Highly penetrant amniogenesis in a Glass-3D+BMP culture system.

(A,B) hPSC-derived pluripotent cyst (hPSC-pluripotent cyst, Glass-3D in (A)) and amnion cyst (Glass-3D+BMP in (B)) assays: IF images show hPSC-pluripotent (A) and -amnion (B) cysts stained for PODXL (lumen, magenta) and DNA (nuclear shape, blue). BMP4 treatment (20ng/mL) leads to uniformly squamous amnion cysts by Day 4. (C) Confocal optical sections of d2 cysts harvested prior to (0-min) and 10-, 20-, 30- and 60-min after BMP4 treatment, stained with indicated markers. Staining for phosphorylated SMAD1/5 reveals a prominent nuclear pSMAD1/5 enrichment within 10-min of BMP4 treatment in all cells. (D) Quantification of nuclear pSMAD1/5: five independent samples were counted, more than 50 cells were counted per sample. * indicates statistically significant changes (p<0.05) compared to 0-min timepoint. (E) Abundant GATA3 expression (in green), but not TFAP2A (red), is seen by 3 hours after BMP4 treatment while SOX2 expression is maintained. (F-H) Pluripotency (NANOG, SOX2) and amnion (ISL1/2, GATA3, TFAP2A) marker expression analyses of d4 cysts grown in the Glass-3D (top) or in Glass-3D+BMP (bottom) conditions. (I,J) Confocal micrographs of d4 cysts grown in the Glass-3D+BMP system stained for phosphorylated EZRIN, RADIXIN and MOESIN (pERM), an apical membrane marker, as well as with indicated amnion markers (top – cysts were treated with BMP4 for 24 hours between d2 and d3; bottom – treated with BMP4 for 48 hours from d2 to d4). (K,L) Quantification of GATA3 and ISL1/2 fluorescent intensities in 24- (blue, n = 115 cells) and 48-hr (red, n = 76 cells) BMP4 treatment samples (K), as well as of nuclear aspect ratios of d4 hPSC-cyst (black, n = 63 cells), and 24- (blue, n = 45 cells) and 48-hr (red, n = 52) BMP4-treated cysts (harvested at d4), (L). (M) pSMAD1/5 staining (green) shows similar nuclear enrichment between 24- and 48-hr BMP4 treatment at d4 (also stained with indicated markers).

Scale bars = 50μm.

Time-course analysis of Glass-3D+BMP hPSC-amnion development.

(A) Timeline of the Glass-3D+BMP hPSC-amnion assay. Twenty four-hr BMP treatment is sufficient to generate uniformly squamous hPSC-amnion cysts. (B,C) Optical sections of time-course Glass-3D+BMP4 hPSC-amnion cysts (harvested at indicated timepoints after BMP4 treatment) stained with indicated markers (inverted black and white fluorescent signals shown for individual channels, to aid visualization). GATA3, an immediate BMP target is seen by 3-hr (yellow arrowheads). hPSC-amnion cysts with prominent nuclear TFAP2A (B) and ISL1/2 (C) are seen by 12- and 24-hr, respectively (yellow arrowheads), while SOX2 shows gradual decrease. Consistent changes are also observed at mRNA transcript levels using quantitative RT-PCR (D-F, also see the expression of additional markers: HAND1 (amniotic) and NANOG (pluripotent); *indicates statistically significant changes (p<0.05) compared to 0-hr timepoint). (G) Relative mRNA expression analyses of indicated pluripotency (top) and amniotic (bottom) markers between d4 Glass-3D hPSC-pluripotent cyst (black), and d4 Glass-3D+BMP hPSC-amnion with 24-hr (blue) or 48-hr (red) BMP treatment; * indicates statistically significant changes (p<0.05). (H) BMP inhibitor (LDN-193189) treatment outline as well as confocal micrographs of d4 Glass-3D+BMP hPSC-amnion (top, control – 24-hr BMP4 treatment between d2 and d3) and hPSC-amnion treated with LDN-193189 between d3 and d4 after BMP4 treatment (between d2-d3). Scale bars = 50μm.

Temporally-resolved transcriptomic characterization of Glass-3D+BMP hPSC-amnion.

(A) Experimental timeline (from d2) of the time-course bulk RNA sequencing analysis. Colored arrowheads indicate timepoints at which samples were harvested. (B) A heatmap showing a hierarchical clustering analysis of all detected genes (12,940): bulk transcriptomes from triplicate samples are shown at each timepoint. A gradient scale indicates Z-score. (C) Principal component analysis (PCA) plot of time-course bulk transcriptomes, using the most variable genes, revealing progressive transcriptomic changes overtime (indicated by the dotted arrow). Note that coloring is consistent with the arrowheads in (A). (D-G) Time-course plots of normalized expression values for selected known immediate BMP target (D), general amnion (E), advanced amnion (F) and pluripotency genes (G). (H) A volcano plot analysis showing up-(red) and down- (blue) regulated genes in Glass-3D+BMP hPSC-amnion cysts (gene expression compared to d4 pluripotent hPSC-cysts): black lines represent cutoffs for p-value <0.05 and 2-fold difference.

Transcriptome comparison of developing Glass-3D+BMP hPSC-amnion cysts, a Carnegie stage (CS) 7 human embryo, and two GD14 cynomolgus macaque peri-gastrula.

(A) A UMAP plot displaying the original single cell transcriptome coordinates of the CS7 human embryo described in Tyser et al., shown with the original annotations. The inset (i) is used for expression analyses in (B). (B) Expression of amnion markers – GATA3, TFAP2A and ISL1 – superimposed onto the UMAP plot in (A – i). (C) Transcriptome similarity analysis comparing the CS7 human embryo single cell dataset and the Glass-3D+BMP time-course bulk RNA-seq dataset, showing the human CS7 embryo UMAP plot (inset) with colored cells that are transcriptomically similar to each timepoints as indicated (orange – pluripotent cysts/monolayer and 0-, 0.5-, 1-, 3- and 6-hr post-BMP4; green – 12-hr post-BMP4; blue – 24-hr; magenta – 48-hr). While pluripotent and earlier timepoints (orange colored cells) overlaps with Epiblast cells, some 12-hr (green) and all 24-hr (blue) samples are similar to a subpopulation of Primitive Streak cells, and 48-hr (magenta) samples overlap exclusively with Ectoderm (see Materials and Methods for detailed bioinformatics pipeline). (D) TBXT expression transposed onto the CS7 human embryo UMAP plot, revealing TBXTlow sub-population that show transcriptomic similarities to 24-hr post-BMP samples (indicated by dotted circle). (E) An integrated UMAP plot for two in vitro cultured gestation date (GD) 14 (8 days after culturing 6-day old blastocyst) cynomolgus macaque peri-gastrula (replotted and reclustered from Yang et al.). (F) Transcriptome similarity analysis comparing the GD14 cynomolgus macaque peri-gastrula dataset and the Glass-3D+BMP time-course dataset, showing the Yang et al. GD14 UMAP plot (inset “i”) with colored cells that are transcriptomically similar to each timepoints as indicated (orange – pluripotent cysts/monolayer and 0-, 0.5-, 1-, 3-, 6- and 12-hr post-BMP4; blue – 24-hr; magenta – 48-hr). While pluripotent and early datapoints (orange colored cells) overlap with epiblast cells, the 24-hr samples share similarities with TBXTlow population, and the 48-hr samples overlap with ISL1high amnion cells ((G), expression analysis of indicated markers). Gradient scales indicate expression level (orange = high, gray = low). Uncropped plots are found in Fig. S2.

Transcriptional characterizations of amnion-epiblast squamocolumnar boundary of the CS6/7 cynomolgus monkey amniotic sac.

(A) RNA velocity analysis (scvelo) of the Tyser et al. CS7 human embryo dataset, showing predicted lineage progression trajectories from Epiblast to Ectoderm traversing the TBXTlow population. (B-G) Optical sections of a CS6/7 cynomolgus macaque embryo stained for indicated transcription factors (GATA3, ISL1/2, pSMAD1/5 and SOX2 (B,D,F)), as well as quantitation for relative fluorescent intensity in proximal (closer to epiblast) to distal (closer to amnion) cells of indicated markers at the amnion-epiblast boundary (C, E and G are quantitation of images in B, D and F, respectively). Each dot represents a single cell. (i), (ii) and (iii) indicate insets in (F). Half of the amniotic sac shown. Quantitation shows that, while GATA3+/TFAP2A+ cells are also SOX2+, ISL1+ cells are only seen after SOX2 expression is no longer detected: ISL1 expression is exclusive to squamous amnion cells. Moreover, the expression of pSMAD1/5 and TFAP2A is reduced in most distal amnion (ii, iii). Scale bars 200μm. (H) Nuclear aspect ratio quantitation of amnion-epiblast boundary cells in (B), (C) and (D), mean+SEM shown. Dotted vertical line in each plot indicates amnion-epiblast boundary (C,E,G,H). (I) A schematic representation of the CS6/7 cynomolgus macaque amniotic sac: the confocal micrographs in (B) were edited to represent SOX2+ epiblast (pseudocolored in blue), primitive streak-derived disseminating (green), GATA3+/TFAP2A+/SOX2low transition boundary (magenta) and GATA3+/TFAP2A+/ISL1+ lineage specified amnion (red) cells.

Characterization of differentially expressed genes identified through the Glass-3D+BMP hPSC-amnion time-course sequencing analysis.

(A-E) Combined and selected genes are shown for each category (A – immediate; B – early; C – intermediate-1; D – intermediate-2; E – late). Genes for each category are selected based on significant upregulation (FDR<0.05, fold>2, based on Z-scores) by 1- (immediate, n = 5), 3- (early, n = 9), 6- to 12- (intermediate-1, n = 30), 24- (intermediate-2, n = 178) and 48-hr (late, n = 157) post-BMP4 treatment, compared to the previous timepoint. Vertical lines indicate timepoints at which significant changes are seen.

Establishment of DLX5 as a new marker of amniogenesis similar to TFAP2A.

(A,B) Expression analyses for ISL1 (A) and DLX5 (B) in the time-course bulk RNA sequencing (top) and in the single cell CS7 human embryo (bottom) datasets. (C) Confocal optical sections of Glass-3D+BMP4 hPSC-amnion cysts (at 0-hr, 24-hr (d3) and d4) stained with indicated markers. (D) Histological section of a cynomolgus monkey amniotic sac at CS7 stained with indicated markers (SOX2+ epiblast; TFAP2A+/DLX5+/SOX2- amnion). Insets (i and ii) indicate the amnion-epiblast boundaries. DLX5 and TFAP2A share a similar expression pattern at the boundary. Similar to pSMAD1/5 and TFAP2A (shown in Fig. 5F), most distal amnion cells show reduced DLX5 expression. Surface ectoderm bridging neural ectoderm and amniotic ectoderm is not seen at this stage. Amniotic mesenchyme is observed underlying the TFAP2A+/DLX5+ amniotic epithelium at this stage. Scale bars = 500μm.

Analyses of Glass-3D+BMP hPSC-amnion in control and TFAP2A-KO background.

(A,B) Optical sections of control and TFAP2A-KO cysts in Glass-3D (A, pluripotent) as well as in Glass-3D+BMP (B). While control-like pluripotent cysts are formed in Glass-3D, several NANOG+ cells are still seen in Glass-3D+BMP in the absence of TFAP2A. (C) Confocal micrographs of d4 control, TFAP2A-KO and TFAP2A-KO carrying a DOX-inducible human TFAP2A transgenic construct with or without DOX treatment, stained with indicated markers. (D) Transcriptome similarity analysis comparing the CS7 human embryo single cell dataset with the time-course (0-, 12-, 24- and 48-hr post-BMP) bulk dataset from control and TFAP2A-KO Glass-3D+BMP hPSC-amnion. Orange, green, blue and magenta colored cells are transcriptomically similar to 0-, 12-, 24- and 48-hr post-BMP bulk RNA sequencing samples, respectively. While a clear differentiation trajectory from Epiblast to Ectoderm is seen in control (left), a halted amnion lineage progression is seen in the TFAP2A-KO background. (E) Optical sections of control and TFAP2A-KO cysts in Glass-3D+BMP stained for NANOG (green), ISL1/2 (red) and TBXT (magenta). Scale bars = 50μm.