Integrative Analysis of Transcriptomic and Epigenomic Dynamics of Induced Pluripotent Stem Cell Derived Hepatic Organoids Using Single Cell RNA-Seq and ATAC-Seq

Abstract

Previously, we developed a novel method by which to generate functionally mature human hepatic organoids derived from pluripotent stem cells (PSCs), and the functionality and maturity of the organoids were validated by various experiments including bulk RNA-seq. In this study, to identify the heterogeneity and dynamics of transcriptomes and epigenomes of the organoids at the single-cell level, we applied single-cell RNA-seq (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) to analyze liver organoids treated with two medium conditions—hepatic medium (HM, expandable condition) and differentiation medium (DM, more matured condition). The cells were separated into 13 distinct clusters corresponding to stages ranging from early to late by both scRNA-seq and scATAC-seq, and DM liver organoids were more similar in more differentiated liver cells compared to those in HM. Mitochondrial gene expression was increased in differentiated hepatocytes, with ATAC-seq peaks enriched near the mitochondrial control region. Many transcription factors (TFs)—both activators and repressors—were revealed throughout the differentiation of liver organoids. Different patterns of expression of alpha-fetoprotein (AFP) and albumin (ALB) were observed between liver organoids and adult liver tissues, and several open chromatin regions (OCRs) near AFP and ALB genes were found to be differentially regulated between the liver organoids and tissues. HNF4A, YY1 (Yin Yang 1) and CTCF binding sites near AFP and ALB genes were differentially occupied between embryo and adult liver tissues. Our integrative analysis of transcriptomic and epigenetic dynamics of the liver organoids can contribute to the understanding of transcriptional regulatory networks during liver development and to the further development of mature in vitro human liver models.