Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors
Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors
复制标题
通过优化重组腺相关病毒载体对肝导管类器官进行基因操作
DOI：
10.1074/jbc.ra119.008616
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发表时间：
2019-09-20
影响因子：
4.8
通讯作者：
Zhao, Bing
中科院分区：
文献类型：
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作者：
Wei, Jinsong;Ran, Gai;Zhao, Bing
了解肝导管细胞（胆管细胞）如何分化为肝细胞的机制将允许肝脏再生医学。新兴的肝导管类器官提供了研究胆管细胞向肝细胞分化的离体系统。然而，由于目前的基因操作方法需要类器官解离成单细胞并且效率低，因此难以剖析这些类器官中的特定基因功能。在这里，我们开发了腺相关病毒（AAV）载体AAV-DJ作为一个强大的工具，以小鼠和人类肝导管类器官。通过AAV-DJ介导的靶基因的上调或下调，我们成功地操纵了胆管细胞向肝细胞的分化。我们通过过表达肝细胞特异性调节因子肝细胞核因子4α（HNF 4 α）诱导分化，并通过刺激Notch信号或干扰Smad信号来阻断分化。进一步筛选对胆管细胞向肝细胞分化至关重要的转录因子，确定HOP同源框（HOPX）、T-box 15（TBX 15）和转录因子CP 2样1（TFCP 2L 1）为主要调节因子。我们的结论是，我们开发的这种高效方便的基因操作系统可以促进对参与细胞谱系转换的基因的研究，并使工程类器官在再生医学中的应用成为可能。
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.