The emergence of human gastrulation upon in vitro attachment

Abstract

While studied extensively in model systems, human gastrulation remains obscure. The scarcity of fetal biological material as well as ethical considerations limit our understanding of this process. In vitro attachment of natural blastocysts shed light on aspects of the second week of human development in the absence of the morphological manifestation of gastrulation. Stem cell-derived blastocyst models, blastoids, provide the opportunity to reconstitute pre- to post-implantation development in vitro. Here we show that upon in vitro attachment, human blastoids self-organize a BRA⁺ population and undergo gastrulation. Single-cell RNA sequencing of these models replicates the transcriptomic signature of the human gastrula. Analysis of developmental timing reveals that in both blastoid models and natural human embryos, the onset of gastrulation as defined by molecular markers, can be traced to timescales equivalent to 12 days post fertilization. In all, natural human embryos and blastoid models self-organize primitive streak and mesoderm derivatives upon in vitro attachment.

Keywords: blastoids; gastrulation; human embryo; in vitro attachment; scRNA-seq.

Graphical abstract

Figure 1

In vitro attached human blastoids self-organize primitive streak and mesoderm populations (A) Blastoid immunostaining displays NANOG (yellow), GATA4 (gray), and GATA3 (blue) marker genes distinguishing epiblast, primitive endoderm, and trophectoderm before in vitro attachment. Scale bar, 100 μm. (B) In vitro attached human blastoids analyzed by immunostaining show OCT4⁺/BRA⁺ cells at 7 days post attachment (dpa). OCT4⁺ cells label the epiblast territory, GATA6⁺ the primitive endoderm, and BRA⁺ the primitive streak and mesoderm populations. Immunostaining pseudocolors: OCT4 (green), BRA (red), GATA6 (magenta), and merge with DAPI (gray). Scale bar, 100 μm. (C) Immunostaining at 10 dpa displays OCT4⁺-epiblast, BRA⁺-primitive streak and mesoderm, and GATA6⁺-endoderm cells. Immunostaining pseudocolors: OCT4 (green), BRA (red), GATA6 (magenta), and merge with DAPI (gray). Scale bar, 100 μm. (D) Scaled area quantification of OCT4⁺ and BRA⁺ cells at 7 and 10 dpa. Data are displayed as a box plot (center line displays the median, and box limits are the 25^th and 75^th percentiles) (n = 51 from three independent differentiations). Student’s t test, unpaired with two tails. ^∗∗∗p < 0.001. (E) Pie chart of in vitro attached blastoids containing at least five BRA-positive cells at 7 and 10 dpa. (F) Area quantification is displayed as a scatterplot. x axis reports the scaled OCT4 area, while the y axis is the BRA scaled area. Each dot represents an individual attached blastoid that has been color coded according to the time of analysis. Gray dots represent blastoids at 7 dpa and blue dots blastoids at 10 dpa.

Figure 2

Single-cell transcriptomics of in vitro attached human blastoids distinguish embryonic and extraembryonic populations (A) Heatmap displays Z-score scaled expression values for marker genes that distinguish extraembryonic (GATA3, GATA2, HSD3B1, KRT7), epiblast/ectoderm (POU5F1, NANOG, DPP5, UTF1), mesoderm (ZEB2, VIM, HAND1, CDH11), and endoderm (FOXA2, SOX17, HNF1B, HNF4A) populations at 7 and 10 dpa (7 dpa, 7,354 cells; 10 dpa, 5,489 cells). (B) UMAP plot shows the identified cell populations: cytotrophoblast, syncytiotrophoblast, amnion, epiblast, ectoderm, primordial germ cells, primitive streak 1–2, mesoderm, extraembryonic mesoderm, endothelial, definitive endoderm, hypoblast, and yolk sac endoderm. (C) Heatmap of average scaled expression displaying marker genes that distinguish individual cell populations. (D) Immunostaining of 10-dpa human blastoids. OCT4 (green) and DAPI (gray) are used to identify blastoids. MIXL1 (yellow), HAND1 (cyan), GATA4 (magenta), FOXA2 (hot cyan), and SOX17 (blue) identify mesoderm and endoderm populations. Scale bar, 100 μm.

Figure 3

In vitro attached human blastoids recapitulate the cellular populations of the human gastrula (A) UMAP shows the integration of 7–10 dpa blastoid scRNA-seq and the human gastrula dataset at 16–19 days post fertilization (dpf) (Tyser et al., 2021). In vitro attached blastoids are color coded according to their lineage. The human gastrula dataset preferentially integrates with the epiblast/ectoderm, mesoderm, and endoderm lineages, though not with the trophectoderm/amnion population. (B) Correlation analysis using the commonly detected highly variable genes between the human gastrula dataset (Tyser et al., 2021) and the in vitro attached human blastoid datasets displayed as a Z-score heatmap. The clusters from the human gastrula dataset are shown with their original annotations followed by an “in vivo Tyser et al.” label and highlighted in light purple. (C) Hierarchical clustering is displayed as a dendrogram. The human gastrula (Tyser et al., 2021) labels are highlighted in light purple.

Figure 4

In vitro attached human embryos self-organize a BRA-positive axial population at 12 dpf (A) Immunostaining of the in vitro attached human embryo at 12 dpf. A low-magnification field of view shows DAPI (cyan), GATA6 (magenta), OCT4 (green), and BRA (red) signals, all overlaid on the F-ACTIN (gray) staining. OCT4 localizes the epiblast territory and GATA6 the primitive endoderm cells. BRA is used as a primitive streak and mesoderm marker. Scale bar, 100 μm. (B) Immunostaining of the in vitro attached human embryo at 12 dpf. Zoomed-in field of view shows the merged image of OCT4 (green), BRA (red), GATA6 (magenta), and F-ACTIN (gray) signals. Scale bar, 50 μm. (C) 3D orthogonal projections of the in vitro attached human embryo at 12 dpf. Top and lateral views show the merge of OCT4 (green), BRA (red), GATA6 (magenta), and DAPI (gray). Scale bar, 50 μm.