Modelling Human Post-Implantation Development via Extra-Embryonic Niche Engineering

Abstract

Implantation of the human embryo commences a critical developmental stage that comprises profound morphogenetic alteration of embryonic and extra-embryonic tissues, axis formation, and gastrulation events. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons. Additionally, human stem cell models of early post-implantation development with both embryonic and extra-embryonic tissue morphogenesis are lacking. Here, we present iDiscoid, produced from human induced pluripotent stem cells via an engineered a synthetic gene circuit. iDiscoids exhibit reciprocal co-development of human embryonic tissue and engineered extra-embryonic niche in a model of human post-implantation. They exhibit unanticipated self-organization and tissue boundary formation that recapitulates yolk sac-like tissue specification with extra-embryonic mesoderm and hematopoietic characteristics, the formation of bilaminar disc-like embryonic morphology, the development of an amniotic-like cavity, and acquisition of an anterior-like hypoblast pole and posterior-like axis. iDiscoids offer an easy-to-use, high-throughput, reproducible, and scalable platform to probe multifaceted aspects of human early post-implantation development. Thus, they have the potential to provide a tractable human model for drug testing, developmental toxicology, and disease modeling.

Extended Data Figure 1:. 3D iDiscoid cultures.

(A) The gene circuit used to create inducible GATA6-expressing iPSCs. pConst is a constitutively active promoter. (B) Heterogeneity of EGFP (GATA6) activation in iGATA6 cells, detected via flow cytometry analysis. Higher gene circuit copy numbers leads to higher expression level of EGFP and GATA6. (C) 3D culture of iGATA4 and WT showing co-expression of the amnion marker ISL1 and the pluripotency marker NANOG spread fully throughout the WT layer without D-V polarization. Middle slice shows the development of a central lumen. (D) 3D culture of iGATA4 and WT showing expression of the pluripotency marker NANOG throughout the WT layer but lack of ISL1 expression in notable subset of these 3D tissues. These spheres do not exhibit polarization as well. (E) Schematic depicting iDiscoid development in 3D versus from 2D>>3D in comparison to embryo morphology. iDiscoid is similar to a flattened yolk sac cavity with epiblast interface and amniotic cavity.

Extended Data Figure 1:. Sorting and symmetry breaking events following GATA6 induction.

(A) Immunofluorescence images of fixed cultures demonstrating cell organization of iGATA6 (green) and WT (NANOG) hiPSCs between day 0 and day 3 after GATA6 induction. PDGFRα rises within the iGATA6 cells as they acquire a more yolk sac endoderm-like morphology. (B) Time lapse images of a single position within the iGATA6/WT co-culture from day 0 to day 3 after GATA6 induction. Top cropped images correspond to positions within the white boxes in the images below. Scale bar = 200 μm. (C) Time lapse images showing the initial confinement of red WT cells with green iGATA6 cells, followed by migration of iGATA6 cells over the WT disc prior to D2 after iDiscoid induction. (D) Z-slices of two representative iDiscoids showing localization of OCT4, GATA4, and SOX17 within the bilaminar disc-like area of iDiscoid culture, as well as development of a central lumen by day 5.

Extended Data Figure 3:. Lumen development within WT of iDiscoids

(A) Immunofluorescence images showing dynamic of LAMA1 deposition on days 1, 3, and 5 of iDiscoid development after induction. Dotted boxes show the areas of inset in the fourth panels of the day 3 and day 5 images. (B) Timecourse graphs showing the increase in LAMA1 signal in immunofluorescence images over the first five days of iDiscoid development. Dots represent randomly sampled areas from a single experiment harvested at each day (C) Immunofluorescence image showing the deposition of laminin around a WT cluster with a central lumen as well as polarization of PODXL . (D) Immunofluorescence showing horizontal and lateral slices of a representative WT cluster with polarization of PODXL and ZO-1 towards a central lumen. (E) A representative cluster of WT iPSCs at day 3. No laminin deposition is observable in the vicinity of the cluster. (F) ELISA comparing secreted AFP and APOA1 detected on D0 and D5 after GATA6 induction with Dox. Individual dots represent biological replicates. Scale bars = 100μm

Extended Data Figure 4:. Lumen development within WT of iDiscoids

(A) Distribution of WT cluster areas versus the number of lumens observed in iDiscoids with iGATA6 coverage. Shaded area indicates the areas of the bilaminar disc between E9 and E17 as recorded by Hertig and Rock, 1949 and Heuser et al. 1945; 64% of discs observed have areas that fall into this range of values, and 39.8% have a single lumen. 33.2% fall into both of these categories. n = 331 total. (B) Distribution of WT cluster circularity versus the number of lumens observed in iDiscoids with iGATA6 coverage, with a representative image of an iDiscoid with the indicated characteristics. n = 331, Scale bar = 100μm. (C) Heatmaps displaying the average area of iDiscoids resulting from different initial seeding densities of iGATA6 and WT. Dotted box shows optimized seeding density used for iDiscoid experiments. (D) Heatmaps displaying the average circularity and average resulting disc number pre mm² of iDiscoids resulting from different initial seeding densities of iGATA6 and WT. Dotted box shows optimized seeding density used for iDiscoid experiments. (E) Distribution of WT cluster areas versus the number of lumens observed in iDiscoids with iGATA6 coverage when seeded at the optimized ratio indicated in C. Shaded area indicates the areas of the bilaminar disc between E9 and E16-19 as recorded by Hertig and Rock, 1949 and Heuser et al. 1945; 74% of discs that fall into this area range. 89.7% of discs have a single lumen. 70.9% of discs fall into both of these categories. n = 79 total.

Extended Data Figure 5:. Merged clustering of D0-D5 iDiscoid single cell populations

(A) Individual UMAP projections and clustering for each time point recorded through day 5 in iDiscoid development. (B) Violin plots showing a curated set of genes in iDiscoid day 0, day 2, and day 4 clusters. iDiscoid clusters are ordered by lowest to highest GATA6 expression level. “W” clusters are clusters with putative wild-type lineage; “G” clusters are clusters with putative iGATA6 lineage.

Extended Data Figure 6:. Hypergeometric statistical comparison of iDiscoid time points to human and NHP embryo data.

(A) Hypergeometric statistical comparison of each iDiscoid day to the annotated human embryo populations from Tyser et al. 2022. Blue dots above each column indicates the relative GATA6 expression level of each population indicated per day. (B) Hypergeometric statistical comparison of each iDiscoid day to the annotated human embryo populations from Xiang et al. 2020. Scale used is the same as shown in panel A. (C) Hypergeometric statistical comparison of each iDiscoid day to the annotated cynomolgus embryo populations from Ma et al. 2019. Scale used is the same as shown in panel A. iDiscoid clusters correspond to those in the individual day-by-day clustering in Extended Data Figure 5 and are ordered from left to right on the x-axis by lowest to highest GATA6 expression level. “W” clusters are clusters with putative wild-type lineage; “G” clusters are clusters with putative iGATA6 lineage. Dotted outlines indicate fate comparisons of the most interest for each cluster. Abbreviations from other datasets: NNE = Non-neural ectoderm, DE (P) = Definitive endoderm (proliferative), DE (NP) = Definitive endoderm (not proliferative), YS = Yolk sac, PGC = Primordial germ cell, CTB = Cytotrophoblast, STB = Syncytiotrophoblast, EVT = Extravillous trophoblasts, PSA-EPI = Primitive streak anlage in the epiblast, EXMC = Extraembryonic mesoderm cells, E- = Early, L- = Late, Gast = Gastrulating cells, AM = Amnion, VE/YE = Visceral endoderm/Yolk sac endoderm

Extended Data Figure 7:. Merged clustering of Day 0 - Day 5 iDiscoid single cell populations

(A) UMAP showing the merged iDiscoid clusters annotated by day. (B) UMAP showing the merged iDiscoid clusters with unsupervised clustering applied. (repeated figures: Cluster 14 is the amnion-like cluster shown in Figure 2B; Cluster 6 is the D2 anterior-like cluster shown in Figure 3A; Cluster 2 is the D3-5 anterior-like cluster shown in Figure 3A; and Cluster 15 is the posterior-like cluster shown in Figure 3B.) (C) Heatmap showing the top 20 genes corresponding to each cluster of the merged day 0 through day 5 dataset.

Extended Data Figure 8:. Major signaling interactions between day 5 iDiscoid cell clusters.

(A) Immunofluorescence staining for the amnion markers ISL1 and AP-2α at day 4. Top-down widefield image of a flattened coverslip. Scale Bar = 100 μm (B) Dot plot of marker genes from the BMP pathway from day 4 iDiscoid scRNA-seq. BMP4 expression and a number of associated genes (boxed in red) are highest in the amnion-like population.

Extended Data Figure 9:. Effect of BMP4 inhibition on anterior-like and posterior domains in iDiscoids.

(A) Control iDiscoids showing development of TBXT⁺ posterior domains and LHX1⁺ areas expressing CER1. (B) iDiscoids with 100ng/mL Noggin added at day 2 showing presence of LHX1⁺ areas expressing CER1, but no expression of TBXT⁺ domains. TBXT expression is seen in iGATA6-lineage cells at the periphery of the WT disc. Scale bars = 100 μm

Extended Data Figure 10:. Examples of TBXT and CER1 observed in iDiscoids.

(A) A representative WT cluster showing syn-polarity of TBXT and CER1 within the WT cluster and iGATA6 layers, respectively. Filled arrow indicates CER1-expressing cells within the iGATA6 layer; empty arrow indicates CER1 and TBXT co-expressing cells within the WT layer. Z-slices are representative slices from the center of two different WT discs. (B) Representative WT clusters showing anti-polarity of TBXT and CER1 within the WT cluster and iGATA6 layers, respectively. Filled arrow indicates CER1-expressing cells within the iGATA6 layer; empty arrow indicates CER1 and TBXT co-expressing cells within the WT layer. Z-slice is a representative slice from the center of a WT disc. (C) Top shows a merged UMAPs of all D0-D5 scRNA-seq data showing the posterior compartment expressing the inhibitor CER1. Dotted boxes show WT lineages. Posterior region was illustrated in Figure 3B. Bottom shows a scatterplot from merged D0-D5 iDiscoid scRNA-seq showing a population of cells derived from D3-D4 iDiscoid that co-express CER1 and TBXT. Axes represent scaled expression levels of each marker; dots represent individual cells colored by day. Scale Bars = 100 μm

Extended Data Figure 11:. Positioning of TBXT and CER1 expression in transcriptomic human, NHP and mouse datasets.

(A) Spatial expression of TBXT and CER1 from the CS5 and CS6 marmoset embryo from Bergmann et al. 2022. In marmoset CS5, CER1 has high and unpolarized expression in the extraembryonic endoderm overlaying the embryonic disc; by CS6, overall expression has decreased, and a very slight polarization away from the putative posterior is observed. From CS5 to CS6, TBXT moves from unregionalized/bipolar expression within the embryonic disc to strong expression at one pole of the embryonic disc. (B) Regional expression of TBXT and CER1 in the E16-19 (CS7) human embryo from Tyser et al. 2022. Highest expression of TBXT and CER1 is observable in the caudal portion of the embryo, which corresponds to the area of the primitive streak and the hypoblast overlaying this area, as shown in the diagram. (C) Pseudospatial representation of TBXT and CER1 expression in the cynomolgus E18-E20 embryo from Cui et al. 2022. Diagram to the right shows the microdissected embryo sections with labels corresponding to each identified tissue layer. CER1 expression strongly overlays TBXT expression in the lower embryonic (EM-Low) and middle embryonic (EM-Mid) regional layers. (D) Expression levels of T and Cer1 from the E6.5-E8.5 mouse embryo. There is a pronounced co-expression within the anterior domain of the primitive streak. (E) Expression levels of T and Cer1 from an anterior primitive streak metacell from 153 mouse embryos spanning E6.5 – E7.5. Within this representative cell/cell type, T and Cer1 rise together over time at the anterior primitive streak. (F) Spatial expression levels of T and Cer1 within the ~E6.5-E7.5 mouse embryo. These markers are co-expressed in the area near the anterior tip of the posterior domain. Boxed area in the diagram corresponds highlights this range. Right diagrams show expression patterns of each marker at the index 3 plane in the Y axis of left diagrams.

Extended Data Figure 12:. Expression of yolk sac mesoderm and extracellular matrix genes in day 4 iDiscoid scRNA-seq.

(A) Violin plots showing the distribution of ECM genes as well as the yolk sac mesoderm marker gene BST2 in day 5 iDiscoid scRNA-seq clusters. (B) Dot plot showing a subset of day 5 yolk sac mesoderm and endothelial marker genes. The highest GATA6 population expresses the highest levels of YSM marker genes. The GATA6 expression is shown to the left with different scaling of circle sizes than the marker list to the right.

Extended Data Figure 13:. Identification of endothelial/hematopoietic populations.

(A) Scatterplots showing the distribution of markers obtained via image analysis of 3 independent experiments at day 5. Percentages correspond to the fraction of cells recorded with corresponding marker expression levels above the thresholds represented by the dotted lines. (B) Immunofluorescence image showing cells expressing hematopoietic markers in iDiscoid culture. Cells expressing the hematopoietic marker TAL1 (Scl) localize between the yolk sac endoderm compartment and the tissue culture dish. Scale bar = 100 μm (C) Immunofluorescence image showing cells expressing RUNX1 and TAL1 positioned against the dish. Scale bar = 100 μm (D) Image analysis of z-slices from 5 areas in 3 biological replicates. The peak of ERG expression is underneath the peak of EGFP expression representing the iGATA6 endoderm layer. Dotted curves represent SEM calculated at each point. (E) Immunofluorescence image showing cells expressing VE-cadherin and VEGFR2 positioned against the dish. Arrow indicates likely differentiating endothelial cell from the overlaying iGATA6 layer. Scale bar = 100 μm (F) Flow cytometry plots showing the presence of hematopoietic markers on day 14 after a CFU assay initiated CD34-expressing cells (enriched from iDiscoids). (G) Results from image analysis demonstrating the change in area of CD34⁺ cells between day 5 and day 12 of iDiscoid culture, assessed via analysis of immunofluorescence images. Individual dots represent biological replicates. **: P value=0.0055 (H) Representative flow cytometry plot on day 5 and day 12 of iDiscoid showing expansion of the CD34⁺ population by day 12 (n=3).

Extended Data Figure 14:. Hematopoietic cells in iDiscoid.

(A) Erythroid markers (CD235a and Hemoglobin) expressed in a subset of CD43⁺ round cells in day 12 iDiscoids. Scale bar = 100μm (B) Cells expressing myeloid (CD31, CD33) and macrophage (CX₃CR1) markers in a representative cluster in day 12 iDiscoid. Scale bar = 100μm (C) Cells expressing megakaryocyte markers in day 12 iDiscoid. Scale bar = 100μm (D) UMAP showing unbiased clustering of scRNA-seq performed on day 21. Boxed area shows populations expressing hematopoietic marker genes. (E) Hypergeometric statistical comparison of day 21 iDiscoid populations to the E16-19 human embryo populations from Tyser et al. 2022. The red box shows populations of interest with similarity to in vivo hematopoietic lineages. iDiscoid clusters are grouped by similar fates on Y axis. (F) Expression of hematopoietic marker genes within the red boxed population in panel D. The four clusters show distinct marker gene expression profiles associated with yolk sac-derived hematopoietic populations. (G) Flow cytometry analysis on day 21 iDiscoid. Cells were gated for positive CD43 expression prior to gating for the markers shown (n=2, representative sample shown).

Extended Data Figure 15:. Comparison of iDiscoids with and without supplementation with high iGATA6 expressing (iGATA6-hi) cells.

(A) Expression of CD43⁺ and Hemoglobin⁺ cells in day 12 iDiscoid without additional supplementation of GATA6-hi cells. Red color in high Hoechst areas is nonspecific staining. Scale bar = 500 μm. Insets show areas of CD43⁺ cells. Dotted outlines on right show zoomed in areas shown within the image. Scale bar = 100 μm (B) Expression of CD43⁺ and Hemoglobin⁺ cells in day 12 iDiscoid with supplementation of 25% GATA6-hi cells at initial seeding. A significant expansion of CD43-expressing cells is observable Scale bar = 500 μm. A representative image of 2-4 biological replicates.

Extended Data Figure 16:. Structure of blood island-like associated tissues within iDiscoid.

(A) Z-slices of a day 12 iDiscoid at the indicated height above the bottom focal plane. Expression of the mesodermal marker Desmin is regionalized close to the bottom of the tissue; endothelial marker CD34 is expressed near the bottom and middle of the tissue, with highest expression just above Desmin (z = 6-8); endoderm marker FOXA2 is exclusively expressed above the other tissues. Scale bar = 50 μm (B) Z-slices around the blood island-like area shown in Figure 4E. Desmin is observed regionalized below the endothelial (CD31⁺) and hematopoietic (CD43⁺) tissues. No stain for endoderm is shown. Scale bar = 100 μm

Extended Data Figure 17:. iDiscoid formation from hiPSCs to model human early post-implantation development in vitro.

(A) From an initially mixed state, iDiscoid cells segregate into WT clusters surrounded by iGATA6 cells. These iGATA6 cells migrate laterally to create a bilaminar boundary on top of the WT clusters. These clusters then undergo lumenogenesis, specification of amnion-like cells, and formation of anterior hypoblast-like and posterior domains. In iGATA6-only areas, yolk sac mesoderm specifies and hematopoietic cell specification is observed.

Extended Data Figure 18:. Investigation of WT fates in day 12 iDiscoid.

(A) Most structures corresponding to former WT clusters at day 12 in iDiscoid have taken on expression of ISL1 and lost expression of the pluripotency markers SOX2 and NANOG. A limited number of cells express SOX2 at the core of former WT clusters, potentially indicating the specification of an ectoderm-like fate in a small number of WT-lineage cells. (B) A subset of former WT clusters have taken on markers of ectoderm differentiation. Scale bars = 500 μm

Extended Data Figure 19:. Mixed iDiscoid cell lines exhibit consistent cluster formation after repeated passaging and cryostorage prior to induction.

(A) Schematic showing the creation of the iDiscoid parental cell line. iGATA6 cells with heterogeneous copy numbers of the inducible GATA6 circuit are mixed with wild-type. This cell combination is then maintained together or frozen prior to induction for iDiscoid experiments. (B) iDiscoid morphology and characteristics following cryostorage and defrosting. Scale Bar = 500 μm (C) Characteristics and morphology of iDiscoid cultures induced immediately after mixing (passage 1) or maintained together and passaged for two months (passage 15). Scale Bars = 500 μm

Extended Data Figure 20:. iDiscoid engineering in a separate iPSC cell line results in the generation all major features of interest

Day 4 immunofluorescence images of iDiscoid engineered using the iPSC cell line PGP9. (A) A portion of PGP9 iGATA6 cells expressing high levels of GATA6 (EGFP) also express the anterior endoderm marker HHEX near the edge of a WT cluster. (B) A ring of PODXL expression lines the inside of a cavities formed in PGP9 WT clusters. (C) ISL1⁺ cells specify away from NANOG⁺ cells, along the base of a cavity formed in PGP9 WT. (D) TBXT⁺ cells develop polar domains at the edge of WT clusters. Representative islands show that both syn- and anti-polar arrangement of cells are observable. Scale Bars = 100 μm

Extended Data Figure 21:. Example of FACS gating strategy.

(Cited in Methods) Cell debris was excluded via an SSC-A vs FSC-A gate; aggregates were excluded by comparing FSC-A and FSC-H; dead cells were gated out using the 7-AAD stain to identify positive cells.

Figure 1:. Engineering co-development of embryonic and extra-embryonic endoderm tissues in iDiscoids

(A) Schematic demonstrating cell mixing and subsequent organization of iGATA6 (green) and WT (strongly orange) hiPSCs after GATA6 induction by Dox. (B) Immunofluorescence staining demonstrating self-organization of iDiscoids in the cultures. Scale bar = 1000 μm (C) Live time lapse images of one iDiscoid showing growth of a central lumen from a rosette indicated by an arrow. Scale bar = 50 μm. (D) Phase and fluorescence images from live day 4 iDiscoid cultures showing developed iDiscoid morphologies with EGFP-expressing iGATA6 around a WT cluster. Scale bar = 100 μm (E) Quantification of characteristics of WT clusters possessing different iGATA6 coverage and lumen formation characteristics. No islands without coverage were observed possessing a lumen (−/+ = 0%) (F) Single cell UMAP and hypergeometric statistical comparisons of differentially expressed gene (DEG) lists between day 2 iDiscoid and human and NHP embryo single cell datasets. Labels above heatmaps indicate pre-implantation versus post-implantation embryo sample comparisons; gray bars under labels indicated NHP comparisons. (G) Single cell UMAP and hypergeometric statistical comparisons of differentially expressed gene (DEG) lists between day 4 iDiscoid and human and NHP embryo single cell datasets. Black boxes highlight high DEG similarity (high P values) to relevant populations of interest.

Figure 2:. Amniotic-like cavity formation and expansion

(A) Merged UMAP of all WT lineages from iDiscoid labeled by day of development. (B) The merged WT population showing the compartment expressing markers of amnion. ISL1, TFAP2A (AP-2^α), and GATA3 are expressed in this area, while NANOG is negative and OCT4 is low. (C) An orthogonal slice of an individual iDiscoid showing top-bottom compartmentalization of ISL1⁺ (cyan) and NANOG⁺ (red) cells while iGATA6 cells cover the top (white). (D) Horizontal z-slices at the indicated distance from the dish of the WT cluster from (C). (E) Schematic showing the position of each population within a single iDiscoid. Dotted lines indicate the area of slices shown. (F) Expression patterns of BMP4 effectors (phosphorylated SMAD1, SMAD5, and SMAD8/9) in iDiscoid. Lower images show a lateral slice of the WT disc shown. (G) Immunofluorescence staining for the BMP4 effectors (phosphorylated SMAD1, SMAD5, and SMAD8/9) in iDiscoid at day 4 after application of the inhibitor Noggin at Day 2 of development. Lower images show a lateral slice of the WT disc shown. (H) Immunofluorescence staining for ISL1 and NANOG in iDiscoid at day 4 after application of Noggin at Day 2 of development. Lower images show a lateral slice of the WT disc shown. (I) Scatterplot showing ISL1 expression (D4) intensities within WT clusters; control (Ctrl) iDiscoid versus BMP4 inhibition (Noggin) conditions. n[control] = 87, n[Noggin] = 134, **** p<0.0001 Scale bars = 100 μm. n represents separate biological samples from a single experiment harvested on the corresponding day.

Figure 3:. Anterior hypoblast-like domain and posterior axis in iDiscoids

(A) Merged UMAP of all iGATA6 lineages showing the compartments expressing markers of anterior hypoblast. Two separate domains of these markers were observed, one within day 2 iDiscoid cells and one within day 3-5 iDiscoid cells. (UMAP labeled by day can be seen in Supplementary Figure 5) (B) Merged UMAP of all WT lineages showing the compartment expressing markers of the posterior axis and primitive streak. (C) Immunofluorescence staining showing a z-slice of an iDiscoid with a HHEX/LHX1/CER1 co-positive domain adjacent to a WT clusters. Dotted line indicates boundaries of the WT cluster in each image, arrow indicates co-positive domain. (D) Immunofluorescence staining showing a TBXT/MIXL1 co-positive domain within the WT clusters of the iDiscoid. (E) Examples of WT clusters with polarized TBXT/MIXL1 domains (top) or without co-expression of markers (bottom). Venn diagram shows proportions of cluster types observed in iDiscoid cultures, n = 746 total. (F) Venn diagram showing the proportion of clusters with a particular TBXT/CER1 polarity type when CER1 confined to one pole is present. n = 169 total. (G) Representative WT clusters showing TBXT and CER1 expression domains at opposing poles of the cluster (anti-polar, top) or at the same pole of the cluster (syn-polar, bottom). (H) Diagrams indicating the average radial expression patterns of TBXT in each polarity pattern from WT clusters with a polarized CER1-expressing domain indicated in (F). All diagrams are scaled to the same expression intensity value (1 a.u.). Degrees indicate radial distance around the circularized perimeter of a WT cluster from the CER1 peak shown. Shaded areas indicate region of highest average TBXT polarity corresponding to the polarity types. Scale bars = 100 μm. n represents separate biological samples from a 3-4 separate experiments harvested on day 4 after iDiscoid induction.

Figure 4:. iDiscoid hematopoietic lineages and blood island-like structures

(A) Heatmap showing day 5 iDiscoid scRNA-seq populations compared to hematopoietic populations from the human E16-19 embryo. (B) Dot plot showing the expression pattern of hematopoietic markers in day 5 iDiscoid scRNA-seq populations. (C) Immunofluorescence image showing the distribution of cells expressing CD34 and TAL1 (scl) in iDiscoid culture. Cells expressing TAL1 localize between the yolk sac endoderm compartment and the tissue culture dish and form arrangements of spindle-shaped cells. Orthogonal slice shows the position of spindle cells against the dish. Dashed line indicates the position from which the slice was taken. (D) Live phase image taken at day 12 showing cells spherical cells generated within iDiscoid tissue. Dotted box indicates area of inset. (E) Immunofluorescence image of day 12 iDiscoid showing the generation of CD43⁺ spherical cells within CD31⁺ endothelial cells. (F) Immunofluorescence image of a day 12 iDiscoid with 25% GATA6-high cells substituted into the normal iDiscoid ratio. (G) Histogram showing the number of CD43⁺ cells detected in experiments in which the given percentages of GATA6-hi cells were substituted. n[0%] = 4 experimental replicates, n[10%] = 2 experimental replicates, n[25%] = 2 experimental replicates. (H) Two slices from a single blood island-like structure demonstrating Desmin⁺ mesoderm cells and CD34⁺ endothelial cells localized underneath a FOXA2⁺ endoderm. (I) Histogram showing the z-distribution of the indicated markers between the bottom of the dish and the top of the culture. Bimodal distribution of FOXA2 indicates areas of expression outside of blood island-like foci. Distributions are averaged from 9 foci from 3 technical replicates. (J) 3D reconstruction of one blood island area showing positioning of endoderm (FOXA2), endothelial (CD34), and mesoderm (Desmin) markers (K) Schematic depicting in vivo embryonic yolk sac blood islands and in vitro iDiscoid yolk sac blood island-like structures. Scale bars = 100 μm.