Extensive reprogramming of the nascent transcriptome during iPSC to hepatocyte differentiation

Leena E Viiri,Henri Niskanen,Mikko Oittinen,Katriina Aalto-Setälä,Mostafa Kiamehr,Tommi Rantapero,Minna U Kaikkonen,Matti Nykter,Keijo Viiri,Anna Alexanova

doi:10.1038/s41598-019-39215-0

Abstract

Hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs) provide a renewable source of cells for drug discovery, disease modelling and cell-based therapies. Here, by using GRO-Seq we provide the first genome-wide analysis of the nascent RNAs in iPSCs, HLCs and primary hepatocytes to extend our understanding of the transcriptional changes occurring during hepatic differentiation process. We demonstrate that a large fraction of hepatocyte-specific genes are regulated at transcriptional level and identify hundreds of differentially expressed non-coding RNAs (ncRNAs), including primary miRNAs (pri-miRNAs) and long non-coding RNAs (lncRNAs). Differentiation induced alternative transcription start site (TSS) usage between the cell types as evidenced for miR-221/222 and miR-3613/15a/16-1 clusters. We demonstrate that lncRNAs and coding genes are tightly co-expressed and could thus be co-regulated. Finally, we identified sets of transcriptional regulators that might drive transcriptional changes during hepatocyte differentiation. These included RARG, E2F1, SP1 and FOXH1, which were associated with the down-regulated transcripts, and hepatocyte-specific TFs such as FOXA1, FOXA2, HNF1B, HNF4A and CEBPA, as well as RXR, PPAR, AP-1, JUNB, JUND and BATF, which were associated with up-regulated transcripts. In summary, this study clarifies the role of regulatory ncRNAs and TFs in differentiation of HLCs from iPSCs.

Highlights

Primary human hepatocytes (PHH) are extensively needed for both basic and translational science but their use is restricted by the lack of donors, and the limited number of cells since functional primary human hepatocytes (PHHs) cannot be expanded in vitro and they rapidly lose their functionality during culture[1]
We were interested in identifying active regulatory RNAs such as pri-miRNAs and long non-coding RNAs (lncRNAs) that could contribute to the differentiation of functional hepatocyte-like cells (HLCs) from induced pluripotent stem cells (iPSCs)
HLCs were generated from patient-derived iPSCs using two protocols derived from the ones described by Si-Tayeb et al.[9] (Method 1, M1) and Hay et al.[10] (Method 2, M2), with minor modifications

Summary

Introduction

Primary human hepatocytes (PHH) are extensively needed for both basic and translational science but their use is restricted by the lack of donors, and the limited number of cells since functional PHHs cannot be expanded in vitro and they rapidly lose their functionality during culture[1]. Producing hepatocytes from iPSCs offers a way to study different aspects of hepatocyte function in a patient-specific cell model, in the context of the genetic background of the patient. GRO-seq identifies the genes that are being transcribed at a given time point in a cell. It provides extensive information on the location and quantity of coding and non-coding transcripts, including promoter- and enhancer associated long non-coding RNAs (lncRNAs) and primary microRNAs (pri-miRNAs5,6). We performed GRO-seq to investigate the nascent transcription in the HLCs to gain better understanding of gene regulatory processes controlling hepatic differentiation. We were interested in identifying active regulatory RNAs such as pri-miRNAs and lncRNAs that could contribute to the differentiation of functional HLCs from iPSCs

Methods

Results

Discussion

Conclusion