RESUMO
The outer surface of chorionic villi in the human placenta consists of a single multinucleated cell called the syncytiotrophoblast (STB). The unique cellular ultrastructure of the STB presents challenges in deciphering its gene expression signature at the single-cell level, as the STB contains billions of nuclei in a single cell. There are many gaps in understanding the molecular mechanisms and developmental trajectories involved in STB formation and differentiation. To identify the underlying control of the STB, we performed comparative single nucleus (SN) and single cell (SC) RNA sequencing on placental tissue and tissue-derived trophoblast organoids (TOs). We found that SN was essential to capture the STB population from both tissue and TOs. Differential gene expression and pseudotime analysis of TO-derived STB identified three distinct nuclear subtypes reminiscent of those recently identified in vivo . These included a juvenile nuclear population that exhibited both CTB and STB marker expression, a population enriched in genes involved in oxygen sensing, and a fully differentiated subtype. Notably, suspension culture conditions of TOs that restore the native orientation of the STB (STB out ) showed elevated expression of canonical STB markers and pregnancy hormones, along with a greater proportion of the terminally differentiated mature STB subtype, compared to those cultivated with an inverted STB polarity (STB in ). Gene regulatory analysis identified novel markers of STB differentiation conserved in tissue and TOs, including the chromatin remodeler RYBP, that exhibited STB-specific RNA and protein expression. Finally, we compared STB gene expression signatures amongst first trimester tissue, full-term tissue, and TOs, identifying many commonalities but also notable variability across each sample type. This indicates that STB gene expression is responsive to its environmental context. Our findings emphasize the utility of TOs to accurately model STB differentiation and the distinct nuclear subtypes observed in vivo , offering a versatile platform for unraveling the molecular mechanisms governing STB functions in placental biology and disease.
RESUMO
The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii. We grew human trophoblast stem cells (TSCT) under conditions leading to either syncytiotrophoblast (TSSYN) or cytotrophoblast (TSCYT) and infected them with T. gondii. We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TSSYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TSSYNs were highly refractory to parasite adhesion and replication, while TSCYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TSSC-derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes. We demonstrate that TSSYNs are highly resistant to L. monocytogenes, while TSCYTs are not. Like T. gondii, TSSYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus's tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii. Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes, at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication.