Matsui Satoshi, Ochiai Miyuki, Yasuda Katsutaro, Mae Shin-Ichi, Kotaka Maki, Toyoda Taro, Yamamoto Takuya, Osafune Kenji
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Stem Cell Res. 2019 Mar;35:101400. doi: 10.1016/j.scr.2019.101400. Epub 2019 Jan 31.
Cholangiocytes are the epithelial cells that line bile ducts, and ductal plate malformation is a developmental anomaly of bile ducts that causes severe congenital biliary disorders. However, because of a lack of specific marker genes, methods for the stepwise differentiation and isolation of human induced pluripotent stem cell (hiPSC)-derived cholangiocyte progenitors at ductal plate stages have not been established. We herein generated an AQP1-GFP reporter hiPSC line and developed a combination treatment with transforming growth factor (TGF) β2 and epidermal growth factor (EGF) to induce hiPSC-derived hepatoblasts into AQP1 cells in vitro. By confirming that the isolated AQP1 cells showed similar gene expression patterns to cholangiocyte progenitors at the remodeling ductal plate stage around gestational week (GW) 20, we established a differentiation protocol from hiPSCs through SOX9CK19AQP1 ductal plate-like cells into SOX9CK19AQP1 remodeling ductal plate-like cells. We further generated 3D bile duct-like structures from the induced ductal plate-like cells. These results suggest that AQP1 is a useful marker for the generation of remodeling ductal plate cells from hiPSCs. Our methods may contribute to elucidating the differentiation mechanisms of ductal plate cells and the pathogenesis of ductal plate malformation.
胆管细胞是衬于胆管的上皮细胞，而胆管板畸形是一种胆管发育异常，可导致严重的先天性胆道疾病。然而，由于缺乏特异性标记基因，尚未建立在胆管板阶段对人诱导多能干细胞（hiPSC）来源的胆管细胞祖细胞进行逐步分化和分离的方法。我们在此生成了一种水通道蛋白1（AQP1）-绿色荧光蛋白（GFP）报告基因hiPSC系，并开发了一种联合使用转化生长因子（TGF）β2和表皮生长因子（EGF）的处理方法，以在体外将hiPSC来源的肝母细胞诱导为AQP1细胞。通过确认分离出的AQP1细胞在妊娠第20周左右的重塑胆管板阶段显示出与胆管细胞祖细胞相似的基因表达模式，我们建立了一种从hiPSC经SOX9⁺CK19⁺AQP1胆管板样细胞分化为SOX9⁺CK19⁺AQP1重塑胆管板样细胞的方案。我们还从诱导的胆管板样细胞生成了三维胆管样结构。这些结果表明，AQP1是从hiPSC生成重塑胆管板细胞的有用标记物。我们的方法可能有助于阐明胆管板细胞的分化机制以及胆管板畸形的发病机制。
