Abstract:
The exome sequencing study of cholangiocarcinoma (CCA) revealed SMAD4 mutation was frequently observed. The role of SMAD4 to cholangiocarcinogenesis as well as cell origin of CCA are not fully understand. According to current CCA model including cell lines and animal models, it still limited in resembling human cells and early event during carcinogenesis. The objective of this study was to study the role of SMAD4 signaling disruption in cholangiocyte progenitors. To clarify these questions, we used induced pluripotent stem cells (iPSCs) to differentiate into cholangiocyte organoids by using growth factors induction in this study. The result demonstrated that cholangiocyte organoids were revealed cholangiocyte progenitor potential to long-term propagate (>8 months) and cholangiocyte-associated genes including hepatocyte-like cell conversion capacity. Our findings indicated that cholangiocyte organoids could represent as modelling for further CCA study. Additionally, we generated inducible SMAD4 knockout iPSC for SMAD4 knockout study in cholangiocyte organoids by using CRISPR/Cas9. Loss of SMAD4 in cholangiocyte organoids significantly affected on proliferation capacity, DNA damage-induced radiation response as well as tubular morphogenesis of cholangiocyte progenitors. The knowledge of SMAD4 knockout in cholangiocyte progenitors will provide more understanding of early event mechanism during cholangiocarcinogenesis and beneficial to develop CCA model in further study.
