Generation of functional ciliated cholangiocytes from human pluripotent stem cells

Abstract

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) would provide a model for studying the pathogenesis of cholangiopathies and for developing novel therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we describe a new strategy to generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of CFTR and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of new cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies showed that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice providing the first indication that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

Perspectives on the translation of in-vitro studies to precision medicine in Cystic Fibrosis

“…This accomplishment enabled studies of CFTR function in a relevant environment, namely, in the presence of cilia-bending sheer force. Interestingly, sheer force alone was sufficient to stimulate F508del-CFTR channel function after correction of its trafficking defect without the need of exogenous potentiators [83]. These findings highlight the importance of generating high fidelity models, capable of responding to natural mechanical stimuli as these models will better predict disease progression and therapeutic needs.…”

“…iPSC-derived models of cholangiocytes promise to provide insight into CF related liver disease. Notably, a recent publication describes a protocol that is highly efficient in generating mature, ciliated cholangiocytes [83]. This accomplishment enabled studies of CFTR function in a relevant environment, namely, in the presence of cilia-bending sheer force.…”

A Fluorescence-based Assay of Membrane Potential for High-throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

“…Although not an issue for Calu-3 and Caco-2 cells, other cells or tissues may not adhere well to the plates and may require precoating of the wells. For example, in previous studies, 2D cholangiocyte cultures were attached to the plates using collagen coating 16 .…”

Unraveling the Complexity of Liver Disease One Cell at a Time

“…These comparisons have been applied to induced pluripotent stem cell (IPSC)-derived hepatic endothelial cells, cholangiocytes, and hepatocytes. 89,[92][93][94] For example, a comparison of IPSC-derived two-dimensional (2D) monocultures to three-dimensional (3D) multicellular organoids containing mesenchymal, endothelial, and IPSC-derived hepatic parenchymal cells using scRNA-seq determined that while monocultures are able to recapitulate many features of in vivo hepatogenesis, organoid systems are better able to reconstruct liver cell intercellular cross-talk during development. 95 Within the organoid system, an epithelial migratory signature reminiscent of the liver bud during development is seen, potentially coordinated by intracellular cross-talk, such as VEGF signaling, that promotes endothelial cell network formation and hepatoblast differentiation.…”