Single nucleus sequencing is essential to capture the STB lineage in full-term tissue and TOs.

A Cartoon of placenta villous tree and cross section, with the multinucleated STB highlighted in green, progenitor CTB cells in magenta, and extravillous cytotrophoblasts in blue. B Schematic of organoid generation via isolation of trophoblast progenitor cells from full-term placental tissue. C Schematic of experimental setup. Both tissue and TOs were processed into either single cells/syncytial fragments or single nuclei and sequenced. UMAP of integrated SC and SN datasets collected from primary tissue (D) and TOs (G). Cell/nucleus types are annotated as follows: cytotrophoblast (CTB), cytotrophoblast pre-fusion (CTB-pf), syncytiotrophoblast (STB), extravillous trophoblast (EVT), dendritic stem cell (DSC), vascular endothelial cell (VEC), fibroblasts (Fib), natural killer cell (NK), and macrophage (MC). Cell/nucleus types that contained multiple clusters are identified with -number after the name. E and H UMAP of the integrated SC and SN dataset separated into individual UMAPs by single cell or nucleus processing type for primary tissue (E) and TOs (H). F and I Expression of key trophoblast markers, including PAGE4 (CTB), CYP19A1 (STB), and HLA-G (EVT) in primary tissue (F) and TOs (I).

Comparison of TO gene expression in different culture conditions by SN RNA sequencing.

TOs were grown with the STB facing inward (STBin), outward (STBout), or induced to differentiate into EVT (EVTenrich). Schematic of TO nucleus type composition (left), UMAP of the individual SN datasets (middle), and barplot showing the proportions of each nucleus type (right) are shown for STBin (A), STBout (B), or EVTenrich (C). UMAP of the integrated datasets (left), integrated dataset split by culture condition (middle), and barplot showing the proportions of each nucleus type (right) are shown for the STBin+STBout integrated dataset (D) or STBin+EVTenrich dataset (E). Prolif in E refers to proliferative subtype encapsulating both EVT-p and CTB-p.

STB subtype analysis and expression differences in STBin vs STBout TOs.

A Proportion of each STB subtype in the integrated STBin+STBout dataset. B Top differentially expressed genes (DEGs) of each STB subtype is shown as a dot plot. Mean expression is shown as a color scale and percent of nuclei expressing each gene is demonstrated by size of the dot. C Top Biological Process GO terms associated with DEGs in each STB subtype was determined with clusterProfiler. D Feature Plots of PAPPA2 in STBin and STBout TOs. E Feature Plots of ADAMTS6 in STBin and STBout TOs. F RNA FISH of PAPPA2 in STBin and STBout TOs. G RNA FISH of ADAMTS6 in STBin and STBout TOs. H DEseq2 was used to find DEGs between STBin and STBout datasets in STB-1, STB-2, and STB-3 clusters and plotted as a volcano plot. GO terms associated with DEGs in either STBin or STBout were found with clusterProfiler and colored on the UMAP with select representative genes highlighted with text. I Feature Plot of representative target genes from E.

Trajectory and gene regulatory network analysis reveals transcription factors and chromatin remodelers associated with STB differentiation.

A Pseudotime was performed with Slingshot and assigned pseudotime value for each nucleus type is overlayed as a color scale on the UMAP from time 0 (blue) to time 100 (red). Arrow demonstrates directionality of the trajectory. Top genes associated with pseudotime (B) or enriched in STBout compared to STBin (C) are plotted on a barplot with nuclei ordered with the pseudotime seen in A. Logcounts and pseudotime are demonstrated as a color scale bar and the identity of each nucleus is labeled with a color as indicated in the legend. D TF/CR and TG pairs were identified via Velorama and plotted as a similarity matrix to show the overlap in TGs for each TF/CR. Each module discussed in the text is labeled as 1, 2, or 3 and colored with blue, orange, or pink respectively. E Network graphs of TF/CRs and their respective TGs were plotted with igraph. Each TF/CR and respective arrows were false colored as per the module labels in D. Grey circles represent the target genes. Target genes that are shared among all three sample types are bolded. The width of the arrow from each TF/CR to each TG represents the interaction strength score determined with Velorama and the color of the arrow represents the module from which the interaction arises.

RYBP is a marker of STB and its deletion downregulates STB-2 marker genes.

A Immunofluorescence of full-term tissue was performed with E-cadherin (ECAD), cytokeratin (CYTO), and RYBP and stained with DAPI to label nuclei. Immunofluorescence of STBin TOs (B) or STBout TOs (C) was performed with chorionic gonadotropin (CGBs) to label STB, RYBP, and E-cadherin (ECAD) to mark cell boundaries. TOs were further stained with DAPI to label nuclei. Green stars represent CTB cells, as marked by E-cadherin, and are negative for RYBP. D Immunofluorescence of WT or RYBP-/- STBin TOs performed with RYBP and CGBs and stained with DAPI and Actin. E Bulk RNA sequencing of WT or RYBP(-/-) STBin TOs was performed and analyzed with DESeq2 to determine differential gene expression. Dotted line represents a log2 fold change greater than 1. Genes enriched in the STB-2 subtype are labeled in green.

Similarities and differences between STB in first trimester and full-term tissue and TOs.

A UMAP of the integrated SN dataset including first trimester, full-term tissue, STBin TOs, and STBout TOs. B Relative proportion of each nucleus type for each sample in the integrated dataset in A. C UMAP of the integrated dataset split by sample type. The five STB subtypes were merged into a single STB subtype while the two non-proliferative CTB subtypes were merged. D Dot plot of key trophoblast marker genes in the STB lineage with heatmap representing mean gene expression and size of the dot representing the percent of nuclei expressing each marker. The labels on the top of the graph refer to the established marker genes used to identify the identity of each cluster. E The top DEG in the STB of each sample type were analyzed for biological process GO term enrichment with clusterProfiler and plotted as a dotplot. F DEseq2 was used to find DEGs between different sample types for the merged STB cluster. GO terms for DEGs enriched in each sample type were found with ClusterProfiler and colored on the UMAP with select representative genes highlighted in text. G Feature plots demonstrating RNA expression of selected genes.