Citation:

Saili, K., T. Zurlinden, T. Antonijevic, I. Shah, AND T. Knudsen. Transcriptome profiling to identify ATRA-responsive genes in human iPSC-derived endoderm for high-throughput point of departure analysis (SOT Annual Meeting). Presented at SOT Annual Meeting, Baltimore, MD, March 11 - 16, 2017. https://doi.org/10.23645/epacomptox.5176909

Impact/Purpose:

Poster presentation at the SOT 2017 annual meeting.This work demonstrates that iPSC-derived endoderm is a suitable human cell-based platform for high-throughput screening to identify toxicological tipping points in a differentiating system. The genes occurring within the identified pathways are being used for targeted, transcriptomic tipping point analysis of ToxCast chemical libraries.

Description:

Toxicological tipping points occur at chemical concentrations that overwhelm a cell’s adaptive response leading to permanent effects. We focused on retinoid signaling in differentiating endoderm to identify developmental pathways for tipping point analysis. Human induced pluripotent stem cell (iPSC)-derived endodermal cells (Allele) were exposed to 0.1% DMSO (vehicle-control) or five concentrations (0.001 - 10 µM) of all-trans retinoic acid (ATRA) renewed daily for 8 days (Vala Sciences). Total RNA samples were collected at 6 hours (h), 4 days (d), and 8 d and prepared for RNA sequencing (Cofactor Genomics). Log2(RPKM+1) values were used for differential gene expression analysis (ANOVA) and filtered using a conservative false discovery rate (FDR) of 2 or <-2). The resulting list comprised 6033 genes, including Foxa2, an endoderm-specific biomarker for which protein expression was confirmed via high-content imaging. Based on Foxa2 expression, differentiation occurred between 6 h and 4 d in the vehicle-control samples. ATRA significantly reduced this marker in a concentration-dependent manner in the 0.1 - 10 µM range. Pathway analysis of the 6033 gene list resulted in 8 pathways with enrichment scores above 9 (p-values < 0.0001). These pathways included protein digestion and absorption, which foreshadows differentiation of endodermal tissues. Stage-specific expression of ATRA-responsive genes returned two highly enriched pathways associated with 4 d ATRA exposure that were driven predominantly by increased expression of 29 genes encoding histones. This effect followed a concentration-response with undifferentiated controls between 0.1 and 1 µM. Overall, gene expression effects were most consistently observed following 1 µM exposure, which elicited time-dependent effects on the expression of Foxa2, Wnt6, Shh, and Hand1, genes known for their role in development. Taken together, iPSC-derived endoderm is a suitable human cell-based platform for high-throughput screening to identify toxicological tipping points in a differentiating system. The genes occurring within the identified pathways are being used for targeted, transcriptomic tipping point analysis of ToxCast chemical libraries. This work was conducted under EPA contract EPD13054 but does not represent US EPA policy.

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