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Human archetypal pluripotent stem cells differentiate into trophoblast stem cells via endogenous BMP5/7 induction without transitioning through naive state – Scientific Reports

Derivation of proliferative TSCs from primed human PSCs without exogenous BMP4

While previously described primed hiPSC differentiations to trophoblast have included BMP4 activation, evidence has shown that hiPSCs can differentiate to trophoblast in the absence of exogenous BMP418. We first asked whether primed human embryonic stem cells (hESCs) and hiPSCs can be differentiated to TSCs in the absence of exogenous BMP4 (Fig. 1a, Supplemental Fig. 1). hESC line H119 colonies demonstrated tight packing with defined edges characteristic of primed cells, 24 h after passaging in feeder-free primed conditions. Once self-organized colonies were established, cells were switched directly to the differentiation medium which activates WNT and EGF while inhibits TGFβ, HDAC and ROCK (TS condition). By brightfield imaging over the subsequent six days, we observed that cells proliferated rapidly and adopted a flatter appearance with some cells adopting a cobblestone appearance (Fig. 1b). After subsequent passaging, two morphologically distinct populations emerged. Circular colonies with an epithelial-like appearance were surrounded by phase bright fibroblastic cells (Fig. 1c). The inner epithelial-like cells continued to proliferate and appeared morphologically similar to TSCs derived from human villus cytotrophoblasts (primary TSCs)3 (Fig. 1d,d′,e). Immunostaining revealed that subpopulations of the inner cells expressed TP63 (a CTB marker) and/or KRT7 (a pan-trophoblast marker) and minimally expressed VIM (a stromal marker), while the surrounding fibroblastic cells strongly expressed KRT7 and VIM (Fig. 1f). TSCs were passaged up to 32 times (Fig. 1g) by maintaining the expression of KRT7 and TP63. (Fig. 1h). These TSCs did not express the pluripotency markers, SOX2 and NANOG (Supplemental Fig. 1c and d). The majority of cells at passage 30 highly expressed CD49f./ITGA6 (89.3%), a common cell surface marker for CTBs and human TSCs3 (Fig. 1i).

Figure 1
figure 1

Derivation of trophoblast stem cells (TSCs) from human pluripotent stem cells (hPSCs). (a) Schematic overview of TSC derivation from hPSC. (b) Representative brightfield images of differentiating TSCs from hESC H1. Days after addition of TS media are indicated. (c) Images of TSCs derived from hESC H1 at 24- and 72-h following passage 1 (P1) on day 6. (d) Images of mature TSCs derived from hESC H1 after ten passages. Magnified image is shown as d′. (e) Image of primary TSCs (CT29)3. (f) Representative images of immunofluorescence staining of TSCs after one passage for VIM, KRT7 and TP63. Nuclei were stained with Hoechst 33342 (blue). TSCs were derived from hiPSC (LIBD7c6). (g) Schematic representation of the protocol for TSC derivation from hPSC. (h) Representative bright and immunofluorescent images for TSCs derived from hPSC lines. Nuclei were stained with Hoechst 33342 (blue). (i) Flow cytometry data showing the expression of CD49f/ITGA6 in mature TSCs at passage 30 derived from hESC H1. Scale bars, 100 mm (be,h: brightfield images); 50 mm (f,h: immunofluorescent images).

Given that pluripotent stem cell lines vary in their propensity to differentiate to different cell types20, we also asked whether TS condition could specify a variety of primed hPSCs from different sources to TSCs. We confirmed TSC specification of a hiPSC line reprogrammed from dermal fibroblasts by Sendai virus, named 2014.06 (Supplemental Fig. 2a–c), and two hiPSC lines derived from postmortem dura fibroblasts by reprogramming with episomal vectors, LIBD1c8 and LIBD7c621 (Supplemental Fig. 1a and b; Supplemental Fig. 2), by immunostaining with VIM, KRT7 and TP63 (Supplemental Fig. 1c and d; Fig. 1f and h).

In summary, our data suggest that primed hPSCs can differentiated to proliferative TSCs in the absence of exogenous BMP4.

Differentiation of TSCs into multinucleated STBs and EVTs

Proliferative CTBs are bipotential stem cells with the capacity to differentiate into STBs and EVTs3. To investigate the differentiation potential of TSCs derived from primed hPSCs, we induced differentiation of TSCs to differentiate into mature trophoblast cell types, EVTs and STBs.

EVT differentiation of the hESC H1-derived TSCs (Fig. 2a) was induced with the TGFβ inhibitor, A83, NRG1α and matrigel, as previously described for differentiation of primary TSCs into EVTs3. After 9 days in EVT differentiation, TSCs acquired EVT morphology (Fig. 2b), and 88% of cells were positive for the EVT specific marker, HLA-G (Fig. 2c and d), thereby resembling previously described EVTs derived from primary TSCs3.

Figure 2
figure 2

hPSC-derived TSCs differentiate into syncytiotrophoblasts (STBs) and extravillous cytotorophoblasts (EVTs). (a) Representative image of TSCs derived from hESC H1. (b) Representative image of EVTs differentiated from hESC H1-derived TSCs. (c) Immunofluorescence image of EVTs showing the expression of HLA-G. Nuclei were stained with Hoechst 33342. (d) Proportion of EVTs positive or negative for HLA-G after 6 days of differentiation (n = 300 cells, 12 ROI). (e) Representative image of STBs derived from hESC H1-derived TSCs. (f) Fusion efficiency of STBs and EVTs derived from hESC H1-derived TSCs. Ten ROIs for both STBs and EVTs were analyzed; n = 30–50 nuclei per ROI. Data is presented by mean ± SEM. ****p < 0.0001 (Mann–Whitney test). (g–j) Immunofluorescence images of STBs showing the expression of hCGb (h) and SDC1 (i). Scale bars, 200 mm (a); 100 mm (b,c,e,gj).

To confirm the differentiation potential into STBs, we induced the differentiation of hESC H1-derived TSCs to STBs with Forskolin for 3 days as previously described3. STBs are a terminally differentiated multinucleated epithelial layer that infiltrates the maternal endometrium. The multinucleated cell forms from multiple CTB cell fusions. We observed cells exhibiting the STB morphology of multinucleation (Fig. 2e). The fusion index revealed that more than 80% of the cells were multinucleated (Fig. 2f), similar to STBs derived from human primary TSCs3 and TSCs derived from naive hPSCs22. These multinucleated cells expressed STB specific markers, human chorionic gonadotropin beta (hCGβ) and Syndecan-1 (SDC1) (Fig. 2g–j), which were not detected in hiPSCs (Supplemental Fig. 1a). Taken together, these findings indicate that primed hPSC-derived TSCs are bipotential stem cells capable of efficient differentiation into both HLA-G-positive EVTs and hCGβ- and SDC1-positive STBs.

scRNA-seq reveals unique transcriptional programs for primed hiPSCs specification to TSCs in the absence of BMP4

To define the molecular events involved in the specification of primed hiPSCs to TSCs, we performed temporal scRNA-seq analysis of hiPSCs that were specified into TSCs using the previously described TS condition. Due to previous reports of BMP4 and WNT involvement in this process, we compared specification with TS condition to BMP4 Alone (BA) condition14 and BMP4 + IWP2 (BI) condition17. Twenty-four hours after passaging of hiPSCs, the medium was changed to differentiation medium for each condition (BA, BI and TS). Single cells were sequenced at the iPSC stage before differentiation (day 0); at days 4 and 6 for the BA condition; at days 2, 4 and 6 for the BI condition, and at days 2, 4, 6 and 8 for the TS condition, resulting in 10 single-cell transcriptomes (Fig. 3a). In the TS condition, day 8 is a timepoint two days after the first passage annotated as passage 1 (P1) in Fig. 3a. In the BA and BI conditions, no proliferative cells survived passaging, therefore the P1 time point was not sequenced. We sought to compare the effects of altering BMP4 and WNT, but did compare previously described iPSC specification methods. All differentiations began with hiPSCs in StemFlex media after clump passaging that gave rise to TSCs from multiple hiPSC lines (Fig. 1), a different iPSC media from previous BMP4 protocols (see Discussion). Using the highly parallel droplet based single cell sequencing method, Drop-Seq23, 9821 high-quality cells were obtained after removing cells with less than 1000 genes detected and more than 20% of mitochondrial mapping rate (Supplemental Fig. 3a–d). The average rate of mapping reads for mitochondrial genes in all cells was 3.9%, indicating good viability (Supplemental Fig. 3a). The total number of genes detected ranged between 21,804 at day 4 in the BI condition (BI D4) to 27,134 at day for in the TS condition (TS D4) (Supplemental Fig. 3b). Total number of high-quality cells ranged between 563 (TS D2) to 1572 (iPSC) (Supplemental Fig. 3c).

Figure 3
figure 3

Temporal single cell RNA sequencing (scRNA-seq) of hiPSCs differentiation into trophoblast differentiation. (a) Schematic of trophoblast differentiation conditions. hiPSCs were plated and cultivated for 24 h before addition of differentiation media. Cells were collected for scRNA-seq at indicated time points. (b) Uniform Manifold Approximation and Projection (UMAP) embedding of 9821 single cell transcriptomes from three distinct differentiation conditions with groupings based on sample identity (b) or gene expression clusters (c) calculated by k nearest neighbors using the Euclidean distance of the 30 first PCs which identifies 19 clusters. Cells from the BA condition almost exclusively formed four clusters (BA1–BA4) indicated in blue. Cells from the BI condition were found predominantly in a group of five heterogeneous clusters (BI1–BI5) indicated in purple. hiPSCs are indicated in green and cells from TS conditions were found in clusters (T1–T7) indicated in orange. (d) Proportion of the cells at the most mature state in each differentiation condition (T7, T6, BI5 and BA4, presented in c). (eh) UMAP showing the normalized expression of marker genes for iPSCs (SOX2, e), placental stromal cells (PITX2, f), trophoblasts (KRT7, g and CGA, h). (i) Dot plot showing genes upregulated in iPSCs and the most mature cells in each differentiation condition (T7, T6, BI5 and BA4), compared to all other cell clusters presented in (c). Non-parametric Wilcoxon rank sum test (adj.p-value < 0.05; log2FC > 0.25). Average normalized expression levels are indicated.

We performed dimensionality reduction for the most variable genes across all cells with Principal Component Analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) embedding. Single cells are separated by differentiation stage and condition, with UMAP dimension 2 generally capturing the differentiation condition (Fig. 3b). To identify transcriptionally similar groups of cells (hereafter referred to as clusters), we performed a graph-based clustering analysis of k nearest neighbors using the Euclidean distance of the 30 first PCs in Seurat24. As a result, we identified 19 clusters (Fig. 3c). The clusters contained mostly a single time-point and differentiation condition (Fig. 3d; Table S1). The iPSC clusters (iPSC1-3) are primarily composed of iPSCs with minor contributions from the initial days of all three differentiation conditions. Cells from the BA condition almost exclusively formed four clusters (BA1-4). BA1 and BA3 were predominantly composed of cells at day 4 of the differentiation (BA D4), while BA2 and BA4 were predominantly from cells at day 6 (BA D6). Cells from the BI condition were found predominantly in a group of five heterogeneous clusters (BI1-5). BI2 and BI3 clusters were predominantly derived from cells at day 2 of the differentiation (BI D2), while cells at day 4 (BI D4) contributed to BI4 cluster, and cells at day 6 (BI D6) contributed to BI5 cluster. BI1 cluster was mixture of cells in the BI condition at different time points, accompanied by a smaller population from the BA condition. Furthermore, within cluster BI5, there was a minor presence of cells derived from the last day of differentiation under the TS condition (TS P1), indicating the ability of cells in this cluster to differentiate into TSCs. Cells from the TS condition almost exclusively populated 7 clusters (T1–T7). T1 cluster was predominantly composed of cells at day 2 (TS D2) and T2-5 clusters were composed of a mixture of cells at days 2–6 (TS D2, TS D4 and TS D6). The cells from the TS P1 condition separated into two clusters, T6 and T7, providing further support for the brightfield observations indicating the presence of two distinct cell populations (Fig. 1c and f). Small portion of cells at day 6 from the BI condition (BI D6) and the BA condition (BA D6) also contributed to T7 cluster (Fig. 3d; Table S1).

To understand transcriptional changes during the differentiation, we asked which cells express canonical pluripotent, trophoblast and mesoderm specific genes. The PSC marker SOX2 was highly expressed in the iPSC stage and was absent from the most differentiated clusters in all conditions (BA4, BI5, T6 and T7) (Fig. 3e). SOX2 expression was maintained at a higher level in the initial days of the TS condition compared to the BI and BA conditions. PITX2, identified as a marker gene for a stromal-fetal communicating cell type in scRNA-seq of the human placenta25,26, exhibited elevated expression during the later stages of cells under the BA condition (BA2 and BA4). This indicates that the BA condition generates cells with mesenchymal lineage (Fig. 3f). KRT7 and CGA are recognized for their expression in both stem cell-derived CTBs and placental tissue in vivo27. We observed the expression of these genes in cells at the later stages of the BI condition (BI4 and BI5) and the TS condition (T6 and T7), indicating a specification towards trophoblast lineage (Fig. 3g and h; Supplemental Fig. 4a and b). Furthermore, cells in both the TS and BI conditions exhibited an overall increase in the expression of trophoblast markers, such as GATA3 and TFAP2C (Supplemental Fig. 4c). Conversely, the BA condition displayed a specification towards mesodermal lineage, characterized by the expression of GATA4, TBXT and PDGFRA (Supplemental Fig. 4d). In the mouse, Cdx2 is involved in the segregation of the inner cell mass and trophoblast lineages at the blastocyst stage by repressing Pou5f1/Oct-4 and Nanog in the trophoblast. Overexpression of Cdx2 in mouse ESCs causes differentiation to TSCs28,29. In humans, CDX2 expression is initiated after blastocyst formation and has variable expression patterns in trophoblast30. Similar to human embryo and primary TSCs3, we observed a transient expression of CDX2 during the intermediate stages of specification (day2–4) for all conditions and is not expressed in the most differentiated state in the TS condition (Supplemental Fig. 4e).

We next investigated differentially expressed genes across the clusters. Primed PSC marker genes, DUSP6 and THY1, are among the top genes upregulated in iPSCs. Cells in the BA4 cluster showed a significant upregulation of genes in WNT signaling pathway, such as a WNT agonist, RSPO231 and PITX2, which interacts with WNT signaling and regulates collagen expression32. Upregulated genes in the BI5 cluster cells included a serine protease involved in Snail-dependent epithelial to mesenchymal transition, PRSS2333 and insulin-like growth factor signaling IGFBP734 and PAPPA235. Cells in the T7 cluster exhibited the upregulation of genes regulating EGFR signaling, such as IFI6, and MUC1536, and the YAP downstream effector ARHGAP1837, which was recently implicated in the specification iPSCs to bipotent TSCs38 (Fig. 3i; Tables S2 and S3). To summarize, our comparative analysis of the TSC differentiation conditions reveals that the TS condition—activating WNT and EGF while inhibiting TGFβ, HDAC, and ROCK—elicits transcriptional patterns in primed hiPSCs closely resembling trophoblast cells in vivo.

Trophoblast expression signatures are enriched in hiPSC-derived TSCs

To compare the primed hiPSC-derived cell types from all differentiation condition to peri-implantation human embryos, we calculated the scaled transcriptional similarity of our scRNA-seq data to previously annotated gene signatures for epiblast, hypoblast and trophoblast from cultured human embryos39 (Fig. 4a–d). Two iPSC clusters exhibited the highest similarity to epiblast marker gene expression, with average score of 0.1 (Fig. 4a). Interestingly, a population of cells from the BA2 cluster were most similar to hypoblast tissue profiles with a similarity score of 0.3 (Fig. 4b). The T6 and T7 clusters, representing the most mature trophoblast stage, displayed a specific expression profile associated with trophoblast tissue, exhibiting a transcriptional similarity of 0.6 (Fig. 4c). Moreover, the T7 cluster exhibited the highest level of trophoblast gene expression, determined through a pairwise comparison of the average trophoblast gene expression across all clusters (Wilcoxon Rank Sum Test with Holm-middle combined p value = 3.5e−41). Additional validation of the trophoblast transcriptional similarity via gene set enrichment analysis (GSEA) demonstrated that the T7 cluster displayed the most pronounced enrichment for the expression of trophoblast-signature genes (adjusted p value = 0.001, Supplemental Fig. 5a). Collectively, the results obtained from our single-cell RNA sequencing analyses suggest that cells derived from hiPSCs, when subjected to the TS condition, develop a trophoblast identity within eight days after specification and passaging.

Figure 4
figure 4

hiPSC-derived TSCs without activating BMP4 exhibit similar gene expression patterns to trophoblasts from cultured human embryos. (ac) UMAP showing the expression similarity scores for single cells based on gene expression signatures in epiblast (a), hypoblast (b) and trophoblast (c) of human embryo39. (d) Mean expression of genes identified in amnion42 and trophoblast39 in each cell cluster. The T7 cluster shows the highest average expression of trophoblast genes (p = 3.5e−41, Wilcoxon Rank Sum Test with Holm-middle combined) and the lowest average expression of amnion genes (p = 4.8e−06), compared to all other cell clusters. (eg) Violin plots showing the expression of amnion markers POSTN (e), IGFBP5 (f) and ITGB6 (g) in each cell cluster.

We further compared the specification map with in vivo amnion. Recent studies suggest that primed PSCs patterned with exogenous BMP4 give rise to amnion-like cells22,40,41. Consistent with transcriptional trophoblast identity, the T7 cluster exhibited the highest enrichment for genes identified in in vivo trophoblast39. Notably, the T7 cluster had the lowest average expression of genes expressed in in vivo amnion42 (Wilcoxon Rank Sum Test with Holm-middle combined p value = 4.8e−06) (Fig. 4d; Tables S2S6). Moreover, we did not observe a significant enrichment of genes expressed in the T7 cluster for human amnion-signature genes (adjusted p value = 1, Supplemental Fig. 5b and c). Conversely, cells in the earlier stages of all conditions exhibited expression of the amnion genes.

For example, POSTN is significantly upregulated in the BA4 cluster (Wilcoxon Rank Sum test; adjusted p value < 2.225074e−308) (Fig. 4e). IGFBP5 showed significant upregulation in both the BI and TS conditions, except the most mature clusters T6 and T7 (BI4: adjusted p value = 4.389994e−95; BI5: adjusted p value = 1.909090e−147; T3: adjusted p value = 4.301357e−148) (Fig. 4f). A recent study reported high expression of the amnion gene ITGB6 in primed hiPSC-derived TSCs40. Although clusters from the BI5 cluster showed significant upregulation of this gene (adjusted p value = 8.890e−286), it was nearly absent from all cells under the TS condition (Fig. 4g). Taken together, these findings suggest that after eight days of specification and passaging in the TS condition, primed hiPSCs become specified as trophoblasts without expressing the amnion genes.

Primed hiPSC-derived TSCs resemble first trimester placental CTBs

Next, we explored the similarity between primed hPSC-derived TSCs and cells from human early placentas. By GSEA using Cell-Specific Expression Analysis (CSEA)43, we identified genes that were preferentially expressed in each of the 19 clusters of hiPSC-derived cells (Fig. 3c), with a specificity index probability (pSI) statistic at thresholds of p < 0.05. We then tested whether cell type-specific genes previously identified by scRNA-seq of human placental25,26 are over-represented in the cell type-specific genes for the 19 clusters, by hypergeometric test and applied the Bonferroni correction for multiple comparisons, considering all the tested gene lists [α = 0.05/(19 × (38 + 14)) = 5.1 × 10–5].

The transcriptional profiles from all iPSC clusters (iPSC1-3) and clusters from the earlier timepoints (days 2 and 4) in all differentiation conditions did not show significant enrichment for any of the placental cell type clusters from first-trimester placentas26 (Supplemental Fig. 6a and b). The T6 and T7 clusters, which are composed of cell at the most mature state in the TS condition, were significantly enriched for the STB and CTB clusters from first-trimester placentas26 (Fig. 5a and Supplemental Fig. 6a). The expression profile of the T7 cluster was highly specific for STB and CTB with no significant enrichment for any other cell-type in the maternal fetal interface. To visualize placental cell-type specific expression patterns among the hiPSC-derived cell types, we generated a heatmap of the top unique markers of the fetal cell types identified in Vento-Tormo et al.26 and polar trophectoderm markers identified in Petropoulos et al.44 (Supplemental Fig. 6c). Consistent with the GESA, we observed high expression of a majority of the unique marker genes for CTBs and STBs in the T7 cluster. Conversely, we observed the significant enrichment of the BA4 cluster for two fibroblast cell types in first-trimester placentas. Vento-Tormo et al. described that these placental fibroblasts as mesenchymal stromal cells of fetal origin that derive from the primitive endoderm expressing GATA4, GATA6, PDGFRA, and SOX17 (Supplemental Fig. 7a). The BI5 cluster did not show strong enrichment for a specific placental cell type and instead had weaker enrichment for several clusters including STBs, and maternal decidua derived fibroblast endodermal cell type and decidual stromal cells (Fig. 5a and Supplemental Fig. 6a). We also compared the expression profiles of the 19 clusters to cells from first trimester (8 weeks of gestation) and second trimester (24 weeks of gestation)25 (Fig. 5b and Supplemental Fig. 6b). Consistent with the findings in Fig. 5a, we observed a strong enrichment of the T6 and T7 clusters for the fusion competent CTBs (CTB1-3). The BA and BI clusters were enriched for two different mesoderm cells (Stromal 1 and 2). In summary, TSCs derived from primed hiPSCs are highly enriched for the expression of genes specific to STBs and CTBs from human early placentas.

Figure 5
figure 5

hiPSC-derived TSCs exhibit similarity to human placental cells during the first trimester. (a,b) Gene set enrichment analysis (GSEA) of cells at the most mature state in each differentiation condition (T7, T6, BI5 and BA4) to cell types identified in first trimester placenta25,26. (c) PCA plot showing the similarity of TSCs derived from hPSCs (primed TSC) in this study to TSCs from human primary tissues (primary TSC)3,10, TSCs derived from naive hPSCs (naive TSC)10,41 and primed TSCs in the previous studies10.

Figure 6
figure 6

The process of primed hiPSC specification into TSCs begins with TFAP2A rewiring, without activating programs associated with naive hPSCs. (a) Dot plot showing the expression of marker genes for naive and primed hPSCs in cells derived from each differentiation condition across various time points. (b) Dot plot displaying the expression of genes in BMP signaling in each cell cluster. (c) Developmental trajectory from hiPSCs to cells differentiated in TS condition at day 2 (TS D2). Cell proportion along the smoothed pseudotime is shown. Nodes are labeled with numbers S0–S4. Branches are defined as the cells between 2 nodes. (d) Expression of top ranked genes showing the significant correlation with the pseudotime transition from S0 to S1 (TSC lineage).

Additionally, we compared the expression profiles of the 19 clusters of hiPSC-derived cells to lineage and marker genes from pre-implantation datasets45. We found the highest expression of trophoblast genes such as KRT18, TEAD3, GATA3 and GATA2 in the TS clusters (T6 and T7), with moderate expression in BI clusters (BI4 and BI5) (Supplemental Fig. 7a). Primitive endoderm genes such as SOX17, GATA4, GATA6 showed the highest expression in the BA clusters. When compared to primary trophoblast cells, we confirmed that the T7 cluster has the highest-level expression of CTB, TSC and EVT enriched genes (Supplemental Fig. 7b).

We then sought to evaluate the biological similarity of primed hPSC-derived TSCs, specifically those differentiated under the TS condition, and several other types of TSCs obtained from different sources. These included primary TSCs from human blastocysts and early-stage placentas3 as well as TSCs derived from both naive and primed hPSCs in the previous studies10,12,41. Principal component analysis of bulk RNA-seq data illustrated that TSCs obtained from primed hPSCs under the TS condition display a comparable transcriptional profile to TSCs from human primary tissues, as well as those derived from both naive and primed PSCs (Fig. 5c).

Altogether, TSCs obtained from primed hPSCs under the TS condition exhibit a transcriptional profile, which is highly similar to CTBs from human early placenta. These cells are transcriptionally indistinguishable from TSCs derived from human blastocysts and early-stage placentas and from both naive and primed PSCs. Our data also suggest that hPSC-derived cells differentiated under the BA condition exhibit more heterogeneous placental cells types with evidence of the amnion gene expression (Fig. 4).

Specification of primed hiPSC to TSC initiates with TFAP2A rewiring without activation of naive hPSC programs

It has been reported that primed hPSCs are restricted in their potency and unable to differentiate to TSCs, while naive PSCs readily differentiate to TSCs41. Therefore, we asked whether the primed hiPSC or cells differentiating from these cells under the BA, BI and TS condition adopt a naive stem cell program during differentiation. Across the differentiation condition, expression of marker genes of the naive PSCs46 was nearly absent (Fig. 6a). Especially, cells from any conditions did not express HORMAD1, ALPPL2, KHDC3L, TRIM60, and HYAL4 (Fig. 6a, Table S7). Less than 1% of cells from any condition showed detectable expression for six other marker genes ALPP, OLAH, LYZ, MEG8, KHDC1L, and FAM151A. ZNF729. In contrast, marker genes of primed PSCs were consistently expressed in iPSCs and often throughout the differentiation state. These findings suggest that the transition of primed hiPSCs into TSCs does not necessitate the activation of transcriptional programs typically associated with naive hPSCs.

Next we investigated the contribution of BMP signaling to the TSC differentiation processes from primed hiPSCs. We observed that only a small proportion of cells expressed BMP4 under the TS conditions, whereas its expression was notably increased in various clusters from the BA and BI conditions compared to iPSCs and cells originating from the TS condition (Fig. 6b; Table S7). Conversely, BMP5 and BMP7 were expressed in a larger portion of the cells under TS condition compared to iPSCs (BMP5: 0–0.67% iPSC vs 24.72% T1 cells and BMP7: 1.01–4.71% iPSC vs 29.06% T1 cells). Genes encoding the BMP effector proteins (transducers) SMAD1,4, and 5 are expressed during the earlier days in all conditions, which may indicate that the endogenous BMP signaling is important during the initial differentiation process.

To identify the most significant gene expression changes associated with the initial stages of hiPSCs specification to TSCs under the TS condition, we applied a single-cell trajectory inference and pseudotime estimation (STREAM)47 to iPSC and cells at day 2 under the TS condition (TS D2). Briefly, single cells were ordered along probabilistic trajectories and a numeric value referred to as pseudotime was assigned to each cell to indicate how far it progresses along the dynamic differentiation.

STREAM identified an initial branch composed of both iPSCs and TS D2 cells (S3-S0), which transition to three branches: S0-S1 branch reflecting the exit from pluripotent state and specification to TSC lineage and two branches reflecting the pluripotency continuum (S0–S2, S0–S4) (Fig. 6c,d; Supplemental Fig. 8a and b; Tables S812). We next identified the genes involved in the specification that are correlated with the transition along the branches (see “Material and methods”). The pluripotency to TSC lineage pathway (branch S0-S1) showed an upregulation of genes encoding cytokeratin (KRT18, KRT19 and KRT8) and calcium binding proteins, such as S100A11 and S100A10, which are known to be expressed in trophoblast48,49 (Supplemental Fig. 8c). The top positively correlated transition genes along the branch S0-S1 included LRRN3, which amplifies MAPK signaling through EGFR50, MEIS251, BMP5, BMPR1B and TFAP2A (Fig. 6c,d) a transcription factor key to the suppression of pluripotency and expression of trophoblast associated genes52. We also found TMSB4X as a top transition gene, which is implicated in stemness of progenitor trophoblast cells of first trimester human placentas by increasing NOTCH1 activity53,54,55 (Tables S812). The top positively correlated transition genes along the branches S3-S0, S0-S2 and S0-S4 included known primed PSC markers such as THY1, LINC00458 and SOX11 (Supplemental Fig. 8d). Collectively, these findings suggest that the activation of trophoblast transcriptional programming takes place early in the hiPSC specification process to TSCs, facilitated by known trophoblast-associated regulatory factors such as TMSB4X, YAP, BMP5, and TFAP2A.

Human endogenous retrovirus-derived genes participate in a regulatory subnetwork within TSCs derived from primed hiPSCs

Endogenous retroviruses have been pivotal in the evolutionary diversification of the mammalian placenta, with numerous instances of human endogenous retroviruses (hERVs) being co-opted for essential functions56,57,58,59. We reasoned that hERV expression may be altered because primed hPSCs, naive hPSCs, and TSCs differ in the regulation of human endogenous retroviruses60. Therefore, we investigated how hERVs are expressed during the specification. While there are potentially 1,500 ERV-derived genes capable of encoding proteins57, we restrict our analysis to the 20 ERV-derived genes that are currently annotated as human genes including ERV3-1, Suppressyn (SUPYN/ERVH48-1), Syncytin-1 (ERVW-1) and Syncytin-2 (ERVFRD-1), which function in antiviral responses and syncytial fusion. We found specific upregulation of five ERV-derived genes, ERVH48-1, ERV3-1, ERVMER34-1, ERVW-1 and ERVFRD-1, in the most differentiated cells in the TS condition, cluster T7 (Supplemental Fig. 9a).

To understand the place of ERV-derived genes in the TSC regulatory network, we analyzed the participation of ERV-derived genes in a gene regulatory network. We built a transcription factor (TF) and target gene network model using the Passing Attributes between Networks for Data Assimilation (PANDA) algorithm on all clusters among the different specification conditions. PANDA integrates information from TF-sequence-motif data, gene expression and protein–protein interaction (PPI) in a message-passing approach61. We found that ERV interactions (TFs + ERV-derived genes) are prominent in the most differentiated TS cluster (TS 7) and in no other cell clusters. We found regulatory interactions for five ERV-derived genes (ERVH48-1, ERV3-1, ERVMER34-1, ERVW-1 and ERVFRD-1) are highly unique to the TS 7 cluster network, while only 0 or 1 ERV-derived gene was found in the other cell clusters (Supplemental Figs. 9b and 10). Interestingly, genes encompassed in ERV regulatory interactions in TSCs were enriched in biological pathways related to hormone metabolism, cell differentiation and the immune system (adj. p value < 0.01) (Supplemental Fig. 9c; Table S13). In addition, ERV regulatory interactions are also enriched for categories such as placenta development, trophoblast cell differentiation and syncytium formation (Table S13). To understand the significance of the ERV-subnetwork interactions, we next evaluated the strength of ERV edges in the TS cluster network. Weights of ERV regulatory interactions were highly ranked among all network connections (Wilcoxon Rank Sum p-value = 1.6778e−31, permuted p-value = 0.001), indicating a significant contribution to the gene regulatory network of the TSC state. In sum, we found that ERV-derived genes participate in a regulatory subnetwork within TSCs derived from primed hiPSCs.

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