Development and characterization of human-induced pluripotent stem cell-derived cholangiocytes. Lab Invest 2015 Jun;95(6):684-96
Date
04/14/2015Pubmed ID
25867762Pubmed Central ID
PMC4447567DOI
10.1038/labinvest.2015.51Scopus ID
2-s2.0-84929851353 (requires institutional sign-in at Scopus site) 63 CitationsAbstract
Cholangiocytes are the target of a heterogeneous group of liver diseases known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a stepwise differentiation strategy toward iDCs, using precise temporal exposure to key biliary morphogens, and we characterized the cells, using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in three-dimensional culture. In vivo, the cells engraft within mouse liver, following retrograde intrabiliary infusion. In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modeling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies.
Author List
De Assuncao TM, Sun Y, Jalan-Sakrikar N, Drinane MC, Huang BQ, Li Y, Davila JI, Wang R, O'Hara SP, Lomberk GA, Urrutia RA, Ikeda Y, Huebert RCAuthors
Gwen Lomberk PhD Professor in the Surgery department at Medical College of WisconsinThiago Milech De Assuncao Research Scientist II in the Surgery department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
AnimalsBile Ducts
Biomarkers
Calcium Signaling
Cell Differentiation
Cell Engineering
Cell Line
Epithelial Cells
Humans
Induced Pluripotent Stem Cells
Liver
Mice
Real-Time Polymerase Chain Reaction