Comparative analysis of the syncytiotrophoblast in placenta tissue and trophoblast organoids using snRNA sequencing
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eLife Assessment
The study is important - not only for its comprehensive transcriptomic analysis of the developmental trajectory of syncytiotrophoblasts (STBs), but also for its comparative evaluation of primary human placental tissues and two human trophoblast organoid models. The study highlights the utility of these organoid models in advancing research on human STB biology. The conclusions of this work are supported by compelling analyses and experimental evidence.
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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 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.
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eLife Assessment
The study is important - not only for its comprehensive transcriptomic analysis of the developmental trajectory of syncytiotrophoblasts (STBs), but also for its comparative evaluation of primary human placental tissues and two human trophoblast organoid models. The study highlights the utility of these organoid models in advancing research on human STB biology. The conclusions of this work are supported by compelling analyses and experimental evidence.
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Reviewer #1 (Public review):
Summary:
This study provides an in-depth analysis of syncytiotrophoblast (STB) gene expression at the single-nucleus (SN) and single-cell (SC) levels, using both primary human placental tissues and two trophoblast organoid (TO) models. The authors compare the older TO model, where STB forms internally (STBin), with a newer model where STB forms externally (STBout). Through a series of comparative analyses, the study highlights the necessity of using both SN and SC techniques to fully understand placental biology. The findings demonstrate that the STBout model shows more differentiated STBs with higher expression of canonical markers and hormones compared to STBin. Additionally, the study identifies both conserved and distinct gene expression profiles between the TO models and human placenta, offering …
Reviewer #1 (Public review):
Summary:
This study provides an in-depth analysis of syncytiotrophoblast (STB) gene expression at the single-nucleus (SN) and single-cell (SC) levels, using both primary human placental tissues and two trophoblast organoid (TO) models. The authors compare the older TO model, where STB forms internally (STBin), with a newer model where STB forms externally (STBout). Through a series of comparative analyses, the study highlights the necessity of using both SN and SC techniques to fully understand placental biology. The findings demonstrate that the STBout model shows more differentiated STBs with higher expression of canonical markers and hormones compared to STBin. Additionally, the study identifies both conserved and distinct gene expression profiles between the TO models and human placenta, offering valuable insights for researchers using TOs to study STB and CTB differentiation.
Strengths:
The study offers a comprehensive SC- and SN-based characterization of trophoblast organoid models, providing a thorough validation of these models against human placental tissues. By comparing the older STBin and newer STBout models, the authors effectively demonstrate the improvements in the latter, particularly in the differentiation and gene expression profiles of STBs. This work serves as a critical resource for researchers, offering a clear delineation of the similarities and differences between TO-derived and primary STBs. The use of multiple advanced techniques, such as high-resolution sequencing and trajectory analysis, further enhances the study's contribution to the field.
Weaknesses:
While the study is robust, some areas could benefit from further clarification. The importance of the TO model's orientation and its impact on outcomes could be emphasized more in the introduction. The differences in cluster numbers/names between primary tissue and TO data need a clearer explanation, and consistent annotation could aid in comparison. The rationale for using SN sequencing over SC sequencing for TO evaluations should be clarified, especially regarding the potential underrepresentation of certain trophoblast subsets. Additionally, more evidence could be provided to support the claims about STB differentiation in the STBout model and to determine whether its differentiation trajectory is unique or simply more advanced than in STBin.
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Reviewer #2 (Public review):
Summary:
In this study, the authors aimed to elucidate the formation and differentiation of syncytiotrophoblast (STB) cells by analyzing placental tissue and trophoblast organoids (TOs) using single-nucleus (SN) and single-cell (SC) RNA sequencing. They identified three distinct nuclear subtypes within the STB and explored the relationship between STB gene expression changes, developmental stages, and environmental contexts. The study emphasizes the utility of TOs as models for understanding STB differentiation and highlights novel gene markers, such as RYBP, involved in STB development.
Strengths:
(1) The use of SN and SC RNA sequencing provides a detailed analysis of STB formation and differentiation.
(2) The identification of distinct STB subtypes and novel gene markers such as RYBP offers new insights …
Reviewer #2 (Public review):
Summary:
In this study, the authors aimed to elucidate the formation and differentiation of syncytiotrophoblast (STB) cells by analyzing placental tissue and trophoblast organoids (TOs) using single-nucleus (SN) and single-cell (SC) RNA sequencing. They identified three distinct nuclear subtypes within the STB and explored the relationship between STB gene expression changes, developmental stages, and environmental contexts. The study emphasizes the utility of TOs as models for understanding STB differentiation and highlights novel gene markers, such as RYBP, involved in STB development.
Strengths:
(1) The use of SN and SC RNA sequencing provides a detailed analysis of STB formation and differentiation.
(2) The identification of distinct STB subtypes and novel gene markers such as RYBP offers new insights into STB development.
Weaknesses:
(1) Inconsistencies in data presentation.
(2) Questionable interpretation of lncRNA signals: The use of long non-coding RNA (lncRNA) signals as cell type-specific markers may represent sequencing noise rather than true markers.
To improve the study's validity and significance, it is crucial to address the inconsistencies and to provide additional evidence for the claims. Supplementing with immunofluorescence staining for validating the distribution of STB_in, STB_out, and EVT_enrich in the organoid models is recommended to strengthen the results and conclusions.
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Reviewer #3 (Public review):
In this report, Keenen et al. present a thoroughly characterized platform for identifying potential molecular mechanisms regulating syncytiotrophoblast cell functions in placental biology. The application of single-cell assessments to identify developmental trajectories of this lineage has been challenging due to the complex, multinucleated structure of the syncytium. The authors provide a comprehensive comparative assessment of term placental tissue and three independent trophoblast organoid models. They use single-cell and single-nucleus RNA sequencing followed by differential gene expression and pseudotime analyses to identify subpopulations and differentiation trajectories. They further compare the datasets generated in this study to publicly available datasets from first-trimester placental tissue. The …
Reviewer #3 (Public review):
In this report, Keenen et al. present a thoroughly characterized platform for identifying potential molecular mechanisms regulating syncytiotrophoblast cell functions in placental biology. The application of single-cell assessments to identify developmental trajectories of this lineage has been challenging due to the complex, multinucleated structure of the syncytium. The authors provide a comprehensive comparative assessment of term placental tissue and three independent trophoblast organoid models. They use single-cell and single-nucleus RNA sequencing followed by differential gene expression and pseudotime analyses to identify subpopulations and differentiation trajectories. They further compare the datasets generated in this study to publicly available datasets from first-trimester placental tissue. The work is timely as optimization of trophoblast organoids is an evolving topic in placental research. Careful characterization of in vitro models has been noted as essential for model selection and result interpretation in the field.
The study elucidates syncytiotrophoblast nucleus subtypes and proportions in three different organoid models and compares subtypes and gene expression signatures to placental tissues. This work advances the field by demonstrating the utility of different trophoblast organoids to model syncytiotrophoblast differentiation. The in-depth characterization of cell types comprising the different organoid models and how they compare to placental tissue will help to inform model selection for future experimentation in the field. Defining cell composition and cell differentiation trajectories will also aid in data interpretation for data generated by these tissue and model sources. Overall, the conclusions presented in the manuscript are well supported by the data. The figures, as presented, are informative and striking.
The authors present outstanding progress toward their aim of identifying, "the underlying control of the syncytiotrophoblast". They identify the chromatin remodeler, RYBP, as well as other regulatory networks that they propose are critical to syncytiotrophoblast development. This study is limited in fully addressing the aim, however, as functional evidence for the contributions of the factors/pathways to syncytiotrophoblast cell development is needed. Future experimentation testing the hypotheses generated by this work will define the essentiality of the identified factors to syncytiotrophoblast development and function. Localization and validation of the identified factors within tissue and at the protein level will also provide further contextual evidence to address the hypotheses generated.
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