O1119: Biliary Epithelial-mesenchymal Transition in Liver Ductal Organoids Is Attenuated by Human Amniotic Fluid Stem Cells

Background: Liver fibrosis in biliary atresia can progress rapidly and turn into an irreversible stage of liver cirrhosis. It has been demonstrated that biliary epithelial-mesenchymal transition (EMT) of reactive ductular cells is associated with liver fibrosis in biliary atresia. In this process, bile duct epithelial cells transform into fibrogenic myofibroblasts resulting in increased collagen deposition in the liver. Human amniotic fluid stem cells (hAFSCs) are pluripotent cells that possess anti-fibrotic potential. We aimed to investigate whether hAFSCs can prevent biliary EMT in liver ductal organoids.

Methods: Liver ductal organoids were generated from the intrahepatic bile duct of healthy neonatal mice. These organoids are composed of cholangiocytes that harbor liver progenitor cells. hAFSCs were co-cultured with liver ductal organoids in Matrigel for 2 days, followed by induction of biliary EMT via administration of the fibrogenic cytokine, transforming growth factor beta-1 (TGF-β1), in culture medium for 48 hours. The expressions of myofibroblast markers and type I collagen were evaluated by RT-qPCR and immunofluorescent staining. Data were compared using one-way ANOVA with Tukey’s post hoc test.

Results: Following administration of TGF-β1, liver ductal organoids were undergoing biliary EMT. They did not lose cell-cell adhesion as indicated by no change in E-cadherin expression; however, they had an increased expression of myofibroblast markers alpha-smooth muscle actin (α-SMA), N-cadherin, and COL1A1 (Fig. A). Administration of hAFSCs to the organoids receiving TGF-β1 resulted in decreased expression of α-SMA, N-cadherin, and COL1A1 (Fig. A). This was also confirmed by immunofluorescent staining of α-SMA and collagen 1 (Fig B).

Conclusion: TGF-β1 is a potent cytokine that induces the development of biliary EMT in liver ductal organoids. hAFSCs significantly attenuated the TGF-β1-induced biliary EMT in liver ductal organoids. This study indicates that hAFSCs have the potential to prevent the progression of liver fibrosis in biliary atresia.