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Single-cell RNA Sequencing of Human Embryonic Stem Cells Reveals Immediate and Deferred Transcriptomic Responses to ATRA Toxicity Across Cell Sub-populations
Citation:
Gallegos, D., S. Jeffay, G. Carswell, B. Chorley, AND S. Hunter. Single-cell RNA Sequencing of Human Embryonic Stem Cells Reveals Immediate and Deferred Transcriptomic Responses to ATRA Toxicity Across Cell Sub-populations. Society of Toxicology, San Diego, CA, March 27 - 31, 2022. https://doi.org/10.23645/epacomptox.19422107
Impact/Purpose:
Presentation to the Society of Toxicology 61st Annual Meeting and ToxExpo March 2022. This study demonstrates the first use of single cell sequencing technology by a US EPA laboratory. In this application, we are identifying the impact of chemical exposure to human embryonic stem cells in vitro that may lead to perturbed differentiation and indicative of develpmental disorders in children. Single RNA sequencing greatly increases our capability and resolution to assess chemical effects on development.
Description:
Developmental disorders, such as heart malformations, affect 3% of children born in the U.S.. There is clear evidence that exposure to environmental chemicals can cause the development of many of these defects. Unfortunately, most commercial chemicals remain un- or under evaluated and there is a need for new approach methodologies (NAMs) that can accurately assess the impact on development at lower cost and in a higher throughput (HT) manner. Mouse embryonic stem cells (mESCs) studies have shown that less than 20% of in vivo developmental toxicants are able to be detected by mESC endpoints in vitro. More specific and sensitive in vitro assessments of early human development that leverage mechanistic understanding are needed to better model these early morphogenetic periods. To this end, we employed single-cell RNAseq (scRNAseq) using the 10XGenomics platform to assess our ability to survey direct, toxicant-induced effects on gene transcription at multiple post-exposure timepoints and putative cell subpopulations of H9 undifferentiated human embryonic stem cells (hESCs). In a proof-of-principle study, we measured scRNAseq profiles of immediate (6h) and persistent (72h) transcriptomic changes elicited by all-trans-retinoic-acid (ATRA) administered at a concentration near the lowest effect level (LOAEL) on hESCs (0.03µM). We identified putative subpopulations of cells within our in vitro system which may indicate very early cellular differentiation. We found a primary response gene program suggesting direct metabolic response to ATRA exposure as well as induction of transcripts involved in the regulation of hLh transcription factor DNA binding and developmental/differentiation processes. Following continued exposure (72h), we observed a qualitative phenotypic increase in cell proliferation, abundance, and morphological range as well as multi-faceted gene programs regulating multiple cellular processes including Wnt signaling, transcription factor binding, metabolic responses, and varied lines of differentiation/proliferation. These early results indicate that scRNAseq may help identify very impacts of environmental chemical exposure on mechanisms of cellular differentiation. Such higher resolution and HT applications will lead to greater understanding of the impact on environmental chemicals to development and children’s health. This abstract does not reflect US EPA policy.
URLs/Downloads:
DOI: Single-cell RNA Sequencing of Human Embryonic Stem Cells Reveals Immediate and Deferred Transcriptomic Responses to ATRA Toxicity Across Cell Sub-populations
DGALLEGOSSOT2022_DKBLUE_44X88V7QR.PDF (PDF, NA pp, 5564.747 KB, about PDF)