Hepatocyte Differentiation From Mouse Liver Ductal Organoids By Transducing Four Liver-Specific Transcription Factors

Abstract

Background & Aims: Hepatocyte sources that are expandable in vitro are required for liver regenerative medicine and to elucidate the mechanisms underlying the physiological functions of the liver. Liver ductal organoids (LDOs) comprise liver tissue stem cells with a bipotential capacity to differentiate into hepatocyte and cholangiocyte lineages, and can thus serve as a hepatocyte source. However, using current differentiation methods LDOs differentiate into immature hepatocytes while retaining strong cholangiocyte characteristics. We thus investigated an alternative differentiation method for LDOs to achieve hepatocyte maturation. Methods: We extracted 12 candidate transcription factors to induce hepatocyte differentiation by comparing their gene expression in LDOs and liver tissues. After evaluating the effects of these transcription factors on LDOs, we analyzed the comprehensive gene expression profile, protein expression, and hepatic function in the transduced organoids. Results: We identified a combination of four transcription factors, Hnf4a, Foxa1, Prox1, and Hlf, which upregulated hepatic lineage markers and downregulated cholangiocyte markers. Differentiation-induced LDOs showed more hepatocyte-specific characteristics than those with the conventional method, enhancing the transition from cholangiocyte to hepatocyte lineage and hepatic functions, such as liver-specific protein synthesis, lipid droplet deposition, and ammonia detoxification. Conclusion: Transduction of the four transcription factors (Hnf4a, Foxa1, Prox1, Hlf) is a promising strategy to promote the differentiation of LDOs to obtain mature hepatocyte-like cells with better functionality.

Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model

“…However, this pro-differentiation effect was not observed when DAPT was combined with FGF19, indicating that the effect of DAPT on hepatocyte differentiation may be context-dependent, potentially requiring specific synergistic interactions with other factors for optimal effectiveness. A combination of DAPT and Dex resulted in the highest albumin-yielding medium, with the addition of FGF19 not improving the albumin yield any further [ 29 ]. FGF19 on the other hand prevented apoptosis [ 42 ].…”

“…Imaging of the organoids was conducted 1-h post-treatment using a Keyence confocal immunofluorescence microscope [ 29 ]. Images were captured under each of the four conditions, focusing on the rhodamine fluorescence in the green channel.…”