GRO-sequencing identifies transcriptional regulators and ncRNAs important for iPSC to hepatocyte differentiation

Hepatocyte-like cells (HLCs) were differentiated from induced pluripotent stem cells (iPSCs) using two different protocols and the genome-wide transcriptomic profile of the iPSC, HLCs and primary human hepatocytes were compared using Global Run-On sequencing (GRO-seq). In addition to protein coding genes, GRO-seq enabled identification of noncoding RNA species including primary miRNAs and long non-coding RNAs (lncRNAs). Altogether, 29 hub miRNAs that could regulate ~3000 target mRNAs related to regulation of transcription, translation, protein metabolism and cell cycle were identified. Alternative transcription start site (TSS) usage between the cell types was detected for several miRNA clusters. Additionally, HLC differentiation included extensive changes in lncRNA expression, which exhibited strong co-regulation with the protein-coding gene expression. Analysis of the motifs within regulated TSSs, allowed identification of transcription factors that might drive the transcriptional changes during hepatocyte differentiation. 3 iPSC cell lines were used and as a reference primary human hepatocytes (PHHs) from 2 donors (Hu8209, male, Caucasian, 50 years and Hu8132, female, Caucasian, 57 years)