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Comparative analysis of the syncytiotrophoblast in placenta tissue and trophoblast organoids using snRNA sequencing

ABSTRACT: 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 finally a subtype enriched in transport and GTPase signaling molecules. Notably, suspension culture conditions of TOs that restore the native orientation of the STB (STBout) showed elevated expression of canonical STB markers and pregnancy hormones, along with a greater proportion of the STB nucleus subtype specializing in transport and GTPase signaling, compared to those cultivated with an inverted STB polarity (STBin). 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. CRISPR/Cas9 knock out of RYBP in STBin TOs did not affect cell-cell fusion, but bulk sequencing demonstrates the downregulation of the pregnancy hormone CSH1 and upregulation of many genes that define the oxygen sensing STB nuclear subtype. 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. The processed datasets can be visualized online at https://gladfelterlab.shinyapps.io/PlacentaRNAsequencing/.

ORGANISM(S): Homo sapiens

PROVIDER: GSE288650 | GEO | 2025/02/08

REPOSITORIES: GEO

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Project description:Purpose: Syncytiotrophoblast (STB) is a multi-nucleated, terminally differentiated syncytium that covers the surface of the villous placenta and forms the major interface with maternal blood. It releases placental hormones and plays a primary role in exchange of gases, nutrients and waste products. Alterations in STB development and turnover have been implicated in placental diseases, including preeclampsia (PE). In vitro cell models are badly needed to study STB development and physiology due to inaccessibility to placental tissues during gestation. To establish in vitro STB model system, we generate STB and its mononucleated precursors from human embryonic stem cells (hESC) and profile for RNA content by RNAseq. Methods: H1 Human ESC (WA01) were treated with BMP4, the ALK4/5/7 inhibitor (A83-01), and the FGF2 signaling inhibitor (PD173074) (BAP) to direct them to the trophoblast lineage and provided both STB and extravillous trophoblast. Syncytial areas emerged at day 8 BAP treatment ranged in diameter from ~40 Âµm to > 100 Âµm. The intact syncytial areas were isolated by sieving successively through 70 Î¼m and 40 Î¼m mesh cell strainers. The captured cells are recovered by inverting the strainer and rinsing with culture medium to separate large (>70 Î¼m) and middle size cell sheets (40-70 Î¼m). The fraction that passes through both sieves represents cells of smallest diameter (< 40 Î¼m), presumably cytotrophoblast. Total 12 RNA samples from triplicate three size-fractioned BAP treated and three untreated hESC cultured in a FGF2 supplemented medium in parallel were analyzed. Results: The larger > 70 Î¼m areas stained positively for STB markers while ultrastructural analysis clearly revealed multi-nuclear cells with an extensive cytoplasm containing many prominent secretion granules. The larger STB areas also had a larger DNA content that > 70 Î¼m fraction contained 37 times more nuclear content and 40-70 Âµm fraction did 16 times more. Compared to the < 40 Î¼m cell fraction, these larger cells over-expressed a full repertoire of genes characteristic of STB, e.g. CGA, CGB, PGF, ERVW1, GCM1. The smallest cell fraction had a DNA content consistent with mononuclear diploid cells, contained few secretory granules, and were only weakly positive for STB markers. Conclusion: The data are consistent with the > 70 Î¼m cells being mature STB, while the intermediate fraction may represent a precursor population. Human ESC directed along the trophoblast lineage by BAP treatment offers a useful model for following STB formation in vitro and suggest that this protocol may have utility in studying the basis of certain placental diseases, especially preeclampsia, where placental tissue isolated at term or after pregnancy terminations can only offer limited information. Three size fraction mRNA profiles of syncytial areas emerged at day 8 BAP treatment of hESC were generated by deep sequencing along with untreated hESC, in triplicate, using Illumina HiSeq 2500.

Project description:Purpose: Syncytiotrophoblast (STB) is a multi-nucleated, terminally differentiated syncytium that covers the surface of the villous placenta and forms the major interface with maternal blood. It releases placental hormones and plays a primary role in exchange of gases, nutrients and waste products. Alterations in STB development and turnover have been implicated in placental diseases, including preeclampsia (PE). In vitro cell models are badly needed to study STB development and physiology due to inaccessibility to placental tissues during gestation. To establish in vitro STB model system, we generate STB and its mononucleated precursors from human embryonic stem cells (hESC) and profile for RNA content by RNAseq. Methods: H1 Human ESC (WA01) were treated with BMP4, the ALK4/5/7 inhibitor (A83-01), and the FGF2 signaling inhibitor (PD173074) (BAP) to direct them to the trophoblast lineage and provided both STB and extravillous trophoblast. Syncytial areas emerged at day 8 BAP treatment ranged in diameter from ~40 µm to > 100 µm. The intact syncytial areas were isolated by sieving successively through 70 μm and 40 μm mesh cell strainers. The captured cells are recovered by inverting the strainer and rinsing with culture medium to separate large (>70 μm) and middle size cell sheets (40-70 μm). The fraction that passes through both sieves represents cells of smallest diameter (< 40 μm), presumably cytotrophoblast. Total 12 RNA samples from triplicate three size-fractioned BAP treated and three untreated hESC cultured in a FGF2 supplemented medium in parallel were analyzed. Results: The larger > 70 μm areas stained positively for STB markers while ultrastructural analysis clearly revealed multi-nuclear cells with an extensive cytoplasm containing many prominent secretion granules. The larger STB areas also had a larger DNA content that > 70 μm fraction contained 37 times more nuclear content and 40-70 µm fraction did 16 times more. Compared to the < 40 μm cell fraction, these larger cells over-expressed a full repertoire of genes characteristic of STB, e.g. CGA, CGB, PGF, ERVW1, GCM1. The smallest cell fraction had a DNA content consistent with mononuclear diploid cells, contained few secretory granules, and were only weakly positive for STB markers. Conclusion: The data are consistent with the > 70 μm cells being mature STB, while the intermediate fraction may represent a precursor population. Human ESC directed along the trophoblast lineage by BAP treatment offers a useful model for following STB formation in vitro and suggest that this protocol may have utility in studying the basis of certain placental diseases, especially preeclampsia, where placental tissue isolated at term or after pregnancy terminations can only offer limited information.

Project description:Prolonged skeletal unloading through bedrest results in bone loss similar to that observed in elderly osteoporotic patients, but with an accelerated timeframe. This rapid effect on weight-bearing bones is also observed in astronauts who lose up to 2% of their bone mass per month spent in Space. Despite important implications for Spaceflight travellers and bedridden patients on Earth, the exact mechanisms involved in disuse osteoporosis have not been elucidated. Parathyroid hormone-related protein (PTHrP) regulates many physiological processes including skeletal development, and has been proposed as a gravisensor. To investigate the role of PTHrP in microgravity-induced bone loss, trabecular osteoblasts (TOs) from Pthrp+/+ and -/- mice were exposed to simulated microgravity for 6 days. Viability of TOs decreased in inverse proportion to PTHrP expression levels. Microarray analysis of Pthrp+/+ TOs after 6 days at 0g revealed expression changes in genes encoding prolactins,apoptosis and survival molecules, bone metabolism and extra-cellular matrix composition proteins, chemokines, IGF family and Wnt-related signalling molecules. Importantly, 88% of 0g-induced expression changes in Pthrp+/+ cells overlap those observed in Pthrp-/- cells in normal gravity. Pulsatile treatment with PTHrP1-36 peptide during microgravity exposure reversed a large proportion of 0g-induced changes in Pthrp+/+ TOs. Our results confirm PTHrP efficacy as an anabolic agent to prevent microgravity-induced cell death in TOs. Total RNA samples extracted from Pthrp+/+and -/- trabecular osteoblasts (TOs) exposed for 6 days to simulated 0g in Synthecon rotating cell, or left 6 days in culture at 1g. Cells had either been treated with a pulsatile treatment (2 h/day) of PTHrP1-36 peptide (10-8M) or received a change in growth medium. In total: 8 different conditions with 2 replicates each, i.e. Pthrp+/+ TOs at 0g or 1g with or without PTHrP1-36 treatment, and Pthrp-/- TOs at 0g or 1 g,with or without PTHrP1-36 treatment.

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Comparative analysis of the syncytiotrophoblast in placenta tissue and trophoblast organoids using snRNA sequencing

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