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Updated: Aug 12, 2025
In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells
Published on: March 16, 2017
HAND1 knockdown disrupts trophoblast global gene expression
Robert Fresch1, Jennifer Courtney2, Heather Brockway3,4
1Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Insights
Disrupting HAND1 gene expression significantly impacts human trophoblast cells, affecting pathways crucial for development, but not placental vascular endothelial cells. This highlights the placenta's role in congenital heart disease (CHD) research.
Area of Science:
- Developmental Biology
- Genetics
- Reproductive Biology
Background:
- Congenital heart disease (CHD) affects nearly 1% of newborns annually.
- Pregnancies with CHD are at higher risk for placental abnormalities.
- Previous studies linked Hand1 gene disruption to developmental issues in mouse models.
Purpose of the Study:
- To investigate the mechanistic effects of HAND1 gene disruption on human placenta trophoblast and vascular endothelial cell gene expression.
- To build upon prior research implicating Hand1 in CHD and placental development.
Main Methods:
- HAND1 gene expression was silenced using siRNA in BeWo cells (human trophoblast model) and human placental microvascular endothelial cells (HPMVECs).
- RNA sequencing was performed after 96 hours of HAND1 knockdown.
- Differential gene expression analysis, pathway overrepresentation, and protein association network analyses were conducted.
Main Results:
- HAND1 knockdown in BeWo cells led to significant downregulation of 664 genes and upregulation of 59 genes.
- Disrupted pathways in trophoblast cells included cell differentiation and localization.
- HAND1 knockdown had minimal impact on gene expression in HPMVECs, with only seven genes altered.
Conclusions:
- HAND1 disruption significantly alters gene expression in human trophoblast cells, impacting key developmental pathways.
- The effect of HAND1 disruption is specific to trophoblast cells, not placental vascular endothelial cells.
- Future research on genetic causes of CHD should consider the role of extra-embryonic tissues like the placenta.
Abstract:
Congenital heart disease (CHD) affects nearly 1% of births annually, and CHD pregnancies carry increased risk of developing pathologies of abnormal placentation. We previously reported significant developmental impacts of disrupting Hand1, a gene associated with CHD, expression in placenta trophoblast and endothelial cells in multiple mouse models. In this study, we aimed to build upon this knowledge and characterize the mechanistic impacts of disrupting HAND1 on human placenta trophoblast and vascular endothelial cell gene expression. HAND1 gene expression was silenced in BeWo cells, a choriocarcinoma model of human cytotrophoblasts, (n = 3-9 passages) and isolated human placental microvascular endothelial cells (HPMVEC; n = 3 passages), with HAND1 siRNA for 96 h. Cells were harvested, mRNA isolated and RNA sequencing performed using the Illumina NextSeq 550 platform. Normalization and differential gene expression analyses were conducted using general linear modeling in edgeR packages. Statistical significance was determined using a log2 fold change of >1.0 or < -1.0 and unadjusted p-value ≤0.05. Panther DB was used for overrepresentation analysis, and String DB for protein association network analysis. There was downregulation of 664 genes, and upregulation of 59 genes in BeWo cells with direct HAND1 knockdown. Overrepresentation analysis identified disruption to pathways including cell differentiation, localization, and cell projection organization. In contrast, only seven genes were changed with direct HAND1 knockdown in HPMVECs. Disruption to HAND1 expression significantly alters gene expression profile in trophoblast but not endothelial cells. This data provides further evidence that future studies on genetic perturbations in CHDs should consider the extra-embryonic tissue in addition to the fetal heart.
