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WO2025006477A2 - Système et procédés de génération de progéniteurs fœtaux humains, de tissus et d'organes à partir de cellules souches cultivées - Google Patents

Système et procédés de génération de progéniteurs fœtaux humains, de tissus et d'organes à partir de cellules souches cultivées Download PDF

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WO2025006477A2
WO2025006477A2 PCT/US2024/035432 US2024035432W WO2025006477A2 WO 2025006477 A2 WO2025006477 A2 WO 2025006477A2 US 2024035432 W US2024035432 W US 2024035432W WO 2025006477 A2 WO2025006477 A2 WO 2025006477A2
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cells
aspects
culture medium
peri
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WO2025006477A3 (fr
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Jun Wu
Lizhong Liu
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University of Texas System
University of Texas at Austin
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2513/00—3D culture

Definitions

  • 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.
  • 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.
  • ESCs embryonic stem cells
  • TSCs trophoblast stem cells
  • XEN extraembryonic endoderm
  • ESCs can differentiate into all three germ layers and germ cells
  • XEN cells can differentiate into visceral endoderm and parietal endoderm
  • TSCs are capable of developing into extravillous trophoblast and syncytiotrophoblast.
  • 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.
  • EPCs pluripotent stem cells
  • the disclosure encompasses a method of generating a peri- gastruloid having embryo-like structures.
  • 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; and maintaining the three-dimensional culture of pluripotent stem cells (PSCs) in the second culture medium, to generate the peri-gastruloids having embryo-like structures.
  • FGF fibroblast growth factor
  • TGF- P transforming growth factor beta
  • WNT Wingless-related integration
  • 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
  • the disclosure further encompasses an in vitro system.
  • 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.
  • PSCs pluripotent stem cells
  • a three-dimensional culture device comprising a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, a WNT activator, and a MEK inhibitor
  • FGF fibroblast growth factor
  • the disclosed PSCs comprise a single cell suspension of PSCs.
  • the PSCs comprise extended pluripotent stem cells (EPSCs).
  • the PSCs comprise human PSCs, primate PSCs, rodent PSCs, porcine PSCs, bovine PSCs, ovine PSCs, or equine PSCs.
  • the three-dimensional culture device is a microwell plate. In some aspects, the three-dimensional culture device is a AggreWellTM plate. In some aspects, about 1-200 PSCs are added to an individual well of a three-dimensional culture device.
  • the disclosed first and second culture medium comprise N2B27 basal medium.
  • the FGF activator is FGF2.
  • the FGF2 activator is present in the culture medium at about 1 ng/ml to about 200 ng/ml.
  • the TGF-p activator is Activin-A.
  • the Activin-A is present in the culture medium at about 1 ng/ml -200 ng/ml.
  • the WNT activator is a GSK3 inhibitor.
  • the GSK3 inhibitor is CHIR99021.
  • the CHIR99021 is present in the culture media at about 0.1 pM to about 5 pM.
  • the MEK inhibitor is PD0325901.
  • the MEK inhibitor is present in the culture medium at about 0.1 pM to about 1 pM of PD0325901.
  • 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.
  • the Matrigel®, Geltrex®, or VitroGel® is present in an amount of about 1-100% (Vol/Vol).
  • KSR is present in the medium at about 1-100% (vol/vol).
  • 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.
  • 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.
  • 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.
  • the disclosure further encompasses a peri-gastruloid or embryo-like structure produced by the method or system disclosed herein.
  • an embryo model comprising a peri-gastruloid or embryo-like structure produced by the disclosed or system is further provided.
  • the disclosure comprises a method of cell replacement therapy.
  • the method comprises administering in a subject in need thereof, cells from a peri-gastruloid or embryo-like structure disclosed herein.
  • 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.
  • an in vitro drug or chemical screening system is provided, the system comprising a peri-gastruloid or embryo-like structure disclosed herein.
  • 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.
  • 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. 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
  • 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.
  • the hEPSCs were treated with tHDM, defined as day -4.
  • day -4 the cells were seeded into the AggreWellTM plate.
  • 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.
  • 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. 1 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%
  • FIG. 1L shows representative immunofluorescence images of a day 9 peri-gastruloid (generated via method shown 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.
  • 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 AggreWellTM 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.
  • selected peri-gastruloids were transferred to low attachment wells.
  • FIG. 1 shows a schematic illustrating the chronological sequence and critical stages of the experimental procedure. Microwells in an AggreWellTM 800 plate are depicted as inverted pyramids.
  • tHDM titrated hypoblast differentiation medium. CHIR99021 was removed on day 2 from tHDM, refer to as tHDM(
  • 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 AggreWellTM 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.
  • 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.
  • 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 hEPSC RUES2 reporter line in the top panels and representative merged fluorescence and brightfield images of days 1-5 aggregates generated from hEPSC RUES2 reporter 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.
  • 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.
  • 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. 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. 4D shows representative immunofluorescence
  • 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.
  • Top right is a schematic illustrating the putative viewing angle for FIG. 4G. Scale bar, 100 pm.
  • 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. 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
  • FIG. 5B shows amnion-like cells are induced in the epiblast compartment, top view.
  • White dotted circle indicates the putative amnion-like cells.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • A-P Anterior-Posterior
  • 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.
  • 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. 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. 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.
  • ect extraembryonic ectoderm (amniotic ectoderm).
  • Ect ectoderm.
  • Meso mesoderm.
  • Endo endoderm.
  • ExE endo extraembryonic endoderm.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, definitive endoderm; 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.
  • DEG differentially expressed genes
  • 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.
  • 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.
  • EPI epiblast
  • PS primitive streak
  • Ect ectoderm (including neural ectoderm; neural plate; neural tube; neural crest);
  • Inter. Meso, intermediated mesoderm; Mes, mesenchyme;
  • 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.
  • 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. 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
  • FIG. 12C shows representative brightfield (left), fluorescence (middle) and merged images of a day 13 peri-gastruloid showing rosette-like structures.
  • 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.
  • FIGs. 12E-12G show time-resolved single-cell RNA-seq analysis of peri- gastruloids.
  • FIGs. 12E-12G show time-resolved single-cell RNA-seq analysis of peri- gastruloids.
  • FIG. 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.
  • FIG. 12G is a heatmap displaying the tissue-specific marker genes for each identified cell type.
  • 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.
  • UMAP Uniform Manifold Approximation and Projection
  • 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.
  • 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 for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.
  • 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.
  • an element means at least one element and can include more than one element.
  • “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.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • 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.
  • 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.
  • treatment refers 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.
  • prevent 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.
  • an effective amount or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • “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.
  • the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals.
  • 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.
  • the subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc.
  • the subject may be a livestock animal.
  • suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas.
  • the subject may be a companion animal.
  • companion animals may include pets such as dogs, cats, rabbits, and birds.
  • the subject may be a zoological animal.
  • 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.
  • the subject can be a human.
  • the subject can be a human in need of repairing or regenerating a tissue or organ.
  • an “amount effective to” refers to an amount that is effective in providing at least one desirable biological result.
  • 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).
  • 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
  • FGF receptor (FGFR) proteins e.g., FGFR1,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • TGF-ps e.g., TGF-pi , TGF-P2, and TGF-P3
  • Activins e.g., Activin-A, Activin-B, and Activin-C
  • BMPs bone morphogenetic proteins
  • TGF-p receptors e.g., type I,
  • TGF-p activators include, but are not limited to, proteins, nucleic acids, small molecules, or any combination 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GSK3 refers to either or both isoforms of GSK3 (GSK3a and GSK3P). Inhibitors that inhibit either or both of these isoforms are of use.
  • the GSK3 inhibitor specifically inhibits GSK3 and does not substantially inhibit the majority of other mammalian kinases.
  • 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).
  • MEK refers to any isoform of MEK (e.g., MEK1 or MEK2). Inhibitors that inhibit either or both of these isoforms are of use.
  • the MEK inhibitor specifically inhibits MEK and does not substantially inhibit the majority of other mammalian kinases.
  • peri-gastruloid encompass embryonic (epiblast) and extraembryonic (hypoblast) tissues.
  • 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.
  • the present disclosure encompasses a method of generating peri-gastruloids having embryo-like structures.
  • 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.
  • PSCs pluripotent stem cells
  • 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.
  • 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.
  • 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.
  • the method further comprises a fourth medium, and maintaining the cells in the fourth culture medium for about 1-14 days.
  • the embryo-like structure or peri-gastruloids having embryo like structure is generated from pluripotent stem cells (PSCs).
  • PSCs may be naive PSC, naive-like PSCs, primed PSCs, formative PSCs, or extended PSCs.
  • the PSCs are naive PSCs.
  • the PSCs are naive-like PSC.
  • the PSCs are primed PSCs.
  • the PSCs are formative PSCs.
  • the PSCs are extended PSCs.
  • PSCs may be conventional human ESCs or induced human PSCs.
  • 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.
  • the PSCs comprise naive human pluripotent stem cells (hPSCs).
  • 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.
  • the PSCs comprise naive-like human pluripotent stem cells (hPSCs).
  • 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.
  • the PSCs comprise primed human pluripotent stem cells (hPSCs).
  • formative PSCs 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.
  • the PSCs comprise formative human pluripotent stem cells (hPSCs).
  • extended (or expanded) PSCs 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.
  • the PSCs comprise extended human pluripotent stem cells (hPSCs).
  • the PSCs comprise a single cell suspension of PSCs.
  • Single cell suspension of PSCs can be made using any known method in the art.
  • 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 TrypLETM.
  • PSCs are added or cultured in a three-dimensional culture device.
  • Any suitable three-dimensional culture vessel can be used.
  • three- dimensional culturing methods including methods using scaffold, and scaffold-free methods.
  • 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.
  • the disclosed three- dimensional culture device is a microwell plate.
  • the disclosed three- dimensional culture device is an AggreWellTM plate.
  • 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).
  • about 1-500 PSCs are added to an individual well of a three- dimensional culture device.
  • 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.
  • about 1-200 PSCs are added to an individual well of a three-dimensional culture device.
  • about 30-50 PSCs are added to an individual well of a three-dimensional culture device.
  • not more than 90 PSCs are added to an individual well of a three-dimensional culture device.
  • 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.
  • FGF activators for any of FGF family members can be used in the media described herein.
  • FGF activators used herein are specific to one FGF family member, e.g., specific to FGF or FGFR.
  • FGF activators are universal to two or more FGF family members, e.g., universal to FGF and FGFR.
  • FGF activators used herein are specific to FGF2.
  • the FGF activator is FGF2.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 used in the media described herein can be between about 1 ng/ml and about 200 ng/ml.
  • 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
  • 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).
  • 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).
  • TGF-p activator used herein is Activin-A.
  • 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.
  • 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.
  • Wild-type activin sequences e.g., sequences of Activin-A, Activin-B, and Activin- C
  • NCBI including human, mouse, and rat.
  • 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.
  • 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.
  • 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.
  • 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 about5,000 ng/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.
  • 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.
  • 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,
  • 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.
  • activators for any of WNT family members can be used in the methods described herein.
  • WNT activators used herein are specific to one WNT family member, e.g., specific to WNT1 , WNT2, WNT3, or WNT3a.
  • WNT activators are universal to two or more WNT family members, e.g., universal to WNT3 and WNT3a.
  • WNT activators used herein are specific to WNT3a.
  • Wild-type WNT sequences e.g., sequences of WNT1 , WNT2, WNT3, or WNT3a
  • NCBI including human, mouse, and rat.
  • 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.
  • the WNT activator is a biologically active WNT protein, e.g., WNT3 a protein.
  • the WNT activator is a glycogen synthase kinase 3 (GSK3) inhibitor.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • MEK inhibitors include, but are not limited to, GSK1120212, XL518, MEK162, CI-1040, PD0325901 , and TAK-733.
  • the MEK inhibitor should be capable of entering cells in sufficient quantities to inhibit MEK therein.
  • 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.
  • 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 5,000 pM, or between about 2,500 pM and about 5,000 pM.
  • 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.
  • 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
  • the medium comprises a cell culture medium.
  • 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.
  • 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).
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Mini
  • 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).
  • GEBCO KnockOut Serum Replacement
  • GIBCO KnockOut SR XenoFree
  • GIBCO KnockOut SR XenoFree Growth Factor Cocktail
  • N2 supplement GIBCO
  • B27 supplement GIBCO
  • 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 NeurobasalTM medium (GIBCO: 21103049).
  • the neurobasal medium can be used at a concentration between 0.1X to 2X.
  • 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.
  • 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).
  • the culture medium comprises DMEM/F-12 medium.
  • DMEM/F12 medium can be, by way of non-limiting example, Gibco’s DMEM/F12TM medium.
  • 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).
  • 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.
  • the culture medium comprises DM EM/F12 at a concentration from about 1% (vol/vol) to about 100% (vol/vol).
  • 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.
  • the culture medium comprises DMEM/F12 at a concentration of at least 40% (vol/vol).
  • 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®, GlutamaxTM, 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.
  • N2 supplement N2 supplement
  • B27 supplement B27
  • CEPT Chroman 1 , Emricasan, Polyamines, and Trans-ISRIB
  • KSR knockout serum replacement
  • Matrigel® Geltrex®
  • VitroGel® GlutamaxTM
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • the culture medium comprises a medium supplemented with B27.
  • B27 includes Gibco’s B27TM.
  • the culture medium comprises a basal medium supplemented with N2.
  • Non-limiting examples of N2 includes Gibco’s N2TM.
  • the culture medium comprises Neurobasal medium supplemented with N2.
  • the culture medium comprises neurobasal medium supplemented with B27.
  • the culture medium comprises a basal medium supplemented with N2 and B27.
  • KSR for e.g, Gibco KnockOut Serum Replacement
  • 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).
  • KSR is added to the medium
  • an extracellular matrix (synthetic, derived from cells, or a combination thereof) can be used in the described medium.
  • Matrigel® reconstituted basement membrane derived from extracts of Engelbreth-Holm-Swarm mouse tumors
  • Geltrex® a basement membrane extract
  • VitroGel® hydrogel system that closely mimics the natural extracellular matrix (ECM) environment
  • the Matrigel®, Geltrex®, VitroGel®, or other similar product can be used in an amount between about 1-40% (vol/vol).
  • 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).
  • 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).
  • FBS for e.g., Gibco Fetal Bovine Serum FBS
  • 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).
  • FBS is added to the medium used in the methods described herein at about 20% (vol/vol).
  • one or more of the additional factors CEPT, GlutamaxTM, ITS-X, or sodium pyruvate can be used in the described media between about 0.1 ml to about 10 ml.
  • the effective amount of one or more of the additional factors CEPT, GlutamaxTM, 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.
  • progesterone can be used in the described media between about 1-500 ng/ml.
  • 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.
  • progesterone is added to the culture medium at about 200 ng/ml.
  • additional factor used in the described media can be D-glucose.
  • D-glucose can be used in the described media between about 1-100 mg/ml.
  • 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.
  • progesterone is added to the culture medium at about 4 mg/ml.
  • 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.
  • 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
  • the medium disclosed herein comprises a first culture medium.
  • the first culture medium is a neurobasal medium.
  • the first culture medium is a neurobasal medium supplemented with N2 and B27.
  • the first culture medium comprises a FGF activator, a TGF-p activator, a WNT activator, or a MEK inhibitor, or any combination thereof.
  • 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.
  • the FGF activator used in the first culture medium is FGF2.
  • FGF2 is present in the first culture medium at about 1-200 ng/ml.
  • the TGF-p activator used in the first culture medium is Activin-A.
  • the Activin-A is present in the first culture medium at about 1-200 ng/ml.
  • the WNT activator used in the first culture medium is a GSK3 inhibitor.
  • the GSK3 inhibitor used in the first culture medium is a CHIR99021.
  • CHIR99021 is present in the first culture medium at about 0.1- 5 pM.
  • the MEK inhibitor used in the first culture medium is a PD0325901 .
  • the MEK inhibitor is present in the first culture medium at about 0.1-1 pM of PD0325901.
  • 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.
  • the first culture medium may be further supplemented with additional factors disclosed herein.
  • the first culture medium is further supplemented with CEPT.
  • the first culture medium is further supplemented with 1 ml of CEPT.
  • the medium disclosed herein comprises a second culture medium.
  • the second culture medium is a neurobasal medium.
  • the second culture medium is a neurobasal medium supplemented with N2 and B27.
  • the second culture medium comprises a FGF activator, a TGF-p activator, a MEK inhibitor, or any combination thereof.
  • 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.
  • the FGF activator used in the second culture medium is FGF2.
  • FGF2 is present in the second culture medium at about 1-200 ng/ml.
  • 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.
  • the medium disclosed herein comprises a third culture medium.
  • the third culture medium comprises In vitro culture medium (IVC).
  • the IVC comprises the basal medium DMEM/F12.
  • the third culture medium comprises IVC supplemented with at least one of Matrigel®, Geltrex®, or VitroGel®.
  • the third culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 1-100% (vol/vol).
  • the third culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 4% (vol/vol).
  • the third culture medium comprises IVC comprising DMEM/F12, GlutamaxTM, ITS-X, B-estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, FBS, or any combination thereof.
  • the third culture medium comprises IVC comprising DMEM/F12, 4% of Matrigel®, 1 ml of GlutamaxTM, 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.
  • the third culture medium comprises IVC comprising DMEM/F12, 4% of Matrigel®, 1 ml of GlutamaxTM, 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.
  • the medium disclosed herein comprises a fourth culture medium.
  • the fourth culture medium comprises IVC.
  • the IVC comprises the basal medium DMEM/F12.
  • 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).
  • the fourth culture medium comprises IVC comprising DMEM/F12, GlutamaxTM, ITS-X, B-estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, KSR, or any combination thereof.
  • the fourth culture medium comprises IVC comprising DMEM/F12, 4% of Matrigel®, 1 ml of GlutamaxTM, 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.
  • the fourth culture medium comprises IVC comprising DMEM/F12, 4% of Matrigel®, 1 ml of GlutamaxTM, 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.
  • 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.
  • the method comprises maintaining PSCs in a first culture medium about 0-10 days.
  • 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.
  • the method comprises maintaining PSCs in a first culture medium about 1- 7 days.
  • the method comprises maintaining PSCs in a first culture medium about 1 days.
  • the method comprises maintaining PSCs in a first culture medium about 2 days.
  • 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 supplemented with 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.
  • 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.
  • the method comprises maintaining PSCs in a second culture medium about 0-10 days.
  • 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.
  • the method comprises maintaining PSCs in a second culture medium about 2-7 days.
  • the method comprises maintaining PSCs in a second culture medium about 1 day.
  • the method comprises maintaining PSCs in a second culture medium about 2 days.
  • the method can further comprise removing, changing and/or replenishing the second culture medium during when PSCs is maintained in the second culture medium.
  • 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.
  • 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.
  • the method comprises maintaining PSCs in a third culture medium about 0-10 days.
  • 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.
  • the method comprises maintaining PSCs in a third culture medium about 1-7 days.
  • the method comprises maintaining PSCs in a third culture medium about 1 day.
  • the method comprises maintaining PSCs in a third culture medium about 2 days.
  • the method can further comprise removing, changing and/or replenishing the third culture medium during when PSCs is maintained in the third culture medium.
  • 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.
  • the method of generating embryo-like structures or peri- gastruloids from PSCs can further comprise maintaining PSCs in a fourth culture medium, in a three-dimensional culture device.
  • the method comprises maintaining PSCs in a fourth culture medium about 0-20 days.
  • 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.
  • 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.
  • the method can further comprise removing, changing and/or replenishing the fourth culture medium during when PSCs is maintained in the fourth culture medium.
  • 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.
  • 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).
  • BMT fluid control solutions Portable Culture Medium Replacement System
  • 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.
  • 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.
  • FGF fibroblast growth factor
  • TGF-P transforming growth factor beta
  • 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.
  • 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.
  • the PSCs is cultured for an appropriate period of time sufficient to form the embryo-like structures or peri-gastruloids.
  • 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.
  • 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.
  • embryo-like structures or peri-gastruloids are generated from a mammalian PSCs.
  • 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.
  • 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 be used herein, non-limiting examples of which can include PCR, RT-PCR, immunostaining, and sequencing.
  • the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise an apical membrane.
  • the apical membrane can be assessed using the presence of apical marker, for e.g., PODXL.
  • the peri-gastruloids or the embryo-like structures generated using the methods disclosed herein comprise yolk sac-like cells.
  • 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.
  • epiblast-like cells express OCT4, SOX2, NANOG.
  • yolk sac-like cells express GATA6, GATA4, SOX17.
  • the hypoblast- like cells express GATA6, GATA4, S0X17.
  • amnion-like cells express GATA3, OCT4, ISL1 , CDX2, TFAP2A, TFAP2C, and/or T (also called Brachyury).
  • 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.
  • peri-gastruloids or the embryo-like structures generated using the disclosed methods comprise yolk sac-like cavity.
  • peri- gastruloids or the embryo-like structures generated using the disclosed methods comprise amniotic-like cavity.
  • peri-gastruloids or the embryo-like structures generated using the disclosed methods comprise yolk sac-like cavity, and amniotic-like cavity.
  • yolk sac-like cavity comprise PDGFRa+ cells and/or PODXL+ cells.
  • cells of the amniotic-like cavity comprise columnar cells.
  • cells of the amniotic-like cavity comprise SOX2, OCT4, and/or NANOG.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise T+ cells marking the site of gastrulation.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise primordial germ cell-like cells (PGCLCs).
  • PGCLCs express NANOG, TFAP2C, PRDM1 , or any combination thereof.
  • PGCLCs express NANOG, TFAP2C, and PRDM1.
  • PGCLCs express TFAP2C, and PRDM 1.
  • PGCLCs are PRDM1+, OCT4+, TFAP2C+, SOX2- or any combination thereof.
  • PGCLCs are PRDM1+, OCT4+, TFAP2C+ and SOX2-.
  • PGCLCs express SOX17, PRDM1 (BLIMP1), NANOS3, or any combination thereof.
  • PGCLCs express SOX17, PRDM 1 (BLIMP1), and NANOS3.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise anterior- posted or (A-P) axis.
  • 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.
  • A-P axis express T at one end of the embryonic disk and/or epiblast.
  • 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.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise bilaminar disk-like structure.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise trilaminar disk-like structure.
  • peri- gastruloids or the embryo-like structures generated using the methods described herein comprise primitive streak like structure.
  • 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.
  • 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.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise neural ectoderm cells.
  • neural ectoderm cells express SOX2, PAX6, or any combination thereof.
  • neural ectoderm cells express SOX2, CDH2 and PAX6.
  • neural ectoderm cells express SOX2, SOX3, OTX2, PAX6, or any combination thereof.
  • neural ectoderm cells express SOX2, SOX3, OTX2, and PAX6.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise non-neural ectoderm cells.
  • non- neural ectoderm cells express TFAP2A, TFAP2C, GATA3, or any combination thereof.
  • non-neural ectoderm cells express TFAP2A, TFAP2C, and GATA3.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise amniotic ectoderm cells.
  • 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.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise surface ectoderm cells.
  • surface ectoderm cells express LHX2, BMP7, EYA1 , RAX, SIX3, PAX6, or any combination thereof.
  • surface ectoderm cells express LHX2, BMP7, EYA1 , RAX, SIX3, and PAX6.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise endoderm cells.
  • endoderm cells express EOMES, SOX17, FOXA2, TDGF1 , or any combination thereof.
  • endoderm cells express EOMES, SOX17, FOXA2, and TDGF1.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise yolk sac endoderm cells.
  • yolk sac endoderm cells express TTR, AFP, APOA1 , APOA2, APOC1 , APOE, or any combination thereof.
  • yolk sac endoderm cells express TTR, AFP, APOA1 , APOA2, APOC1 , and APOE.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise nascent mesoderm.
  • nascent mesoderm express FGF8, MIXL1 , EOMES, WNT3, MESP1 , MESP2, or any combination thereof.
  • nascent mesoderm express FGF8, MIXL1 , EOMES, WNT3, MESP1 , and MESP2.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise advanced mesoderm cells.
  • advanced mesoderm cells express TMEM88, COL3A1 , ACTC1 , or any combination thereof.
  • advanced mesoderm cells express TMEM88, COL3A1 , and ACTC1.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise lateral plate mesoderm.
  • lateral plate mesoderm express HAND1 , MEIS1 , GATA4, PITX1 , PITX2, BMP4, or any combination thereof.
  • lateral plate mesoderm express HAND1 , MEIS1 , GATA4, PITX1 , PITX2, and BMP4.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise cardiogenic mesoderm.
  • cardiogenic mesoderm express cTnT, MYL7, KDR, IGFBP3, or any combination thereof.
  • cardiogenic mesoderm express cTnT, MYL7, KDR, and IGFBP3.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise pre-somitic mesoderm.
  • pre-somitic mesoderm express TBX6, HES7, MLLT3, TCF15, or any combination thereof.
  • presomitic mesoderm express TBX6, HES7, MLLT3, and TCF15.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise hematopoietic progenitors.
  • hematopoietic progenitor cells express CD34.
  • peri-gastruloids or the embryo-like structures generated using the methods described herein comprise rosette-like structures.
  • the rosettelike structure comprise TBX18+ cells.
  • aspects of the present disclosure further provides an in vitro system.
  • the system comprises PSCs, a three-dimensional culture device, a first culture medium, second culture medium, and a third culture medium.
  • the in vitro system further comprises a fourth culture medium.
  • the in vitro system disclosed herein can be used for generation of peri-gastruloids or the embryo-like structures.
  • PSCs are mammalian PSCs.
  • 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.
  • PSCs in the in vitro system may be naive PSCs, PSC, naive-like PSCs, primed PSCs, formative PSCs, or extended PSCs.
  • the PSCs is naive PSCs.
  • the PSCs is naive-like PSCs.
  • the PSCs is primed PSCs.
  • the PSCs is formative PSCs.
  • the PSCs is extended PSCs.
  • PSCs may be conventional human ESCs or induced human PSCs.
  • 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.
  • the PSCs comprise naive human pluripotent stem cells (hPSCs).
  • 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.
  • the PSCs comprise naive-like human pluripotent stem cells (hPSCs).
  • 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.
  • the PSCs comprise primed human pluripotent stem cells (hPSCs).
  • formative PSCs 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.
  • the PSCs comprise formative human pluripotent stem cells (hPSCs).
  • Extended PSCs 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.
  • the PSCs comprise extended human pluripotent stem cells (hPSCs).
  • 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.
  • 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-l, Accutase, or TrypLETM.
  • 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.
  • three-dimensional culturing methods including methods using scaffold, and scaffold-free methods.
  • 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.
  • the disclosed three-dimensional culture device is a microwell plate.
  • the disclosed three-dimensional culture device is an AggreWellTM plate.
  • 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).
  • the in vitro system disclosed herein comprises a first culture medium.
  • the first culture medium is a neurobasal medium.
  • the first culture medium is a neurobasal medium supplemented with N2 and B27.
  • the first culture medium comprises a FGF activator, a TGF-p activator, a WNT activator, or a M EK inhibitor, or any combination thereof.
  • 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.
  • the FGF activator used in the first culture medium is FGF2.
  • FGF2 is present in the first culture medium at about 1-200 ng/ml.
  • the TGF-p activator used in the first culture medium is Activin-A.
  • the Activin-A is present in the first culture medium at about 1-200 ng/ml.
  • the WNT activator used in the first culture medium is a GSK3 inhibitor.
  • the GSK3 inhibitor used in the first culture medium is a CHIR99021.
  • CHIR99021 is present in the first culture medium at about 0.1-5 pM.
  • the MEK inhibitor used in the first culture medium is a PD0325901.
  • the MEK inhibitor is present in the first culture medium at about 0.1-1 pM of PD0325901.
  • 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.
  • the first culture medium may be further supplemented with additional factors disclosed herein.
  • the first culture medium is further supplemented with CEPT.
  • the first culture medium is further supplemented with 1 ml of CEPT.
  • the in vitro system disclosed herein comprises a second culture medium.
  • the second culture medium is a neurobasal medium.
  • the second culture medium is a neurobasal medium supplemented with N2 and B27.
  • the second culture medium comprises a FGF activator, a TGF-p activator, or a MEK inhibitor, or any combination thereof.
  • 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.
  • the FGF activator used in the second culture medium is FGF2.
  • FGF2 is present in the second culture medium at about 1-200 ng/ml.
  • 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.
  • the in vitro system disclosed herein comprises a third culture medium.
  • the third culture medium comprises In vitro culture medium (IVC).
  • the IVC comprises the basal medium DMEM/F12.
  • the third culture medium comprises IVC supplemented with at least one of Matrigel®, Geltrex®, or VitroGel®.
  • the third culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 1-100% (vol/vol).
  • the third culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 4% (vol/vol).
  • the third culture medium comprises IVC comprising DMEM/F12, GlutamaxTM, ITS-X, B-estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, FBS, or any combination thereof.
  • the third culture medium comprises IVC comprising DMEM/F12, 4% of Matrigel®, 1 ml of GlutamaxTM, 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.
  • the third culture medium comprises IVC comprising DMEM/F12, 4% of Matrigel®, 1 ml of GlutamaxTM, 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.
  • the in vitro system disclosed herein comprises a fourth culture medium.
  • the fourth culture medium comprises IVC.
  • the IVC comprises the basal medium DMEM/F12.
  • the fourth culture medium comprises IVC supplemented with at least one of Matrigel®, Geltrex®, or VitroGel®.
  • the fourth culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at ari amount of about 1-100% (vol/vol).
  • the fourth culture medium comprises IVC supplemented with Matrigel®, Geltrex®, or VitroGel® at an amount of about 4% (vol/vol).
  • the fourth culture medium comprises IVC comprising DMEM/F12, GlutamaxTM, ITS-X, B-estradiol, progesterone, N-acetyl-L-cysteine, sodium pyruvate, KSR, or any combination thereof.
  • the fourth culture medium comprises IVC comprising DMEM/F12, 4% of Matrigel®, 1 ml of GlutamaxTM, 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.
  • the fourth culture medium comprises IVC comprising DMEM/F12, 4% of Matrigel®, 1 ml of GlutamaxTM, 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.
  • the fourth culture medium may be supplemented with D-glucose.
  • D-glucose may be supplemented in the fourth medium at about 4 mg/ml.
  • 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).
  • BMT fluid control solutions Portable Culture Medium Replacement System
  • 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.
  • the present disclosure further provides peri-gastruloid or embryo-like structure produced by the methods or system disclosed herein.
  • the disclosure encompasses an embryo model comprising a peri-gastruloid or embryo-like structure produced by the method or system described herein.
  • embryo models can be used for studying imprinting, reprogramming, rejuvenation, and other biochemical, metabolic, and physiological phenomena associated with embryogenesis and development.
  • embryo models can be used for studying effect of mutations in embryogenesis and development.
  • 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.
  • 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.
  • 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.
  • a method of cell replacement therapy 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.
  • 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.
  • a desired target site e.g., heart, brain, liver, bone marrow, kidney or other organ that requires cell therapy.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GFP green fluorescent protein
  • the method may further comprise repeatedly administering the cells, for e.g., in the case of chronic diseases such as autoimmune disorders or cancer.
  • 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.
  • 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.
  • a candidate molecule e.g., a compound, or a drug
  • 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.
  • 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.
  • 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.
  • 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.
  • high throughput platform comprising a peri-gastruloid, embryolike structure, or cells of the peri-gastruloid, or embryo-like structure
  • a candidate molecule or could comprise a mutation.
  • 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.
  • 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.
  • 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).
  • kits for use in generating the peri-gastruloid, or embryo-like structure described herein 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.
  • 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.
  • the instructions may comprise a description of methods for culturing the PSCs and generating the peri-gastruloid, or embryo-like structure described herein.
  • the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • RUES2-GLR human embryonic stem cell line
  • Nodal-mCitrine reporter cell line (ESI017 background)
  • H9 WA09, human ESC
  • 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.
  • 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.
  • hEPSCs Human extended pluripotent stem cells
  • 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 x10 4 /cm 2 ) plates pre-coated with 0.1% gelatin.
  • PD0325901 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
  • 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 the co-culture of the epiblast and hypoblast-like cells. The differentiated cells were passaged as a mixture.
  • EPSCs extended pluripotent stem cells
  • 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 AggreWellTM plate at a density of 45 cells per microwell, for instance, 5.4x10 4 per well of AggreWellTM 400 (24-well format) or 1.35x10 4 per well of AggreWellTM 800 (24-well format).
  • CEPT 50 nM Chroman 1 , 5 pM Emricasan, IxPolyamines, and 0.7 pM Trans-ISRIB
  • Cells were counted using a hemocytometer and then seeded into an AggreWellTM plate at a density of 45 cells per microwell, for instance, 5.4x10 4 per well of AggreWellTM 400 (24-well format) or 1.
  • 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.
  • 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 2.7x10 4 cells per well.
  • 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.
  • RCF Relative Centrifugal Force
  • microwells 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.
  • ICV1 76 ml Advanced DMEM/F12, 1 ml GlutamaxTM (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.
  • ITS-X Insulin-Transferrin-Selenium- Ethanolamine
  • 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.
  • This step allowed the gel formation of the Matrigel component, which allowed the peri-gastruloids to be suspended in the medium instead of sinking to the bottom and attaching to the plate.
  • 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.
  • 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.
  • 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.
  • 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.
  • PFA paraformaldehyde
  • 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) .
  • PBST PBS + 0.1% T riton X
  • CUBIC-L a delipidation and decoloring reagent
  • 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.
  • 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.
  • 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.
  • 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 medium in 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).
  • DMEM/F12 + 10% FBS 5 ml fibroblast medium
  • 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).
  • 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.
  • Example 1 A 3D culture method enables robust self -organization of human EPSCs into peri-gastruloids.
  • HDM hypoblast differentiation medium
  • 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.
  • ICM inner cell mass
  • 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 (hEPSCs RUES2 ) were used to facilitate tracking of epiblast and hypoblast-like lineages.
  • hEPSCs RUES2 are SOX2-Citrine positive and SOX17-tdTomato negative (FIG. 1A). It was found that tHDM not only could induce HLCs from hEPSCs RUES2 , 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).
  • TFs key pluripotency transcription factors
  • OTX2 a gene known to be upregulated in formative epiblast-like cells
  • 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).
  • day 4 HLCs also expressed OTX2, N-CAD, and PRDM1 (or called BLIMP1) (FIG. 1E, FIG. 1G, and FIG.
  • the amnion functions as a signaling hub, secreting morphogen molecules to the neighboring epiblast to facilitate the initiation of gastrulation.
  • the induction of amnion cells in peri-gastruloids was investigated.
  • Day 4 aggregates were subjected to attachment culture and determined their cell lineage composition.
  • GATA6+ yolk saclike cells
  • OCT4+ epiblast-like cells
  • 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, SOX2 Low NANOG Low squamous epithelial cells expressing amnion markers CDX2 and TFAP2C, and exhibiting high BMP signaling were observed (FIG. 5A-5D, FIG. 5F-5G, FIG. 6A-6C).
  • amnion cells secrete BMP4, which acts on nearby epiblast cells to activate the expression of WNT and NODAL
  • BMP4 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.
  • NODAL production was augmented in epiblast-like cells (FIG. 5F, FIG. 5I, and FIG. 10M).
  • primordial germ cells 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.
  • PRDM1+/ OCT4+/TFAP2C+/SOX2- were observed in day 6 BTAG peri-gastruloids (FIG. 6A, and FIG. 6C-6E).
  • A-P anterior- posterior
  • AVE anterior visceral endoderm
  • 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.
  • the migrating cells 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.
  • 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-well plates (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).
  • 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
  • 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).
  • FIG. 2E 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).
  • FIG. 2E, FIG. 9C-9D and FIG. 10D 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).
  • scRNA-seq single cell RNA sequencing
  • UMAP Uniform Manifold Approximation and Projection
  • 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).
  • 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).
  • OCT4 core pluripotency transcription factors
  • SOX2 SOX2
  • NANOG NANOG
  • Adjacent to the primitive streak-like population (cluster s, T, TDGF1 , CDH1 and FGF8) (Figure S6B)
  • Figure S6B we found a group of cells expressing node signature genes like GSC, FOXA2, FST, CER1 , DKK1 , HHEX and CHRD ( Figure S6C).
  • ectoderm cells 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.
  • SOX2, SOX3, OTX2, PAX6 non-neural ec
  • Definitive endoderm cells cluster 6, EOMES, SOX17, FOXA2, TDGF1 etc.
  • yolk sac endoderm cells cluster 7, TTR, AFP, APOA1 , APOA2, APOC1 , APOE, etc.
  • a cell cluster (cluster 12, PECAM1 , HHEX, ETV2, CD34, HBZ, HBA1 , HBE1 and PDPN etc.) was found situated near 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).
  • PDCLCs Primordial Germ Cell-Like Cells
  • nascent mesoderm cluster 4, FGF8, MIXL1 , EOMES, WNT3, MESP1 , MESP2
  • advanced mesoderm cells cluster s, TMEM88, COL3A1 , ACTC1
  • 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).
  • rosette-like structures were detected in day 13 peri-gastruloids that contained TBX18+ cells (FIG. 12C-12D).
  • 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.
  • RNA-seq analysis of peri-gastruloids was undertaken.
  • Heatmap displaying the tissue-specific marker genes for each identified cell type is shown in FIG. 12G.
  • FIG. 12H 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).
  • AVE human anterior visceral endoderm
  • 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.
  • peri-gastruloids recapitulated several crucial stages of human peri-gastrulation development, including the formation of amniotic and yolk sac cavities, the development of bilaminar 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.
  • 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 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.
  • 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.
  • peri-gastruloids 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 of BMP4 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.
  • visceral endoderm which is known to secrete BMP2.
  • 2D and 3D human gastruloids models derived from primed hPSCs require treatments with BMP4 and a WNT agonist, CHIR99021 , respectively.
  • 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.
  • 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.
  • stem cell derived models for yolk sac development in humans.
  • 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.
  • peri-gastruloid formation potentially provided an accessible platform to model primary and secondary yolk sac development in humans.
  • 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.
  • 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).
  • 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.
  • 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.
  • it enables high-throughput genetic and chemical screens, thereby offering insights that could potentially mitigate early pregnancy loss and congenital defects.
  • 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 advanced development 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.

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Abstract

La présente divulgation concerne de manière générale des procédés et un système in vitro de génération de structures de type embryon. La divulgation concerne en outre des méthodes d'utilisation des structures de type embryon en médecine régénérative et un criblage de médicament.
PCT/US2024/035432 2023-06-27 2024-06-25 Système et procédés de génération de progéniteurs fœtaux humains, de tissus et d'organes à partir de cellules souches cultivées Pending WO2025006477A2 (fr)

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