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A protocol for generating biliary epithelial cells from human pluripotent stem cells facilitates disease modeling and drug screening. Although bile duct disorders are well-recognized causes of liver disease, the molecular and cellular events leading to biliary dysfunction are poorly understood. To enable modeling and drug discovery for biliary disease, we describe a protocol that achieves efficient differentiation of biliary epithelial cells (cholangiocytes) from human pluripotent stem cells (hPSCs) through delivery of developmentally relevant cues, including NOTCH signaling. Using three-dimensional culture, the protocol yields cystic and/or ductal structures that express mature biliary markers, including apical sodium-dependent bile acid transporter, secretin receptor, cilia and cystic fibrosis transmembrane conductance regulator (CFTR). We demonstrate that hPSC-derived cholangiocytes possess epithelial functions, including rhodamine efflux and CFTR-mediated fluid secretion. Furthermore, we show that functionally impaired hPSC-derived cholangiocytes from cystic fibrosis patients are rescued by CFTR correctors. These findings demonstrate that mature cholangiocytes can be differentiated from hPSCs and used for studies of biliary development and disease.

Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4 , which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4 -related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump ( ABCB11 ) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection. Neonatal cholestasis is a result of elevated bile acid levels, and is associated with mutations in genes regulating bile acid homeostasis. Here the authors identify mutations in the bile acid sensing farnesoid X receptor in four individuals with neonatal cholestasis from two unrelated families.

Tom Karlsen and colleagues report an association study for primary sclerosing cholangitis (PSC), a severe liver disease, using the Immunochip array. They identify nine loci newly associated with PSC and examine pleiotropy with other autoimmune disorders. Primary sclerosing cholangitis (PSC) is a severe liver disease of unknown etiology leading to fibrotic destruction of the bile ducts and ultimately to the need for liver transplantation 1 , 2 , 3 . We compared 3,789 PSC cases of European ancestry to 25,079 population controls across 130,422 SNPs genotyped using the Immunochip 4 . We identified 12 genome-wide significant associations outside the human leukocyte antigen (HLA) complex, 9 of which were new, increasing the number of known PSC risk loci to 16. Despite comorbidity with inflammatory bowel disease (IBD) in 72% of the cases, 6 of the 12 loci showed significantly stronger association with PSC than with IBD, suggesting overlapping yet distinct genetic architectures for these two diseases. We incorporated association statistics from 7 diseases clinically occurring with PSC in the analysis and found suggestive evidence for 33 additional pleiotropic PSC risk loci. Together with network analyses, these findings add to the genetic risk map of PSC and expand on the relationship between PSC and other immune-mediated diseases.

Biliary atresia (BA) is a progressive inflammatory fibrosclerosing disease of the biliary system and a major cause of neonatal cholestasis. It affects 1:5,000–20,000 live births, with the highest incidence in Asia. The pathogenesis is still unknown, but emerging research suggests a role for ciliary dysfunction, redox stress and hypoxia. The study of the underlying mechanisms can be conceptualized along the likely prenatal timing of an initial insult and the distinction between the injury and prenatal and postnatal responses to injury. Although still speculative, these emerging concepts, new diagnostic tools and early diagnosis might enable neoadjuvant therapy (possibly aimed at oxidative stress) before a Kasai portoenterostomy (KPE). This is particularly important, as timely KPE restores bile flow in only 50–75% of patients of whom many subsequently develop cholangitis, portal hypertension and progressive fibrosis; 60–75% of patients require liver transplantation by the age of 18 years. Early diagnosis, multidisciplinary management, centralization of surgery and optimized interventions for complications after KPE lead to better survival. Postoperative corticosteroid use has shown benefits, whereas the role of other adjuvant therapies remains to be evaluated. Continued research to better understand disease mechanisms is necessary to develop innovative treatments, including adjuvant therapies targeting the immune response, regenerative medicine approaches and new clinical tests to improve patient outcomes. Biliary atresia is a devastating paediatric inflammatory disease of the bile ducts that restricts flow of bile from the liver. In this Primer, Tam et al. summarize current research on biliary atresia, covering its epidemiology, mechanisms, diagnosis and management, quality of life, and future directions for research.

Cholestatic liver injuries, characterized by regional damage around the bile ductular region, lack curative therapies and cause considerable mortality. Here we generated a high-definition spatiotemporal atlas of gene expression during cholestatic injury and repair in mice by integrating spatial enhanced resolution omics sequencing and single-cell transcriptomics. Spatiotemporal analyses revealed a key role of cholangiocyte-driven signaling correlating with the periportal damage-repair response. Cholangiocytes express genes related to recruitment and differentiation of lipid-associated macrophages, which generate feedback signals enhancing ductular reaction. Moreover, cholangiocytes express high TGFβ in association with the conversion of liver progenitor-like cells into cholangiocytes during injury and the dampened proliferation of periportal hepatocytes during recovery. Notably, Atoh8 restricts hepatocyte proliferation during 3,5-diethoxycarbonyl-1,4-dihydro-collidin damage and is quickly downregulated after injury withdrawal, allowing hepatocytes to respond to growth signals. Our findings lay a keystone for in-depth studies of cellular dynamics and molecular mechanisms of cholestatic injuries, which may further develop into therapies for cholangiopathies. A comprehensive time series characterization of a mouse model of cholestatic liver injury with spatial enhanced resolution omics sequencing and single-cell RNA sequencing identifies zonal responses to insult, such as cholangiocyte signaling recruiting lipid-associated macrophages.

Carl Anderson and colleagues report a genome-wide association study identifying 13 new susceptibility loci for primary biliary cirrhosis, a chronic autoimmune liver disease. In addition to the HLA locus, six genetic risk factors for primary biliary cirrhosis (PBC) have been identified in recent genome-wide association studies (GWAS). To identify additional loci, we carried out a GWAS using 1,840 cases from the UK PBC Consortium and 5,163 UK population controls as part of the Wellcome Trust Case Control Consortium 3 (WTCCC3). We followed up 28 loci in an additional UK cohort of 620 PBC cases and 2,514 population controls. We identified 12 new susceptibility loci (at a genome-wide significance level of P < 5 × 10 −8 ) and replicated all previously associated loci. We identified three further new loci in a meta-analysis of data from our study and previously published GWAS results. New candidate genes include STAT4 , DENND1B , CD80 , IL7R , CXCR5 , TNFRSF1A , CLEC16A and NFKB1 . This study has considerably expanded our knowledge of the genetic architecture of PBC.

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing 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 generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (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 cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies show that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice indicating that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease. Current protocols to generate cholangiocytes from human pluripotent cells produce immature cells. Here the authors identify retinoic acid, BMP, cAMP and Rho kinase pathways as regulators of cholangiocyte maturation, and generate ciliated cholangiocytes expressing high levels of CFTR that form ductal structures in vivo.

Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholangiopathy. The role of cholangiocytes (biliary epithelial cells) in PSC pathogenesis is unknown and remains an active area of research. Here, through cellular, molecular and next-generation sequencing (NGS) methods, we characterize and identify phenotypic and signaling features of isolated PSC patient-derived cholangiocytes. We isolated cholangiocytes from stage 4 PSC patient liver explants by dissection, differential filtration and immune-magnetic bead separation. We maintained cholangiocytes in culture and assessed for: (i) cholangiocyte, cell adhesion and inflammatory markers; (ii) proliferation rate; (iii) transepithelial electrical resistance (TEER); (iv) cellular senescence; and (v) transcriptomic profiles by NGS. We used two well-established normal human cholangiocyte cell lines (H69 and NHC) as controls. Isolated PSC cells expressed cholangiocyte (eg, cytokeratin 7 and 19) and epithelial cell adhesion markers (EPCAM, ICAM) and were negative for hepatocyte and myofibroblast markers (albumin, α -actin). Proliferation rate was lower for PSC compared with normal cholangiocytes (4 vs 2 days, respectively, P <0.01). Maximum TEER was also lower in PSC compared with normal cholangiocytes (100 vs 145 Ωcm 2 , P <0.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all P <0.01). The proportion of cholangiocytes staining positive for senescence-associated β -galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, P <0.01). Finally, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclusion, we have demonstrated that high-purity cholangiocytes can be isolated from human PSC liver and grown in primary culture. Isolated PSC cholangiocytes exhibit a phenotype that may reflect their in vivo contribution to disease and serve as a vital tool for in vitro investigation of biliary pathobiology and identification of new therapeutic targets in PSC.