Project description:Mutations of isocitrate dehydrogenase (IDH) 1 or 2 occur in 10-30% of intrahepatic cholangiocarcinomas (ICCs). However, their functional roles in biliary carcinogenesis are still unknown. We used microarrays to investigate how mutant IDH1 affect the gene expression of intrahepatic biliary organoids generated from normal murine liver.
Project description:3D cell culture systems (organoids), cultured from liver biopsies, resemble biliary epthelium (cholangiocytes) upon culture. These intrahepatic cholangiocyte organoids (ICOs) maintain their gentic stability, transcription profile and can be maintained propagated during long periods of culturing while still maintaining their functional properties. The data presented in this ArrayExpress submission contained micro array results of 6 from 15 different ICO lines that were analyzed for their functional cholangiocyte ion-channels, involved in secreting a protective bicarbonate layer. It was shown that these data closely resemble the expression profiles of extrahepatic cholangiocyte organoids (ECOs), previously described by Sampaziotis et al. and deposited in the EMBL-EBI ArrayExpress repository (E-MTAB-4591)
Project description:Current model systems for studying intrahepatic biliary disease fail to recapitulate the architecture of intrahepatic bile ducts. Organoids from intrahepatic cholangiocytes can with a new culture method grow with a branching pattern resembling the structure of intrahepatic bile ducts. This dataset contains data from samples of these organoids and from control organoids cultured without this technique. Culture have been performed using isotope labeled amino acids to help distinguish cell derived proteins from the basement membrane extract used as a culture substrate for the organoids.
Project description:Although providing promising and unique tools for studying cholangiocytes, current tissue-derived cholangiocyte-organoid systems do not recapitulate the complex architecture of the intrahepatic bile ducts in vitro. Here, we report a new method for creating branching cholangiocyte organoids (BRCO) from human adult tissue to study the intrahepatic biliary tree and diseases. BRCOs self-organize into large complex tubular structures, while closely resembling primary cholangiocytes on a transcriptomic and functional level. They are capable of mimicking branching bile duct development as well as being used for studying diseases in which the biliary tree does not develop properly (Alagille Syndrome). Furthermore, we deliver evidence that our culture method allows for formation of complex cholangiocyte cancer (cholangiocarcinoma, CCA) organoids. These branching CCA organoids resemble the primary tumor more closely compared to previously published protocols as well as showing unique tumor-specific drug responses. In conclusion, our culture method allows for creation of novel (malignant) cholangiocyte-organoids to study the intrahepatic bile ducts.
Project description:Although providing promising and unique tools for studying cholangiocytes, current tissue-derived cholangiocyte-organoid systems do not recapitulate the complex architecture of the intrahepatic bile ducts in vitro. Here, we report a new method for creating branching cholangiocyte organoids (BRCO) from human adult tissue to study the intrahepatic biliary tree and diseases. BRCOs self-organize into large complex tubular structures, while closely resembling primary cholangiocytes on a transcriptomic and functional level. They are capable of mimicking branching bile duct development as well as being used for studying diseases in which the biliary tree does not develop properly (Alagille Syndrome). Furthermore, we deliver evidence that our culture method allows for formation of complex cholangiocyte cancer (cholangiocarcinoma, CCA) organoids. These branching CCA organoids resemble the primary tumor more closely compared to previously published protocols as well as showing unique tumor-specific drug responses. In conclusion, our culture method allows for creation of novel (malignant) cholangiocyte-organoids to study the intrahepatic bile ducts.
Project description:Background: RNASeq was performed on organoids derived from livers of normal healthy donors and patients with biliary atresia to characterize transcriptomic signatures. Methods: Organoids generated from livers of normal healthy donors and patients with biliary atresia were cultured either in expansion (undifferentiated: 3 NCOs and 11 BACOs) or differentiation medium (differentiated: 3 BACOs). Liver tissues obtained from deceased-donor subjects served as normal controls (N=3). Total RNA was isolated from organoids and liver biopsy tissue specimens. Results: Organoids from patients with biliary atresia showed abnormal cell polarity, loss of tight junctions, increased permeability and decreased expression of genes related to epidermal growth factor (EGF)- and fibroblast growth factor 2 (FGF2)-signaling. When treated with EGF+FGF2, biliary atresia organoids expressed differentiation and functional markers with restored cell polarity. Conclusion: Organoids from biliary atresia are viable and have evidence of halted epithelial development. The induction of developmental markers, improved cell‐cell junction, and decreased epithelial permeability by EGF and FGF2 identifies potential strategies to promote epithelial maturation and function.
Project description:Restitution of the extrahepatic biliary luminal epithelium in cholangiopathies is poorly understood. Prominin-1 (Prom1) is a key component of epithelial ciliary body of stem/progenitor cells. Given that intrahepatic Prom1-expressing progenitor cells undergo cholangiocyte differentiation, we hypothesized that Prom1 may promote restitution of the extrahepatic bile duct (EHBD) epithelium following injury. Utilizing various murine biliary injury models, we identified Prom1-expressing cells in the peribiliary glands (PBGs) of the EHBD. These Prom1-expressing cells are progenitor cells which give rise to cholangiocytes as part of the normal maintenance of the EHBD epithelium. Following injury, these cells proliferate significantly more rapidly to re-populate the biliary luminal epithelium. Null mutation of Prom1 leads to significantly more than ten-fold dilated PBGs following rhesus rotavirus-mediated biliary injury. Cultured organoids derived from Prom1 knockout mice are comprised of biliary progenitor cells with altered apical-basal cellular polarity, significantly fewer and shorter cilia, and decreased organoid proliferation dynamics consistent with impaired cell motility. We, therefore, conclude that Prom1 is involved in biliary epithelial restitution following biliary injury in part through its role in supporting cell polarity.
Project description:3D cell culture systems (organoids), cultured from extrahepatic bile duct biopsies, resemble biliary epthelium (cholangiocytes) upon culture according to the protocol previously established for culturing intrahepatic cholangiocyte organoids from liver biopsies (Huch et al. Cell 2015). These extrahepatic cholangiocyte organoids (ECOs) in Canonical-Wnt culture conditions maintain their genetic stability, and transcription profile and can be maintained and propagated during long periods of culturing while still maintaining their functional properties. The data presented in this ArrayExpress submission contained micro array results of 3 different ECO lines that were analyzed for their functional cholangiocyte ion-channels, involved in secreting a protective bicarbonate layer. It was shown that these data closely resemble the expression profile of intrahepatic cholangiocyte organoids (ICOs) as described by our group. Results of the ICOs are deposited in the EMBL-EBI ArrayExpress repository under number E-MTAB-9044.
Project description:To define the regulatory role of Sox9 in intrahepatic biliary epithelium, we conducted bulk RNA-seq on biliary epithelial cells (BECs) from adult control (Sox9fl/fl) and conditional Sox9 knockout (Sox9cKO; Sox9fl/fl:Albumin-Cre) mice. Livers were dissociated to single cells and BECs isolated by FACS based on CD326 positivity. CD31+ and CD45+ cells were excluded by FACS. Gallbladder and extrahepatic tissues were excluded by dissection. RNA was isolated using Ambion RNAqueous Micro kit and libraries were made using Takara SMARTer Stranded Total RNA-seq Kit v3 Pico.
Project description:RNA-Sequencing was performed on mechanically dissociated, epithelial-enriched samples, of human extrahepatic biliary tissue from Gallbladder, Common Bile Duct, and Pancreatic Duct tissues. Sequencing was also performed on in vitro cultures of Organoid cell lines at passage 5 that were derived from human Gallbladder, Common Bile Duct, Pancreatic Duct, or Intrahepatic Bile Ducts.