Cholangiopathies represent a subset of liver diseases affecting the biliary three which collect and transport the bile. Despite their impact, the cause for the majority of biliary disorders remains elusive and treatment options are limited. Poor access to primary tissue, difficulties in culturing and maintaining primary cholangiocytes in vitro and physiological limitations of animal models restrict our insight in the pathophysiology of cholangiopathies and drug development. Consequently, there is currently no cure for disorders affecting cholangiocytes and end stage disease require liver transplantation.
Human induced pluripotent stem cells (hIPSCs) could provide an advantageous solution to bypass this major challenge. Indeed, these pluripotent stem cells are generated by direct reprograming of somatic cells and they can proliferate almost indefinitely in vitro while maintaining their capacity to differentiate into almost any cell types. Interestingly, hIPSCs can be derived from patients with genetic disorders and then differentiated into the relevant cell types for disease modeling purposes. Of direct interest, we recently developed a protocol to generate cholangiocytes from hIPSCs using chemically define conditions. The resulting cells display functional characteristics of their in vivo counterpart including the capacity to transport bile acid. The goal of the Relieve-Chol project was to improve our culture system for the production of large quantity of hIPSCs derived cholangiocytes in conditions compatible with the pharmaceutical industry requirements.
To reduce the time and costs of generating hIPSCs derived cholangiocytes, we developed protocols to grow these cells in vitro using 2D and 3D culture. To achieve this, we screened a combination growth factors, basal media, extra-cellular matrix and splitting method. We uncovered that WNT signaling control the switch between expansion and maturation. Furthermore, we developed a method to generate frozen stock of hIPSCs derived cholangiocytes. However, more experiments remains to be done to create a self-propagating pool for the generation of hIPSCs derived cholangiocytes. Indeed, in vitro produced cholangicoytes could only be grown for 5 passages in 2 D culture conditions without losing their biliary characteristics.
Despite this drawback, we were able to transplant these cells in immune deficient mice and show that the resulting cells could for bile duct structure after transplantation. Furthermore, to enhance the potential of hIPSCs cholangiocytes for regenerative medicine applications a biodegradable scaffold was used to try to create a 3D tissue mimicking bile duct. The experiments showed that hIPSCs derived cholangiocytes can grow into a tissue format on biodegradable PGA scaffolds whilst maintaining key functional and structural characteristics.
hIPSCs generated cholangiocytes were also used to model Cystic Fibrosis in vitro. hIPSCs derived from CF patients were corrected for the original mutation. The resulting pair of mutant and corrected isogenic hIPSC lines was then differentiated into cholangiocytes. These cells have then been fully characterised to validate their relevance for modelling Cystic fibrosis and for testing small molecules.
Based on these results, two patents have been submitted and the resulting intellectual property has been licensed to the biotechnology company DefiniGEN. The goal is to apply this technology for generating in vitro of model for cholangiopathies. These models will be commercialised to drug screening companies and to develop new therapeutics.
Thus, the Relieve-Chol project has achieved its primary objective to create a novel drug screening platform for the discovery of therapeutics targeting genetic form of cholangiopathies.
