Expansion and characterization of peribiliary gland-resident stem cells using organoid cultures

M.M.A. Verstegen, M. de Wolf, K. Burka, M.J.C. Bijvelds, H. Gehart, J.N.M Ijzermans, H.R. de Jonge, L.J.W. van der Laan

Chair(s): Prof. dr. C.C. Baan & dr. M.R. Rookmaaker

Wednesday 8 march 2017

17:54 - 18:06h at Hendrik Marsmanzaal

Categories: Parallel - Basaal

Parallel session: Parallelsessie V – Basaal 1 - Analyse niet-hematopoietische cellen in orgaantransplantatie

Integrity of the biliary tree is imperative for liver function. Though evidence suggests that peribiliary glands (PBG) harbor stem cells which contribute to bile duct homeostasis and repair during disease and injury, these biliary stem cells are still not well characterized. In addition, the PBG may also play a role in repairing ischemic damage during liver transplantation. Therefore, the aim of this study is to expand and characterize biliary stem cells using 3-dimensional (3D) organoid cultures from bile ducts. Human extra-hepatic bile ducts (n=32) were collected from donor liver grafts or explant patient livers at time of liver transplantation. Biliary organoid cultures were initiated using similar conditions as described for human liver biopsies and propagated by weekly passaging for over 6 months. RNA expression analysis (q-PCR) and immunohistochemistry was performed. Transporter channel function was measured using Ussing chamber technology and Forskolin Induced Swelling (FIS) assays. In addition, the hepatocyte differentiation potential of biliary stem cells was studied.

Organoids were efficiently grown from the common bile duct for many passages (>6 months) and compared to liver parenchyma-derived organoids. As expected, bile duct organoids stain positive for biliary cell markers CK19, Epcam and MUC1. RNA analysis showed expression of stem cell markers LGR5, PDX1 and Sox9 and showed less hepatic differentiation capacity of biliary organoids compared to liver organoids. Preliminary results suggest the presence of functional transport channels in the biliary organoids, as they were responsive to forskolin, vasoactive intestinal peptide (VIP) and bicarbonate. Extensive characterization of the 3D cultures using proteomics and gene-array analysis are ongoing.
This study demonstrates the presence of LGR5-positive stem/progenitor cells in human extra hepatic bile duct. These organoids can be propagated long-term, express hepato-biliary genes and proteins, and show functional transporter channel activity. Biliary organoids could potentially be used to model biliary disease and biliary strictures after liver transplantation.