The development of protocols for pluripotent stem cell (PSC) differentiation into cholangiocytes and cholangiocyte organoids in three-dimensional structures represent a huge advance in both research and medical fields because of the limited access to primary human cholangiocytes and the potential bias induced by animal models used to study cholangiopathies in vivo. PSC-derived cholangiocyte organoids consisting of either cysts with luminal space or branching tubular structures are composed of cells with apico-basal polarity that can fulfill cholangiocyte functions like the transport of bile salts. Several protocols of PSC differentiation have already been published but we added to the detailed protocol we describe here some notes or advice to facilitate its handling by new users. We also propose detailed protocols to carry out some of the characterization analyses using immunofluorescence to study the expression of specific markers and a functionality test to visualize bile acid transport using cholyl-lysyl-fluorescein (CLF).
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