Multi-omics analysis reveals distinct gene regulatory mechanisms between primary and organoid-derived human hepatocytes.

Hepatic organoid cultures are a powerful model to study liver development and diseases in vitro. However, hepatocyte-like cells differentiated from these organoids remain immature compared to primary human hepatocytes (PHHs), which are the benchmark in the field. Here, we applied integrative single-cell transcriptome and chromatin accessibility analysis to reveal gene regulatory mechanisms underlying these differences. We found that, in mature human hepatocytes, activator protein 1 (AP-1) factors co-occupy regulatory regions with hepatocyte-specific transcription factors, including HNF4A, suggesting their potential cooperation in governing hepatic gene expression. Comparative analysis identified distinct transcription factor sets that are specifically active in either PHHs or intrahepatic cholangiocyte organoid (ICO)-derived human hepatocytes. ELF3 was one of the factors uniquely expressed in ICO-derived hepatocytes, and its expression negatively correlated with hepatic marker gene expression. Functional analysis further revealed that ELF3 depletion increased the expression of key hepatic markers in ICO-derived hepatocytes. Our integrative analysis provides insights into the transcriptional regulatory networks of PHHs and hepatic organoids, thereby informing future strategies for developing improved hepatic models.