontrollable variability). This confirmation step is critical because low initial seeding density significantly decreased formation efficiency. If microwell cell numbers were less than about 30-50 cells, the seeding procedure was repeated as needed to reach the desired cell number. CEPT was supplemented to the tHDM on day 0. On day 1 , 1 ml medium was removed from the each well, and 1 ml of fresh tHDM medium without CEPT was added. On day 2, a full medium change was performed. 2 ml of fresh N2B27 basal medium supplemented with 10 ng / ml FGF2, 10 ng / ml Activin A, and 0.3 pM PD0325901 (FAP, self-sorting medium) was added to each well. On day 3, another half medium change was performed with FAP. On day 4, a full medium change was performed with fresh IVC1 supplemented with 4% Matrigel. ICV1 : 76 ml Advanced DMEM / F12, 1 ml Glutamax™ (100x), 1 ml Insulin-Transferrin-Selenium- Ethanolamine (ITS-X) (100x), 10.8 pl B-estradiol stock (73.4 pM, final concentration 8 nM), 1 ml progesterone stock (20 pg / ml, final concentration 200 ng / ml), 10 pl N-acetyl-L-cysteine stock (250 mM, final concentration 25 pM), 1 ml sodium pyruvate (100x), and 20 ml heat inactivated FBS. To perform the complete medium change on day 4, approximately 1.8 ml old medium was removed carefully by pipetting, about 200 pl residual medium was left in each well to prevent perturbation of the aggregate. And then 2 ml room temperature IVC1 4% Matrigel medium was added to each well gently, 200 pl at a time via p200 pipette. Medium change was not performed on day 5. On day 6, peri-gastruloids were first evaluated under bright filed microscope (for cell line without labeling) or Epifluorescence microscope (for reporter cell lines) before being transferred to a low attachment 6-well plate. Peri-gastruloids were then handed picked on stereoscope based on morphology. Those that had well developed epithelial epiblast compartments and fluid-filled yolk-sac cavities were selected for further development. Selected aggregates were manually moved to another low attachment 6-well plate, which was pre-treated with anti-adherence solution (this step is essential to prevent attachment) and filled with 2 ml IVC2, 4% Matrigel medium, at density 15-20 peri- gastruloids per well. IVC2 medium is identical to IVC1 except 20% FBS was replaced with 30% KSR and the basal media adjusted accordingly to accommodate for the increase in serum. Prior to seeding the peri-gastruloids, 2 ml of IVC2, 4% Matrigel medium was added to each well of the pre-treated low attachment 6-well plate and placed in a 37 °C incubator for approximately 1h. This step allowed the gel formation of the Matrigel component, whichallowed the peri-gastruloids to be suspended in the medium instead of sinking to the bottom and attaching to the plate. On day 8, 2 ml IVC2, 4% Matrigel, supplemented with about 7-8 mg / ml additional D-glucose (final concentration was about 3.5-4 mg / ml), was added to each well, while no old medium was removed. On day 10, 2 ml medium was carefully removed from each well, avoiding perturbation of peri-gastruloids, and then 2 ml IVC2, 4% Matrigel, supplemented with 4 mg / ml additional D-glucose, was added to each well. We observed variability in the efficiency of peri-gastruloid development after day 8, which might be dependent on the cell line used for the protocol. Among all the cell lines tested, the RLIES2 line displayed the most robust results.

[0195] To score the peri-gastruloids at different stages, on day 4, the efficiency can be determined based on the presence of epiblast-like cells (ELC, e.g., SOX2+) and hypoblastlike cells (HLC, e.g., SOX17+) compartments; On day 6, efficiency can be determined based on the presence of epiblast-like (EPI, e.g., SOX2+), primary yolk sac-like (PYS, e.g., SOX17+) structures and amniotic cavity (AC, e.g., CDX2+); On day 8, efficiency was determined based on the presence of primary yolk sac cavity (SOX17+ cavity) amniotic tissue (TFAP2C, ISL1 , TFAP2A or CDX2). The presence of above-mentioned lineages should be determined when more than 10 positive cells were observed. Reporter cell line are recommended for evaluation of the peri-gastrulation efficiency.

[0196] For instance, in this study RUES2-GLR hESC and BATG iPSC reporter lines were used for the efficiency evaluation. Cell line-dependent efficiency variation might be observed. For instance, lower efficiency of amnion / amniotic cavity formation with BATG iPSC reporter line was observed (FIG. 1 M) versus RUES2-GLR hESCs (as shown in FIG. 2D-2E).Immunofluorescence staining

[0197] Day 4 peri-gastruloids generated by using tHDM pretreated hEPSCs (in total 8 days since the beginning of the experiment) were cultured on fibronectin- coated ibidi dishes in MEF medium: DMEM / F12 supplemented with 10% FBS. Medium was changed every other day.

[0198] Samples, including regular attached cell culture, micropatterned, and peri- gastruloids at various stages, were washed with PBS before fixation with 4% paraformaldehyde (PFA) in PBS at room temperature for 15 minutes or at 4 °C overnight. Fixed samples were washed with PBS twice before permeabilization in PBST (PBS + 0.2% Triton X) at room temperature for 1 hour. Samples were then blocked with 6% Donkey serum in PBST (0.1% Triton X), at room temperature for 1 hour. For phosphorylated protein staining, samples were incubated in PBS with 1% SDS for 30 min at 37 °C before blocking. Primary antibody dilution was prepared in 3% Donkey serum in PBST (PBS + 0.1% Triton X). Sampleswere incubated in the primary antibody dilution at room temperature for 2 hours or at 4 °C overnight followed by PBST (PBS + 0.1% Triton X) wash 3 times, about 20-30 minutes at a time. After washing, samples were then transferred to a secondary antibody dilution made with 3% Donkey serum in PBST (0.1% Triton X), and incubated at room temperature for 1 hour. After that, samples were washed 3-5 times, 30 minutes at a time, with PBST (PBS + 0.1% T riton X) at room temperature (for attached culture) or at 37 °C (for advanced peri-gastruloids) .

[0199] To perform tissue clearing, CUBIC-L, a delipidation and decoloring reagent, was used according to the manufacturer’s instructions. Specifically, the samples were fixed with 4% PFA in PBS at 4 °C overnight. The fixed samples were then washed 3 times with PBS at room temperature. Samples were then soaked in pre-delipidation solution (a 1 :1 mixture of ddH2O and CUBIC-L) and placed on a rotator at 37 °C overnight. Samples were then moved to a 100% CUBIC-L solution on a rotator at 37 °C for 3-5 days. CUBIC-L was changed on day 1 and 2, and every other subsequent day. After this delipidation process, the samples were washed with PBS 3 times prior to the above-described immunostaining procedures.Tissue sectioning

[0200] Advanced peri-gastruloids, e.g., day 11 , were subjected to frozen and paraffin sectioning for visualization of internal architecture or tissue clearing for better whole mount imaging.

[0201] To perform cryosectioning, the samples were fixed with 4% PFA in PBS at 4 °C overnight. The fixed samples were washed 3 times with PBS at room temperature, followed by embedding in unfrozen OCT (Optimal cutting temperature) compound. The OCT block was then frozen and stored at -80 °C prior to sectioning. Cryosections were made at a thickness of 10 pm, and the resulting tissue section was placed on pre-treated glass slides. Samples slides were then stored at -80 °C until use in immunofluorescence staining experiments. Before conducting the staining procedures, the sample slides were heated to 95 °C for 10 minutes for antigen retrieval. After that, immunostaining was performed as described above. The stained samples were treated with TrueBlack to eliminate autofluorescence caused by lipofuscin prior to fluorescent imaging.

[0202] To perform paraffin sectioning, the fixed peri-gastruloids samples were embedded in 2% low melting point agarose (LONZA SeaKem LE). The embedded samples were placed in histology cassettes, which were immersed in 70% ethanol. The dehydrated samples were cleared and infiltrated with paraffin. 5 pm serial sections were obtained for hematoxylin and eosin (H&E) staining.Fluorescence microscopy

[0203] Attached samples were cultured and imaged on 8 well p-slides (ibidi). Peri- gastruloids were placed in 96-well glass bottom black plate with 1.5 high performance cover glass (0.17±0.005 mm) (Cell vis) for imaging. Phase contrast and epifluorescence images were taken with a hybrid microscope (Echo Laboratories, CA) equipped with 2x (0.06 numerical aperture (NA), air), 4x (0.13 NA, air) and 10x (0.7 NA, air) objectives. Fixed sample confocal fluorescence imaging and time-lapse imaging were performed on a Nikon CSLI-W1 SoRa spinning disk confocal microscope equipped with 4x (0.13 NA, WD 17.1 , air), 20x (0.75 NA, WD 1 , air), 40x (1.3 NA, WD 0.24, oil) objectives, 405, 445, 488, 514, 561 , 640 nm confocal / SoRa laser lines, Piezo z-drive and environmental controller including CO2 and Zeiss LSM880 Airyscan laser scanning microscope equipped with 10x (0.3 NA, air), 20x (0.8 NA, air), 40x (1.4 NA, oil) objectives, 405, 458, 488, 514, 561 , and 633 nm laser lines, and an environmental controller including CO2.Image analysis

[0204] Representative images or quantification results based on imaging experiments were repeated twice with consistent results, ‘n’ numbers in figure captions denote the numbers of biological repeats included in an experiment. Fiji (Imaged) was used to process raw image files such as generated look-up table (LUT), maximal intensity projection (MIP) and representative images. Raw images were converted to .ims files using Imaris File Converter (v10.0) for advanced processing in Imaris (v10.0, Oxford) for creating orthogonal views, 3D rendering, normal shading, volume reconstruction and movie file exportation.Scanning electron microscopy

[0205] Day 8 peri-gastruloids were fixed with 2.5% (v / v) glutaraldehyde in 0.1 M sodium cacodylate buffer overnight at 4 °C and carefully placed into Microporous Specimen Capsules (Electron Microscopy Sciences, catalog #70187-10). After three rinses in 0.1 M sodium cacodylate buffer, they were post-fixed with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 hours. Samples were rinsed with water and dehydrated with increasing concentration of ethanol, followed by increasing concentrations of Hexamethyldisilazane in ethanol. Samples were air dried under the hood, mounted on SEM stubs and sputter coated with gold / palladium in a Cressington 108 auto sputter coater. Images were acquired with a Field-Emission Scanning Electron Microscope (Zeiss Sigma) at an accelerating voltage of 3.5kV in Variable Pressure Mode.Single-cell RNA-Seg library preparation

[0206] Day 11 peri-gastruloids were filtered through a 100 pm cell strainer to remove structures that were undersized. The retained peri-gastruloids were then transferred from culture medium (IVC2 supplemented with 4% Matrigel) into pre-warmed DMEM / F12 mediumin a 10 mm petri dish to wash away excesses Matrigel and then were evaluated on a stereoscope to sort out fused peri-gastruloids. Peri-gastruloids were then collected via centrifugation and were resuspended in 5 ml TrypLE supplemented with 50 pl DNase I (1 ll / pl), in a 15 ml centrifuge tube. The tube was then fixed on a horizontal shaker and placed in 37 °C incubator for 10 minutes, samples were pipetted periodically to ensure complete digestion. After a 10-minute incubation, 5 ml fibroblast medium (DMEM / F12 + 10% FBS) was added to neutralize TrypLE. The cell suspension was passed through a 40pm cell strainer before being centrifuged at 1000 rpm for 3 minutes to pellet the cells. The pellet was resuspended in fibroblast medium and cell viability was evaluated on an automated cell counter (Countess II FL, Invitrogen). After that, cells were washed twice and resuspended in PBS supplemented with 0.04% BSA at a density of 1000 cells / pl, kept on ice, before being loaded to into a 10X Genomics Chromium Chip. Single cell libraries were prepared using Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (CG000389) following the manufacturer instruction and sequenced by Illumina NextSeq 500 / 550 sequencing systems (Illumina).Single-cell RNA-Seq analysis

[0207] A raw gene expression matrix was prepared using the Cell Ranger (7.1.0) pipeline. Cells were excluded if they contained a portion of mitochondrial reads greater than 15% or had fewer than 500 genes detected. Doublets were filtered using the DoubletDetection (4.2) python package. Any genes detected in fewer than 10 cells were removed. The resulting matrix contained 10,684 cells by 21 ,847 genes. Gene expression was CPM normalized and z-scored for all subsequent analysis. Dimensionality reduction was performed using PCA and UMAP, with neighbors assigned using the Scanpy (1.9.1) python package. The resulting cell network was clustered using the Louvain algorithm with 0.75 resolution. PAGA and UMAP were applied subsequently for cluster visualization. The same process was applied to cells annotated to each of the three germ layers to generate the plots of the ectoderm, mesoderm, and endoderm, lineages. Genes differentially expressed between clusters were identified based on the Wilcoxon rank-sum test using the rank_gene_groups module of the Scanpy package.

[0208] Natural embryo scRNA-seq datasets used in the integration analysis were retrieved from previous publications and listed in Tables 2A-2B. Sex ratio of cells was balanced to minimize noises. Cell quality control and screening procedures were applied to these cells using the Seurat v4.1.1 , followed by data normalization. Batch effect removal and data embedding were then performed on all cells using the harmony algorithm, resulting in the generation of a UMAP visualizing the integration of human, and cynomolgus monkey embryo originated cells. Annotation information was retrieved from the original datasets, respectively, was then compared and combined based on similarity between cell types in vivo.

[0209] Table 2A: Data for integration analysisTable 2B: Cell types identifiedExample 1. A 3D culture method enables robust self -organization of human EPSCs into peri-gastruloids.

[0210] A robust method for modeling human peri-gastrulation development was developed. Based on the premise that the trophoblast tissue is physically segregated from the epiblast in early post-implantation human embryos, it was hypothesized that a 3D model comprising both epiblast and hypoblast cells would be adequate for this purpose. Furthermore, an incomplete human embryo model without the trophectoderm derivatives will help assuage ethical concerns. To enable differentiation of human EPSCs into hypoblast-like cells (HLCs), a condition called HDM (hypoblast differentiation medium) was adapted, which supported the differentiation of HLCs from naive human PSCs. HDM contains FGF2, Activin-A, and CHIR99021 (Chir). In mouse embryos, FGF signaling levels are known to influence the lineage fate of inner cell mass (ICM), where high FGF levels direct ICM cells toward the primitive endoderm lineage. To support balanced hypoblast and epiblast differentiation and culture, FGF signaling was optimized by including a low dose of a MEK inhibitor (PD0325901 , 0.3 pM) in the HDM, which was termed titrated HDM or tHDM. hEPSCs converted from RUES2 hESCs harboring SOX2-Citrine and SOX77-tdTomato reporters (hEPSCsRUES2) were used to facilitate tracking of epiblast and hypoblast-like lineages. These hEPSCsRUES2are SOX2-Citrine positive and SOX17-tdTomato negative (FIG. 1A). It was found that tHDM not only could induce HLCs from hEPSCsRUES2, but also supported long term co-culture of both epiblast-like cells (ELCs) and HLCs (FIG. 1A and FIG. 1B). Upon tHDM treatment dome-shaped hEPSC colonies became flattened (FIG. 1A), and ELCs and HLCs robustly self-organized into concentric domains during extended passaging (FIG. 1B).

[0211] Leveraging the ability of hEPSCs to differentiate into cells resembling both epiblast and hypoblast lineages, next structures that can model post-implantation human embryos was engineered. To this end, a three-dimensional (3D) culture using AggreWell™ plates was implemented, beginning with the cultivation of hEPSCs in tHDM in a 2D environment for 4 days, to induce ELCs and HLCs (FIG. 1C). These cells were then placed in an AggreWell™ plate with tHDM without Chir addition, referred to as tHDM (-Chir), to stimulate aggregate formation (FIG. 1C). After a 4-day incubation period, it was observed that the formed aggregates had self-organized into epiblast-like (SOX2, OCT4, NANOG, OTX2High, E- CADHERIN (E-CAD)High) and hypoblast-like compartments (GATA4, E-CADLow, N-CADHERIN (N-CAD), OTX2LOW, FOXA2, CERBERUS1 (CER1), LEFTY) (FIG. 1D-1I).

[0212] Next, the disclosed protocol was optimized and streamlined. The 2D pre-treatment step was eliminated and hEPSCsRUES2 was directly seeded in the AggreWell™ plate in tHDM (FIG. 2A). Following tHDM treatment for two days, a rapid loss of SOX2 expression and a corresponding gain of SOX17 expression in some hEPSCsRUES2 within the aggregates were observed. On day 2, switching to tHDM (-Chir) was done. By days 3-4, the SOX2+ and SOX17+ cells had sorted into separate compartments (FIG. 2A-2B and FIG. 3A). After testing additional culture parameters from day 5 onwards (details provided in Methods), a protocol was developed that supported robust growth and self-organization of these aggregates up to days 11-13, encapsulating the period of peri-gastrulation development in humans (FIG. 2B- 2C). Specifically, the days 3-5 dual-compartment structures underwent significant growth (FIG. 2C) and displayed robust and efficient morphological transformations, with days 5-6 closely resembling those of ex vivo cultured d.p.f. cynomolgus monkey embryos and d.p.f. human embryos and with days 11-13 closely resembling ex vivo cultured d.p.f. 23-25 (equivalent to Carnegie Stage 8, CS8) and in vivo d.p.f. 20 (CS8) cynomolgus monkey embryos (FIG. 2B-2E, and FIG. 1 M). These embryo-like structures were designated as peri- gastruloids.

[0213] In addition to hEPSCsRUES2, it was that found this optimized protocol to be reproducible across several hEPSC lines, derived from both ESCs (ESI017 and H9) and iPSCs (NCMR1 , AICS-0058m, and BTAG) (See Method). However, cell line-specific variations in efficiencies was observed (FIG. 1D-1E and FIG. 1 M). Notably, a large number of advanced (e.g., n=395, day 11) embryo-like structures could be generated in a single experiment, starting with only 3-6 wells of a 24-well AggreWell™ plate (FIG. 2B).

[0214] Taken together, a stepwise protocol was developed and fine-tuned to enable hEPSCs to effectively and efficiently self-organize into three-dimensional structures mimicking the human peri-gastrulation developmental period.Example 2: Cavitation of in hypoblast- and epiblast-like compartments

[0215] By days 4-5, the ELCs within the aggregates displayed expression of key pluripotency transcription factors (TFs) such as SOX2, NANOG and OCT4, as well as OTX2, a gene known to be upregulated in formative epiblast-like cells (FIG. 3A-3F, FIG. 1D-1E and FIG. 1G). In contrast, HLCs demonstrated high expression levels of hypoblast related genes, including SOX17, FOXA2, GATA6, GATA4, LEFTY and CER1 (FIG. 3A-3F, FIG. 1D, FIG. 1G- 1H). Notably, day 4 HLCs also expressed OTX2, N-CAD, and PRDM1 (or called BLIMP1) (FIG. 1E, FIG. 1G, and FIG. 1 M), which are reminiscent of post-implantation hypoblast derivatives.

[0216] Early signs of cavitation were observed in both the epiblast- and hypoblast- 1 ike compartments of days 4-5 aggregates (FIG. 3A, FIG. 3C, FIG. 3E-3F, and FIG. 1F). To substantiate cavity formation, we stained the aggregates using PODXL (Podocalyxin-like protein), an apical marker. Consistently, PODXL was predominately localized to the apical membranes in most of the day 4 structures (FIG. 3E and FIG. 1F). Further evidence showed an epithelium layer marked by high E-CAD and B-CATENIN lining the emerging lumen in epiblast-like compartment (FIG. 3F and FIG. 1J). In contrast, the hypoblast-like lumen was lined by cells exhibiting low E-CAD and high N-CAD levels (FIG. 1E). These findings align with observations made in gastrulating mouse embryos and human non-integrated gastrulation models.

[0217] In summary, on day 4, cells in the aggregates segregated into an epiblast- and a hypoblast- 1 ike compartments, both of which exhibited signs of cavitation.Example 3: Binding specificity of Rb-mAb7

[0218] In natural primate embryos, the amnion functions as a signaling hub, secreting morphogen molecules to the neighboring epiblast to facilitate the initiation of gastrulation. Given this, the induction of amnion cells in peri-gastruloids was investigated. Day 4 aggregates were subjected to attachment culture and determined their cell lineage composition. The results revealed that the attached aggregates consisted of several different cell types, including yolk saclike cells (GATA6+) surrounding epiblast-like cells (OCT4+) (FIG. 1K). Interestingly, a group of cells nested between the epiblast-like and yolk sac-like cells stained positive for both GATA3 and OCT4, which are reminiscent of human amnion epithelial cells. This observation was further corroborated by staining with another amnion marker, ISL1 (FIG. 1K). Consistently, a gastrulation marker, T (also called Brachyury), was expressed among cells juxtaposed to ISL1+amnion-like cells (FIG. 1 K). Alongside the attachment culture, we also maintained an extended culture of day 4 aggregates suspended in basal N2B27 medium embedded within 4% Matrigel. Remarkably, even in the basal medium devoid of any external factors, gastrulating (T+) cells were observed adjacent to an epiblast-like structure on day 9 (FIG. 1L).

[0219] The incidence of T+ cells appearing in the basal medium / 4% Matrigel condition was relatively low, and under these conditions, aggregates could not progress beyond day 9. To improve, we transitioned to cultivating the aggregates in IVC1 medium supplemented with 4% Matrigel post day 4. This medium has proven effective in supporting in vitro development of both non-human primate embryos and human embryo models. Starting from days 4 and 5, paralleling natural embryo development, the primary yolk sac-like cavity expanded at a faster pace compared to the amniotic-like cavity. Consequently, most aggregates rearranged into a configuration where the amniotic-like cavity was oriented upwards (FIG. 3A and FIG. 3G).

[0220] By day 6, bilaminar disk embryo-like structures were robustly formed (FIG. 4A-4E and FIG. 5J). While varying in size, some amniotic-like cavities within day 6 aggregates featured columnar cells on one side, expressing high levels of SOX2, OCT4 and NANOG (FIG. 4B-4C, FIG. 5A-5B). On the opposite side, SOX2LowNANOGLowsquamous epithelial cells expressing amnion markers CDX2 and TFAP2C, and exhibiting high BMP signaling were observed (FIG. 5A-5D, FIG. 5F-5G, FIG. 6A-6C). An enlarged yolk sac-like cavity, outlined by PDGFRa+ cells was discernible beneath the amniotic-like cavity, marked by apically polarized PODXL+ cells (FIG. 4D, FIG. 5C, FIG. 5E). We noted that the inclusion of Matrigel bolstered the formation of amnion-like cavity (FIG. 6H). In line with the presence of amnion tissue, a signaling hub inducing gastrulation in primates, a handful of T+cells were situated at putative posterior side of the embryonic disk, marking the onsite of gastrulation (FIG. 6H). By days 7- 8, the expanded amniotic-like cavity was situated near and encapsulated the embryonic disklike structure, with a yolk sac-like cavity beneath it (FIG. 6G and FIG. 5K). Notably, a primitive streak-like structure emerged at the junction between the embryonic-disk- and amniotic-like epithelia (FIG. 6G).

[0221] In primates, amnion cells secrete BMP4, which acts on nearby epiblast cells to activate the expression of WNT and NODAL The hypoblast-derived visceral endoderm, located beneath the epiblast, serves as another crucial signaling hub, emitting molecules like BMP2 Consistent with this, elevated levels of phosphorylated SMAD1 / 5 / 9 — the intracellular transducers of BMP signaling — in amnion- and hypo blast- 1 ike cells were observed. Concurrently, NODAL production was augmented in epiblast-like cells (FIG. 5F, FIG. 5I, and FIG. 10M).

[0222] Taken together, day 6 peri-gastruloids were characterized by well-defined amnion- and yolk sac-like cavities, and the onset of gastrulation, exhibiting proper lineage allocation and signaling activities.Example 4: Primordial germ cell-like cells specification

[0223] During primate development, primordial germ cells (PGCs) are specified in nascent amnion prior to gastrulation and migrate towards the visceral endoderm. Given the presence of amnion- and hypo blast- 1 ike tissues, the primordial germ cell-like cells (PGCLCs) were examined in day 6 peri-gastruloids. By zooming-in to the region beneath the epiblast-like (NANOG+) and amnion-like (TFAP2C+) tissues, several NANOG, TFAP2C and PRDM1 triple positive cells were spotted (FIG. 6B). A hiPSC line bearing PRDM1 (BL / MP / )-tdTomato and TF4P2C-EGFP (BTAG) reporter alleles were used to generate peri-gastruloids and tracked the movement of PRDM 1+ TFAP2C+ PGCLCs for 6 to 12 hours. In consistent, a handful of PGCLCs (PRDM1+ / OCT4+ / TFAP2C+ / SOX2-) were observed in day 6 BTAG peri-gastruloids(FIG. 6A, and FIG. 6C-6E). By measuring the angle of the long axis, it was observed that these PGCLCs were positioned in an orientation suggestive of migrating towards the PRDM1+ / TFAP2C- hypoblast (yolk sac) plane (FIG. 6E). Moreover, time-lapse imaging revealed high motility of some PGCLCs.Example 5: Primitive streak formation and gastrulation

[0224] Primitive streak formation and gastrulation constitute an important milestone during mammalian development. Next, it was determined whether these critical morphogenetic events occurred in peri-gastruloids. To this end, it was first examined whether anterior- posterior (A-P) axis is established, which is crucial for correct morphogenesis. In natural embryos, the A-P axis is established prior to the onset of gastrulation through the formation of opposing WNT and NODAL gradients, as well as their antagonists CER1 and LEFTY. Specifically, anterior visceral endoderm (AVE) overlying epiblast expresses high levels of CER1 and LEFTY1 , which specify the anterior end and help restrict WNT and NODAL signaling activities to the posterior end. Similar to natural embryos, gradients of CER1 and LEFTY was observed along a putative A-P axis, respectively, and an opposite gradient of NODAL in day 6 peri-gastruloids (FIG. 5H-5I). In consistent with the signaling gradients, posterior marker T was expressed at one end of the embryonic disk / epiblast (FIG. 4F).

[0225] The primitive streak formation, a conserved feature among amniotic vertebrates, plays crucial roles in fate patterning and morphogenesis during specification of the three primary germ layers via conferring anterior-posterior and dorsal-ventral spatial information to differentiating cells. To date, however, the primitive streak formation in humans has not been recapitulated in any stem cell derived embryo models. A study on the primitive streak formation in peri-gastruloids was further undertaken. In humans, the primitive streak forms at the posterior end within the sheet of epithelialized epiblast cells known as the embryonic disk. Cells at the posterior end of the embryonic disk begin to migrate towards the midline, driven by the action of signaling molecules such as NODAL and WNT. As the migrating cells converge at the midline, they form an elongated depression called the primitive groove (FIG. 7B). Some cells in the primitive streak began to delaminate and migrate inwards, forming the endoderm layer. Other cells moved through the primitive streak and occupied the space between the endoderm and the overlying ectoderm layer, forming the mesoderm. Finally, cells at the anterior end of the embryo that do not pass through the primitive streak became the ectoderm layer (FIG. 7B). Embryos at this stage are referred to as a trilaminar disk.

[0226] Following the establishment of A-P axis, peri-gastruloids continued to develop. To ensure consistency and minimize batch effects, quality control was implemented on day 6 before the peri-gastruloids were transferred to suspension culture in low attachment 6-wellplates (FIG. 2A). In particular, peri-gastruloids were selected based on morphology under a microscope, and only the structures with an evident embryonic disk-like structure (-60%) and a fluid-filled yolk sac cavity (-60%) were picked for further cultivation (FIG. 2B). By day 8, a significant growth of the epiblast compartment was observed, which was accompanied by the expansion of extraembryonic tissues and cavities (FIG. 2B and FIG. 7A). Immunofluorescence analysis confirmed the appearance of a trilaminar disk-like structure in day 8 peri-gastruloids (FIG. 7E- 7F).

[0227] In depth characterization revealed a structure similar to the primitive streak at the posterior end of the embryonic disk-like cell sheet, which was identified as SOX2+ (FIG. 7C- 7D). In particular, T+ cells demonstrated an asymmetric localization to the posterior (caudal) end of a SOX2+ cell sheet. Through immunofluorescence analysis, it was discerned that the gastrulating T+ cells were not only altering their cell shape and delaminating, but also starting to express N-CAD, indicative of an epithelial-to-mesenchymal transition (EMT) (FIG. 7D and FIG. 5L). Additionally, a feature bearing resemblance to the primitive groove was noticeable in the center of the embryonic disk-like structure, which was characterized by the alignment and inward migration of T+ cells (FIG. 7F).

[0228] In summary, peri-gastruloids at day 8 could successfully simulate symmetry breaking and the formation of the anterior- posted or (A-P) axis within the epiblast-like compartment. Additionally, these structures can serve as models for understanding the formation of the primitive streak and the process of gastrulation.Example 6: Early neurulation in advanced peri-gastruloids

[0229] In natural embryos, as the process of gastrulation advances, cells that persist at the anterior side of the epiblast differentiate into ectoderm cells. This anterior section of the ectoderm, situated along the midline, undergoes thickening to form the neural plate, marking the commencement of neurulation. We investigated the possibility of a neurulation-like event taking place in more advanced peri-gastruloids.

[0230] From days 8 to 9, the epiblast compartment continued to expand alongside the growth of both the amniotic-like and yolk sac-like cavities. This growth could be attributed to the addition of glucose to the IVC2 medium, a method recently found to support the ex-utero development of both mouse embryoids and monkey embryos (FIG. 2C). At this stage, structures featuring a noticeable amniotic-like cavity enveloping the epiblast-like sheet was consistently detected, with a sizable yolk sac-like cavity positioned below. Both cavities appeared to be filled with fluid (FIG. 8A-8B). By days 10 and 11, neural ectoderm cells, characterized by SOX2 and PAX6 expression, were visible in the anterior region, present in approximately 69% of the peri-gastruloids (FIG. 2E, FIG. 9C-9D and FIG. 10D). On average,between 3 and 6 wells of a 24-well AggreWell™ 800 plate (containing 300 microwells per well) could yield hundreds of day-11 peri-gastruloids, each with distinct epiblasts and fluid-filled yolk sac- and amniotic-like cavities, measuring roughly 0.8-1 mm in diameter (FIG. 2B-2C and FIG. 9A-9B).

[0231] Next, immunostaining was performed to visualize N-CAD, which is known to be expressed in the primitive streak and neural plate. On one end of the peri-gastruloids, N-CAD+ cells displayed a more mesenchymal-like appearance, a characteristic trait of gastrulating cells. In accordance with this, these cells expressed T and were found next to a cluster of SOX2+ cells that were forming a tube-like structure, resembling the onset of tail bud (TB) formation (FIG. 8C, FIG. 8E and FIG. 9E). On the opposing end, N-CAD+ cells presenting high SOX2 expression appeared more epithelial-like, suggesting their likely identification as neural ectoderm (FIG. 8E). It is worth noting that the endodermal cells, characterized by SOX17 and GATA4 markers, were found positioned underneath the neural plate-like structure, mirroring the positioning observed in natural embryos (FIG. 9J).

[0232] To examine whether early neural tube formation occurred in day 10-11 peri- gastruloids, optical sections were made around the neural plate-like region. These cross- sectional planes along the putative dorsal-ventral axis unveiled a structure resembling a closing neural tube (FIG. 9G). Immunostaining with ZO-1 was subsequently conducted, a tight junction component which accumulates in the apical surface of the neural plate during neural tube closure. The result revealed an apical localization of the ZO-1 staining in the SOX2+ neuroepithelial-like cells (FIG. 9F). In line with this, we also noticed an apical concentration of F-actin (FIG. 9H). Due to the increased thickness and complexity of the peri-gastruloids at this stage, whole mount imaging and 3D architecture reconstruction became more challenging with confocal microscopy, even after implementing tissue clearing procedures (See Methods). To address this issue, cryosections and paraffin sections were generated to examine the internal architectures more effectively (FIG. 8D and FIG. 8G). To obtain cryosections, 10 to 15 peri-gastruloids were embedded in a single OCT (optimal cutting temperature compound) block at a time. Consistent with the quantification results derived from whole mount imaging, approximately 71% (10 out of 14) of the peri-gastruloids exhibited PAX6 expression, and 50% of these (5 out of 10) displayed a lumen, suggesting the potential formation of a neural tubelike structure (FIG. 8G and FIG. 9C-9D). In addition, the hematoxylin and eosin staining of days 11 and 12 peri-gastruloids exhibited various embryonic and extraembryonic tissues and structures, which are similar to a CS8 human embryo (retrieved from virtual human embryo, CS8 section #197) (FIG. 8G).

[0233] T aken together, the combined evidence of marker gene expression, morphological characteristics, and axes formation collectively indicates the commencement of neurulation in day 10-12 peri-gastruloids.Example 7: Single cell transcriptomics analysis

[0234] To further characterize various cell types present in advanced peri-gastruloids, single cell RNA sequencing (scRNA-seq) was performed on pooled day 11 peri-gastruloids using 10x Genomics Chromium. After stringent quality control, during which doublets and low- quality cells (mitochondria’ reads > 15% or had < 500 genes detected) were filtered out, 10,684 single cell transcriptomes were obtained with a median unique molecular identifier (UMI) of 7,759 and 3,306 genes detected per cell. Median mitochondrial read portion was 5.6%, suggesting good cell quality.

[0235] Uniform Manifold Approximation and Projection (UMAP) was performed and 13 major cell clusters were identified based on differentially expressed genes (FIG. 11 A). The annotation of these clusters was accomplished based on a combination of putative lineage markers, as established by previous single-cell transcriptomics studies of gastrulating human and cynomolgus monkey embryos 47,48,57,68,69. This annotation revealed the presence of major embryonic and extraembryonic endoderm cell types during the period of human periimplantation development (FIG. 11A-11B).

[0236] Specifically, a post- implantation epiblast-like cluster (cluster 1), characterized by the expression of signature genes such as FGF4, DNMT3B, SOX11 , SFRP2, SALL2, and UTF1 was identified. This cluster also expressed core pluripotency transcription factors POU5F1 (OCT4), SOX2, and NANOG (FIG. 11B and FIG. 12A). Adjacent to the primitive streak-like population (cluster s, T, TDGF1 , CDH1 and FGF8) (Figure S6B), we found a group of cells expressing node signature genes like GSC, FOXA2, FST, CER1 , DKK1 , HHEX and CHRD (Figure S6C). Within the ectoderm cells (cluster 2), several subclusters were identified, such as the neural ectoderm (SOX2, SOX3, OTX2, PAX6), non-neural ectoderm (TFAP2A, TFAP2C, GATA3), and amniotic ectoderm (TFAP2A, TFAP2C, ISL1 , CDX2, GABPR) (FIG. 11A, FIG. 11C, FIG. 12D-12E). Furthermore, a group of cells were observed in the surface ectoderm cluster (cluster 10) expressing LHX2, BMP7, EYA1 , RAX, SIX3, and PAX6 (FIG. 11 A). These genes constitute a signature of the eye primordium, which is suggested to be specified within the anterior neural plate shortly after gastrulation (around E7.5-8.0) in mice.

[0237] Definitive endoderm cells (cluster 6, EOMES, SOX17, FOXA2, TDGF1 etc.) and yolk sac endoderm cells (cluster 7, TTR, AFP, APOA1 , APOA2, APOC1 , APOE, etc.) were detected (FIG. 11 C, FIG. 11F, FIG. 101, and Table 2A-2B). Furthermore, a cell cluster (cluster 12, PECAM1 , HHEX, ETV2, CD34, HBZ, HBA1 , HBE1 and PDPN etc.) was found situatednear yolk sac endoderm and mesoderm clusters, displaying an overrepresentation of genes signifying the initiation of hematopoiesis, including the differentiation of endothelial cells and blood cells (FIG. 11A and FIG. 10K). The expression of CD34, a marker of human hematopoietic progenitors was validated via immunofluorescent staining (FIG. 12A). In addition, consistent with the immunofluorescent staining and live cell imaging results from day 6 peri-gastruloids, the presence of Primordial Germ Cell-Like Cells (PGCLCs), characterized by S0X17, PRDM1 (BLIMP1) and NANOS3 (FIG. 10J) were detected.

[0238] Several mesoderm lineages were identified including nascent mesoderm (cluster 4, FGF8, MIXL1 , EOMES, WNT3, MESP1 , MESP2) and advanced mesoderm cells (cluster s, TMEM88, COL3A1 , ACTC1). Interestingly, within the mesoderm cells, the expression of signature genes associated with the lateral plate mesoderm (HAND1, MEIS1 , GATA4, PITX1 , PITX2, BMP4 etc), cardiogenic mesoderm (cTnT, MYL7, KDR and IGFBP3 etc), and pre- somitic mesoderm (TBX6, HES7, MLLT3, TCF15) were also observed (FIG. 11 A, FIG. 11 E, FIG. 11F, FIG. 12F-12H). These findings suggest that, in consistent with early neurulation, the initiation of organogenesis seems to be commenced in day 11 peri-gastruloids. To provide additional evidence, the expression of HES7, a transcriptional factor regulated by the Notch signaling and governs the pace of somitogenesis was examined. Peri-gastruloids with HES7- Achilles knock-in hEPSCs were generated and the Achilles signal in a putative late primitive streak / tail bud (PS / TB) region between days 12-13 was observed (FIG. 12B). Moreover, rosette-like structures were detected in day 13 peri-gastruloids that contained TBX18+ cells (FIG. 12C-12D).

[0239] Next, peri-gastruloids scRNA-seq dataset was compared with a dataset from human post-conceptional week (PCW) 3 embryos, generated via 10X Genomics platform. Notably, developmental processes such as neurulation, organogenesis, and erythropoiesis were occurring during PCW3. This dataset was chosen instead of the human CS7 embryo dataset (generated using the Smart-seq2 platform) to minimize potential discrepancies arising from sequencing platform differences, which could compromise the accuracy of the analysis. Additionally, since the human PCW3 embryo dataset lacked yolk sac and amnion cells and represented a later stage (CS10) than day 11 peri-gastruloids (CS8-9), in vivo and ex utero cultured CS8 cynomolgus monkey embryo datasets were also included for comparison. The integration analysis yielded highly congruent LIMAP outputs between day 11 peri-gastruloids and both human and cynomolgus monkey embryos. Prominent similarities were observed in several cell lineages including the epiblast, primitive streak, neural ectoderm, non-neural ectoderm, nascent mesoderm, various types of advanced mesoderm, different types of definitive endoderm, blood and vascular progenitors, as well as extraembryonic mesoderm, extraembryonic endoderm, allantois and amniotic cell types (FIG. 11F). These analysesconfirmed that the disclosed human peri-gastruloids accurately mimiced primate peri- gastrulation cell types at the single-cell transcriptomic level.

[0240] In summary, the scRNA-seq analysis corroborated the earlier immunofluorescent staining results and confirmed the presence of derivatives from all three germ layers, evidence of early organogenesis, and the development of yolk sac and amniotic tissues in day 11 peri- gastruloids.

[0241] Further, time-resolved single-cell RNA-seq analysis of peri-gastruloids was undertaken. Uniform Manifold Approximation and Projection (UMAP) visualization representing individual cells derived from days 4-11 peri-gastruloids, respectively, was performed (FIGs. 12E-12F). The starting material, extended pluripotent stem cells (EPSCs), labeled as day 0, served as a control. Heatmap displaying the tissue-specific marker genes for each identified cell type is shown in FIG. 12G. These results shows that peri-gastruloids not only recapitualte the major embryonic and extra-embryonic cell types during human postimplantation development but also the progression of their differentiation.

[0242] UMAP visualization representing individual cells derived from day 4 peri- gastruloids was further undertaken (FIG. 12H). individual cells derived from day 4 peri- gastruloids, displayed signatures of human anterior visceral endoderm (AVE) cells (FIG. 121). This result, serving as an example, shows that early-stage peri-gastruloids replicate the early stage of post-implantation development, which involves the specification of the anterior visceral endoderm, essential for establishing the anterior- posted or body axis. Additionally, heat map analysis of metabolites in different stages of peri-gastruloids formation was performed. FIG. 12J shows the heat-map of metabolites that are differentially enriched in the different stages of peri-gastruloids formation. This indicates that peri-gastruloids are useful for investigating metabolism during early human embryonic development, such as for example metabolic defects that may lead to pregnancy complications.Summary of examples

[0243] Capitalizing on the dual-potential of hEPSCs, both embryonic and extraembryonic, the disclosed study seeked to create an integrated embryo model of the lesser-explored peri- gastrulation developmental phase in humans. It was observed that hEPSC aggregates, under permissive conditions that include the presence of FGF, TGF- ? signaling, and canonical WNT pathway agonists, underwent fate bifurcation to produce epiblast-like and hypoblast- 1 ike cells. By implementing several optimizations, a robust protocol was established that facilitated the generation of early post- implantation human embryo-like structures. These structures, designated as peri-gastruloids, recapitulated several crucial stages of human peri-gastrulation development, including the formation of amniotic and yolk sac cavities, the development ofbilaminar and trilaminar disks, the specification of primordial germ cells, the formation of the primitive streak and gastrulation, as well as the initial stages of neurulation and organogenesis. The peri-gastruloid protocol exhibited reproducibility across different hEPSC lines, regardless of whether they were derived from ESCs or iPSCs. Interestingly, the peri-gastruloid sizes maintained a uniform appearance from days 1 through 8, although they began to show increased heterogeneity from days 11 to 13. The considerable expansion of the peri- gastruloids during these later stages posed a challenge to the accurate quantification of various cell types and their respective allocations. However, quantification by evaluating the expression of several marker genes at different timepoints, namely S0X2, S0X17, PRDM1 , TFAP2C, CDX2, T, and PAX6 was conducted.

[0244] The bidirectional developmental potency of human EPSCs was used, which showed transcriptional similarity to human peri-implantation (-E7-8) epiblast, to develop a 3D culture technique that facilitated the differentiation and self-organization of hEPSCs into an advanced integrated model of human peri-gastrulation development. The disclosed model, the peri-gastruloid, effectively recapitulated critical developmental processes, from the immediate post- implantation stage through to early organogenesis. This encapsulated various events such as cell fate patterning, establishment of body axis, the formation of the primitive streak, gastrulation, and the initial stages of organ development. This model progressed through several distinct stages, initially generating epiblast-like, amniotic cavity-like, and yolk sac-like compartments, followed by gastrulation and early neurulation (FIG. 1). The power of this model stems from its ability to exploit the remarkable self-organizing capacity of hEPSCs with minimal external intervention. Furthermore, the presence of both embryonic and extraembryonic tissues enables researchers to examine the interactions between the epiblast, amnion, and yolk sac — an endeavor previously unattainable in humans.

[0245] While models of human gastrulation (gastruloids) and early organogenesis (such as neural tube models, somitoids and cardioids) have been developed from primed hPSCs, these models lack the extraembryonic cells that play vital roles in embryo patterning and morphogenesis during embryogenesis. By incorporating hypoblast cells, which are recognized as crucial signaling hubs driving epiblast development, peri-gastruloids offer substantial advancements. They are capable of mimicking morphogenetic events associated with the formation of structures such as the yolk sac, blood islands, bilaminar disk, primitive streak, and trilaminar disk. These are complex processes that traditional PSC-only gastrulation models cannot accurately replicate.

[0246] In contrast to PSC-only models, the development of peri-gastruloids depends primarily on self-organization following the initial lineage bifurcation and cell sorting, requiring minimal external signaling inputs. For example, earlier studies have indicated the necessity ofBMP4 supplementation in prompting dorsal — ventral patterning within the epiblast, leading to the differentiation of the squamous amniotic ectoderm in a primed hPSC-derived amnion model. Interestingly, the differentiation of amniotic ectoderm-like cells within peri-gastruloids occurred without the supply of BMP4. Thus, it will be interesting to determine the source of BMPs for specifying amnion cells. One possibility is visceral endoderm, which is known to secrete BMP2. The pSMAD1 / 5 / 9 immunostaining results and scRNA-seq results support this hypothesis. As another instance, 2D and 3D human gastruloids models derived from primed hPSCs require treatments with BMP4 and a WNT agonist, CHIR99021 , respectively. In contrast, peri-gastruloids were capable of initiating the gastrulation-like process without providing signaling molecules. This impressive self-organization ability was likely to be underpinned by endogenous activation of major morphogen pathways involved in peri- gastrulation. For instance, establishment of TGF-p signaling gradients along the perspective A-P axis prior to gastrulation-like events and the tissue-specific expression of variety of BMP, WNT, TGF-p and FGF pathway ligands (FIG. 12H-12J). Moreover, the endodermal expression of Sonic Hedgehog (SHH) captured by scRNA-seq (FIG. 12L), was reminiscent of in vivo ventral morphogenesis instructed by Hedgehog signaling. These findings underscore the importance of extraembryonic cells, particularly hypoblast cells, in embryo patterning during peri-gastrulation development.

[0247] The mammalian yolk sac, derived from the hypoblast, is the first extraembryonic membrane to develop and mainly functions as an absorptive epithelium for nutrient acquisition and the origin for the first blood cells. Despite its indispensable role during embryogenesis, currently there are a lack of stem cell derived models for yolk sac development in humans. Here, it was observed that HLCs segregated from ELCs on days 3-4, and from day 4 yolk sac- like cavities formed within HLCs and continued to expand and reached a diameter about 600- 900 pm by day 11. scRNA-seq analysis revealed the presence of blood cells, suggesting the first wave of hematopoiesis occurred within yolk sac-like structures. Thus, peri-gastruloid formation potentially provided an accessible platform to model primary and secondary yolk sac development in humans.

[0248] Single-cell RNA sequencing analysis validated the cellular complexity of mature peri-gastruloids and highlights their resemblance to major cell types found in human and cynomolgus monkey embryos during analogous developmental stages. Despite the similarity, it should be noted that differences exist in the single cell transcriptomes between day 11 peri- gastruloids and human PCW3 embryos. For instance, the day 11 peri-gastruloid scRNA-seq dataset lacked cells from cluster #6 (Placenta [STB, CTB, EVT]) due to the absence of trophoblast cells. Furthermore, in some clusters (e.g., clusters 0, 2, 13), cells from the two datasets did not overlap well. PCW3 human embryos aligned with CS10, while day 11 peri-gastruloids approximated stages CS8-9, and these observed discrepancies in single cell transcriptomes between PCW3 human embryos and day 11 peri-gastruloids likely resulted from developmental stage differences. Supporting this hypothesis, day 11 peri-gastruloids showed greater overlap with the single-cell transcriptomes of in vivo CS8 stage monkey embryos than with PCW3 human embryos (FIG. 11 F).

[0249] These findings not only confirmed the occurrence of early neurulation but also implied the initiation of organogenesis. For instance, cells that possessed transcriptom ic characteristics of pre-somitic mesoderm, crucial for somitogenesis, were identified and experimentally validated. Additionally, cardiac mesoderm-like cells were identified, indicating the potential utility of the peri-gastruloid system for modeling embryonic heart development. With further refinement, peri-gastruloids may serve as a superior source of lineage progenitors for cell replacement therapies and other downstream applications, surpassing traditional monolayer and 3D PSC differentiation approaches.

[0250] The disclosed method involved culturing hEPSCs on MEFs, and complete removal of MEFs was necessary for efficient peri-gastruloid formation. Chemically defined and feeder- free culture conditions can be further optimized as an alternative approach for hEPSCs. Investigating the potential of feeder-free cultured hEPSCs as starting cells for generating peri- gastruloids would be valuable. Further, to facilitate the continued development of peri- gastruloids, additional enhancements could be considered. For example, the addition of human cord blood serum and the utilization of a roller culture system with controlled oxygenation and pressure have shown promise in mouse studies. These approaches could be explored to optimize the culture conditions and further extend the growth and development of human peri-gastruloids.

[0251] The peri-gastrulation model disclosed herein remarkably recapitulated the intricate morphogenesis involved in human peri-gastrulation development. This robust platform can facilitate investigations into various processes such as cell fate determination, collective cell movement, the formation of body axes, and interactions between embryonic and extraembryonic tissues. Moreover, it enables high-throughput genetic and chemical screens, thereby offering insights that could potentially mitigate early pregnancy loss and congenital defects.

[0252] The peri-gastruloid model offers several advantages, which include a relatively straightforward experimental method requiring no transgene-directed differentiation, a single starting cell type, and minimal manipulation. Furthermore, the peri-gastruloid model demonstrated a high efficiency in developmental progression, robust growth, clearcut representation of the primitive streak, trilaminar disk, neuroectoderm, and advanceddevelopment beyond gastrulation into early organogenesis. Looking ahead, with further refinement, particularly in combination with the roller embryo culture system, human peri- gastruloids could lay the groundwork for in-depth research beyond the early organogenesis stage. This might ultimately lead to the creation of human tissues and organs for applications in regenerative medicine.

Claims

CLAIMSWhat is claimed is:

1. A method of generating peri-gastruloids having embryo-like structures, the method comprising:(a) obtaining a three-dimensional culture of pluripotent stem cells (PSCs) in a first culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, a Wingless-related integration site (WNT) activator, and a Mitogen-activated protein kinase kinase (MEK) inhibitor;(b) maintaining the cells in the first culture medium for about 1 to 7 days;(c) changing the culture medium to a second culture medium, wherein the second culture medium comprises a FGF activator, a TGF-p activator, and a MEK inhibitor; and(d) maintaining the three-dimensional culture of pluripotent stem cells (PSCs) in the second culture medium, to generate the peri-gastruloids having embryo-like structures.

2. A method of generating embryo-like structures from pluripotent stem cells (PSCs), the method comprising:(a) obtaining a three-dimensional culture device, wherein the three-dimensional culture device comprises PSCs and a first culture medium, wherein the first culture medium comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, a WNT activator, and a MEK inhibitor;(b) maintaining the cells in the first culture medium for about 1-7 days;(c) changing the culture medium in the three-dimensional culture device to a second culture medium, wherein the second culture medium comprises a FGF activator, a TGF-p activator, and a MEK inhibitor;(d) maintaining the cells in the second culture medium for about 2-7 days;(e) changing the culture medium in the three-dimensional culture device to a third culture medium, wherein the third culture medium comprises IVC medium supplemented with Matrigel®, Geltrex®, or VitroGel®; and(f) maintaining the cells in the third culture medium for about 1-7 days to generate the embryo-like structures.

3. An in vitro system comprising:(a) pluripotent stem cells (PSCs);(b) a three-dimensional culture device;(c) a first culture medium comprising a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, a WNT activator, and a MEK inhibitor;(d) second culture medium comprising a FGF activator, a TGF-p activator, and a MEK inhibitor; and(e) a third culture medium comprising IVC medium supplemented with Matrigel®, Geltrex®, or VitroGel®; and(f) a means for replacing the first culture medium with the second culture medium and / or the second culture medium with the third culture medium.

4. The method or system of any one of claims 1-3, wherein the PSCs comprise a single cell suspension of PSCs.

5. The method or system of any one of claims 1-4, wherein the PSCs comprise extended pluripotent stem cells (EPSCs).

6. The method or system of any one of claims 1-5, wherein the PSCs comprise human PSCs, primate PSCs, rodent PSCs, porcine PSCs, bovine PSCs, ovine PSCs, or equine PSCs.

7. The method or system of any one of claims 2-6, wherein the three-dimensional culture device is a microwell plate.

8. The method or system of any one of claims 2-6, wherein the three-dimensional culture device is a AggreWell™ plate.

9. The method or system of any one of claims 2-8, wherein about 1-200 PSCs are added to an individual well of a three-dimensional culture device.

10. The method or system of any one of claims 1-9, wherein the first and second culture medium comprise N2B27 basal medium.

11. The method or system of any one of claims 1-10, wherein the FGF activator is FGF2.

12. The method or system of claim 11 , wherein the FGF2 activator is present in the culture medium at about 1 ng / ml to about 200 ng / ml.

13. The method or system of any one of claims 1-12, wherein the TGF-p activator is Activin-A.

14. The method or system of claim 13, wherein the Activin-A is present in the culture medium at about 1 ng / ml -200 ng / ml.

15. The method or system of any one of claims 1-4, wherein the WNT activator is a GSK3 inhibitor.

16. The method or system of claim 15, wherein the GSK3 inhibitor is CHIR99021.

17. The method of system of claim 16, wherein the CHIR99021 is present in the culture media at about 0.1 M to about 5 pM .

18. The method or system of claims 1-3, wherein the MEK inhibitor is PD0325901.

19. The method or system of any one of claims 1-3, wherein the MEK inhibitor is present in the culture medium at about 0.1 pM to about 1 pM of PD0325901 .

20. The method or system of any one of claims 2-3 further comprising a fourth medium, wherein the fourth culture medium comprises IVC medium supplemented with Matrigel®, Geltrex®, or VitroGel® and Knock out serum (KSR) and maintaining the cells in the fourth culture medium for about 1-14 days.

21. The method or system of any one of claims 1-3 or 20, wherein the Matrigel®, Geltrex®, or VitroGel® is present in an amount of about 1-100% (Vol / Vol).

22. The method or system of claims 20, wherein KSR is present in the medium at about 1-100% (vol / vol).

23. The method of any one of claims 1-2 or 4-22, wherein peri-gastruloids or the embryolike structures comprise epiblast-like, hypoblast- 1 ike compartments, or any combination thereof.

24. The method of any one of claims 1-2 or 4-22, wherein peri-gastruloids or the embryolike structures comprise epiblast-like cells, amnion-like cells, hypoblast-like cells, yolk sac-like cells, or any combination thereof.

25. The method of any one of claims 1-2 or 4-22, wherein peri-gastruloids or the embryolike structures comprise yolk sac-like cavity, amniotic-like cavity or any combination thereof.

26. The method of any one of claims 1-2 or 4-22, wherein peri-gastruloids or the embryolike structures comprise primordial germ cell-like cells.

27. The method of any one of claims 1-2 or 4-22, wherein peri-gastruloids or the embryolike structures comprise bilaminar disk-like structure, trilaminar disk-like structure, primitive streak like structure, embryonic disk-like structure, primitive groove like structure, or any combination thereof.

28. The method of any one of claims 1-2 or 4-22, wherein peri-gastruloids or the embryolike structures comprise neural ectoderm cells, non-neural ectoderm cells, amniotic ectoderm cells, surface ectoderm cells, endoderm cells, yolk sac endoderm cells, nascent mesoderm, advanced mesoderm cells, lateral plate mesoderm, cardiogenic mesoderm, presomitic mesoderm, or any combination thereof.

29. A peri-gastruloid or embryo-like structure produced by the method or system of any one of claims 1-28.

30. An embryo model comprising a peri-gastruloid or embryo-like structure produced by the method or system of any one of claims 1-28.

31. A method of cell replacement therapy, the method comprising administering in a subject in need thereof, cells from a peri-gastruloid or embryo-like structure of claim 29.

32. A method of repairing or regenerating a tissue or organ, the method comprising administering in a subject in need thereof, cells from a peri-gastruloid or embryo-like structure of claim 29.

33. An in vitro drug or chemical screening system, the system comprising a peri-gastruloid or embryo-like structure of claim 29.

34. A method of high-throughput genetic or chemical screening, the method comprising testing genetic mutations or chemicals that can cause pregnancy loss or congenital defects using the in vitro drug or chemical screening system of claim 34.

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