Dissecting regulators of hepatocyte identity using a CRISPR activation screening in iPSC-derived hepatocytes
Tchorz, Jan and Lueck-Delgado, Claudia (2025) Dissecting regulators of hepatocyte identity using a CRISPR activation screening in iPSC-derived hepatocytes. n/a, x (x). x-x. ISSN na
Abstract
Induced pluripotent stem cell-derived hepatocytes (iHEPs) hold significant promise for
disease modeling, drug testing, and regenerative medicine. However, their limited
maturity compared to primary human hepatocytes remains a major challenge. This
study employed a CRISPR activation (CRISPRa) system in induced pluripotent stem
cells to investigate the role of selected transcription factors (TFs) in enhancing iHEP
maturation during differentiation. Following sgRNA-mediated activation of 14 target
TFs, bulk RNA sequencing and subsequent transcriptomic analyses—including
UMAP visualization, MuSIC deconvolution, gene set enrichment analysis (GSEA),
and hepatocyte zonation marker assessment—were performed to evaluate changes
in cell identity and function.
Our results demonstrate that activation of AR and CEBPA significantly promotes
hepatocyte differentiation and metabolic function, as evidenced by increased
proportions of hepatocyte-like cells, enrichment of hepatocyte-specific gene sets, and
activation of key liver metabolic pathways. Conversely, TFs such as IRF1 and IRF2
biased differentiation toward cholangiocyte lineage, while NCOA2 and MYC activation
favored mesenchymal cell fate indicating epithelial-to-mesenchymal transition.
Notably, HNF4A activation unexpectedly failed to enhance hepatocyte maturation,
suggesting potential technical or biological complexities. Comparative analyses
revealed that hepatocyte enrichment strongly correlates with the activation of
metabolic pathways, underscoring the functional relevance of AR and CEBPA-driven
maturation.
This comprehensive transcriptomic study highlights the critical influence of TF
selection on iHEP fate decisions and functional maturation. Identifying TFs that
robustly drive hepatocyte differentiation—particularly AR and CEBPA—offers valuable
insights for optimizing differentiation protocols aimed at producing functionally mature
hepatocytes for biomedical applications.
Item Type: | Article |
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Date Deposited: | 14 Aug 2025 00:45 |
Last Modified: | 14 Aug 2025 00:45 |
URI: | https://oak.novartis.com/id/eprint/56711 |