Abstract
Understanding the mechanism of how liver ductal cells (cholangiocytes) differentiate into hepatocytes would permit liver-regenerative medicine. Emerging liver ductal organoids provide an ex vivo system to investigate cholangiocyte-to-hepatocyte differentiation. However, as current gene manipulation methods require organoid dissociation into single cells and have only low efficiency, it is difficult to dissect specific gene functions in these organoids. Here we developed the adeno-associated virus (AAV) vector AAV-DJ as a powerful tool to transduce mouse and human liver ductal organoids. Via AAV-DJ-mediated up- or down-regulation of target genes, we successfully manipulated cholangiocyte-to-hepatocyte differentiation. We induced differentiation by overexpressing the hepatocyte-specifying regulator hepatocyte nuclear factor 4α (HNF4α) and blocked differentiation by stimulating Notch signaling or interfering with Smad signaling. Further screening for transcriptional factors critical for cholangiocyte-to-hepatocyte differentiation identified HOP homeobox (HOPX), T-box 15 (TBX15), and transcription factor CP2-like 1 (TFCP2L1) as master regulators. We conclude that this highly efficient and convenient gene manipulation system we developed could facilitate investigation into genes involved in cell lineage transitions and enable application of engineered organoids in regenerative medicine.
Highlights
Understanding the mechanism of how liver ductal cells differentiate into hepatocytes would permit liver-regenerative medicine
While searching for an ideal tool to deliver genes into liver ductal organoids, we focused on associated virus (AAV) vectors, which have been recognized for primary tissue affinity and biosafety in gene therapy
We found that combination of three point mutations (N498S, Y706F, and Y732F) further enhanced the transduction efficiency of double-stranded AAV-DJ–GFP in liver ductal organoids (80.5%) (Fig. 2, A–C)
Summary
Further screening for transcriptional factors critical for cholangiocyte-to-hepatocyte differentiation identified HOP homeobox (HOPX), T-box 15 (TBX15), and transcription factor CP2-like 1 (TFCP2L1) as master regulators We conclude that this highly efficient and convenient gene manipulation system we developed could facilitate investigation into genes involved in cell lineage transitions and enable application of engineered organoids in regenerative medicine. Screening of 16 hepatocyte-enriched transcriptional factors identified HOPX, TBX15, and TFCP2L1 as master regulators of hepatocyte differentiation This high-efficiency and convenient gene manipulation method facilitates the investigation of gene functions in liver lineage transition and application of engineered organoids in regenerative medicine
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