Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis

LOARCA, LORENA; DE ASSUNCAO, THIAGO M; JALAN-SAKRIKAR, NIDHI; BRONK, STEVE; KRISHNAN, ANURADHA; HUANG, BING; MORTON, LESLIE; TRUSSONI, CHRISTY; BONILLA, LORENA MARCANO; KRUEGER, EUGENE; O?HARA, STEVE; SPLINTER, PATRICK; SHI, GUANG; LORENZO PISARELLO, MARÍA JOSÉ; GORES, GREGORY J; HUEBERT, ROBERT C; LARUSSO, NICHOLAS F

LABORATORY INVESTIGATION

NATURE PUBLISHING GROUP

Año: 2017 p. 1 - 12

0023-6837

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effectivepharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while othersshowed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescenceand macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop andcharacterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal humancholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formedspheroids of ~ 5000 cells with a central lumen of ~ 150 μm. By confocal microscopy and western blot, cholangioidsretained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor andGM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~ 80%. Cholangioidsexposed to hydrogen peroxide exhibited cellular senescence and the senescence-associated secretory phenotype (SASP;increased IL-6, p21, SA‐β-Gal, yH2A.x and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSCpatients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the numberof macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture withnon-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater number of extracellular vesicles(EVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~ 2-fold increase inmacrophage migration. In summary, we developed a method to generate normal and diseased cholangioids,characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, anddemonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus willbe useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.