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

Lorena Loarca, Thiago M. De Assuncao, Nidhi Jalan-Sakrikar, Steve Bronk, Anuradha Krishnan, Bing Huang, Leslie Morton, Christy Trussoni, Lorena Marcano Bonilla, Eugene Krueger, Steve O'Hara, Patrick Splinter, Guang Shi, María José Lorenzo Pisarello, Gregory James Gores, Robert C Huebert, Nicholas F La Russo

Research output: Contribution to journalArticle

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Abstract

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ∼5000 cells with a central lumen of ∼150 μm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ∼80%. Cholangioids exposed 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 PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-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 in macrophage 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, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

Original languageEnglish (US)
Pages (from-to)1385-1396
Number of pages12
JournalLaboratory Investigation
Volume97
Issue number11
DOIs
StatePublished - Nov 1 2017

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Sclerosing Cholangitis
Macrophages
Bile Duct Diseases
Keratin-7
Keratin-19
Phenotype
In Vitro Techniques
Secretin
Cell Aging
Conditioned Culture Medium
Coculture Techniques
Confocal Microscopy
Hydrogen Peroxide
Interleukin-6
Western Blotting
Drug Therapy

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Cell Biology

Cite this

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; Pisarello, María José Lorenzo; Gores, Gregory James; Huebert, Robert C; La Russo, Nicholas F.

In: Laboratory Investigation, Vol. 97, No. 11, 01.11.2017, p. 1385-1396.

Research output: Contribution to journalArticle

Loarca, L, De Assuncao, TM, Jalan-Sakrikar, N, Bronk, S, Krishnan, A, Huang, B, Morton, L, Trussoni, C, Bonilla, LM, Krueger, E, O'Hara, S, Splinter, P, Shi, G, Pisarello, MJL, Gores, GJ, Huebert, RC & La Russo, NF 2017, 'Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis', Laboratory Investigation, vol. 97, no. 11, pp. 1385-1396. https://doi.org/10.1038/labinvest.2017.63
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 ; Pisarello, María José Lorenzo ; Gores, Gregory James ; Huebert, Robert C ; La Russo, Nicholas F. / Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis. In: Laboratory Investigation. 2017 ; Vol. 97, No. 11. pp. 1385-1396.
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abstract = "Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ∼5000 cells with a central lumen of ∼150 μm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ∼80{\%}. Cholangioids exposed 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 PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-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 in macrophage 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, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.",
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AU - Loarca, Lorena

AU - De Assuncao, Thiago M.

AU - Jalan-Sakrikar, Nidhi

AU - Bronk, Steve

AU - Krishnan, Anuradha

AU - Huang, Bing

AU - Morton, Leslie

AU - Trussoni, Christy

AU - Bonilla, Lorena Marcano

AU - Krueger, Eugene

AU - O'Hara, Steve

AU - Splinter, Patrick

AU - Shi, Guang

AU - Pisarello, María José Lorenzo

AU - Gores, Gregory James

AU - Huebert, Robert C

AU - La Russo, Nicholas F

PY - 2017/11/1

Y1 - 2017/11/1

N2 - Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ∼5000 cells with a central lumen of ∼150 μm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ∼80%. Cholangioids exposed 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 PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-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 in macrophage 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, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

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