Defining lineage plasticity of hepatic progenitor cells using transcriptomics and epigenomics

Abstract

Hepatocyte turnover becomes abrogated during chronic liver injury, resulting in an impaired liver function and the eventual onset of cirrhosis, only treatable with organ transplant. In this scenario of repeated damage, an alternative repair mechanism mediated by hepatic progenitor cells (HPCs) of biliary origin replaces the damaged hepatocytes with HPC-derived hepatocytes.

However, HPC into hepatocyte differentiation results in an imperfect repair, probably due to most cells being functionally immature, and the signals and roadblocks involved in this process are still undefined. In this project, we established cholangiocyte organoids from human bile duct HPCs, which can differentiate into hepatocytes, and looked at their chromatin state and transcriptome to identify genes and pathways involved in differentiated organoid maturity. First, we determined by ATAC-seq that differentiated organoids resemble foetal rather than adult hepatocytes at the chromatin level, suggesting an incomplete differentiation. To overcome this, we identified by RNA-seq 13 transcription factors (TFs) failing to upregulate to mature hepatocyte levels after organoid differentiation, and that p53 was wrongly upregulated during that same process. Overexpression of these TFs, combined with p53 knockdown, resulted in more mature differentiated organoids, as tested by RNA, protein and functional studies. Finally, we looked at the abundance of mature hepatocytes in the cultures using single cell RNA-seq. Cholangiocyte organoids contained hepatocyte-primed progenitor populations, while differentiated organoids were very heterogeneous, with only one cluster showing features of differentiation into the hepatocyte lineage. TF overexpression or p53 knockdown increased the number of organoids differentiating into the right fate.

This study defines TFs and pathways promoting and hindering hepatocyte maturation. Translating these targets to a clinical setting will help develop a treatment to promote HPC activation and differentiation, regenerating chronically injured livers at an earlier stage, as well as overcoming the shortage in available organs.