Innovative Research for a Sustainable Future www.epa.gov/research Enriched pathways uniquely associated with 4 d ATRA exposure were driven by differential expression of 29 histone-encoding genes concentration Transcriptome profiling to identify ATRA-responsive genes in human iPSC-derived endoderm for high-throughput point of departure analysis Katerine S. Saili, Todd J. Zurlinden, Todor Antonijevic, Imran Shah, Thomas B. Knudsen National Center for Computational Toxicology, U.S. Environmental Protection Agency, Office of Research and Development, Research Triangle Park, NC, USA Background Methods Results (pathway analysis) Summary and Conclusions References saili.katerine@epa.gov l 919-541-3871 • Toxicological tipping points occur at chemical concentrations that overwhelm a cell’s adaptive response [Shah et al. 2016] • Human iPSC-derived endodermal differentiation (endogenesis) is an in vitro platform for probing the developmental impacts of a toxicological tipping point • Endogenesis is critical for organs such as the stomach, intestine, colon, pancreas, liver, urinary bladder, trachea, lung, pharynx, thyroid, and parathyroid glands, visceral yolk sac • Retinoid signaling is critical for early development and directs morphogenesis, growth, and differentiation of the embryo including endogenesis via retinoic acid gradients • Epigenetic changes mediate retinoid-induced stem cell differentiation [Gudas. 2013] Log2(RPKM+1) values were used for differential gene expression analysis (ANOVA) and filtered (p < 0.00001; -2 > fold-change >2) KEGG Pathway Analysis: two highly enriched pathways (p < 0.0001) • Endodermal differentiation based on FOXA2 expression occurred between 6 h and 4 d • ATRA first suppressed differentiation at 0.1 µM (by 4 d) indicated by multiple pathways such as protein digestion and absorption that characterize the trajectories for tipping point analysis • ATRA increased the expression of 29 histone encoding genes by 4 d, suggesting DNA remodeling is a target process for tipping point analysis • Endogenesis may be a suitable ToxCast platform for molecular-level characterization of toxicological tipping points during cell differentiation • Shah, et al. 2016. Using ToxCast™ Data to Reconstruct Dynamic Cell State Trajectories and Estimate Toxicological Points of Departure. Environmental Health Perspectives. Jul;124(7) • Gudas. 2013. Retinoids induce stem cell differentiation via epigenetic changes. Semin Cell Dev Biol. Dec; 24(0) • The views expressed in this poster are those of the authors and may not reflect U.S. EPA policy Total RNA for each treatment (x2) Human fibroblast-derived iPSCs Low serum + Activin differentiating endoderm Exposures (Vala Sciences) Sample Collection (Vala Sciences) 0d 6h 1d 2d 3d 4d 5d 6d 7d 8d 0.1% DMSO 0.001 µM ATRA 0.01 µM ATRA 0.1 µM ATRA 1 µM ATRA 10 µM ATRA Cells (Allele Biotech) RNA Sequencing (Cofactor Genomics) Data Analysis (US EPA) Vitamin A  All-trans retinoic acid (ATRA) RAR/RXR Total RNA for each treatment (x2) Total RNA for each treatment (x2) >30 million single end reads 26011 genes (75339 transcripts) 0.1% DMSO 10 µM ATRA FOXA2 expression (purple) at 4 d ATRA reduced FOXA2 expression and other endoderm-specific biomarkers in a concentration- dependent manner at 4 d Results (epigenetics) KEGG pathway analysis of differentially expressed genes (6033) returned 8 highly enriched signaling pathways (p<0.0001) Tipping point FOXA2 fold-change (versus time-matched DMSO control) un: undifferentiating control Pathway name Conc. Time OBJECTIVE: Identify biomarkers for high content analysis of tipping points in a developmental context Ectodermal markers (FC versus 6h DMSO control) Endodermal markers (FC versus 6h DMSO control) Histone modifying enzymes Histones ATRA-responsive genes (p<0.00001, 2 < FC < -2) List of differentiation markers provided by Sid Hunter (EPA/NHEERL) Mean FC vs. time-matched DMSO control Mean FC vs. 6h DMSO control Mean FC vs. 6h DMSO control