Fotios Sampaziotis

Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation

The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct–like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

Potential of human induced pluripotent stem cells in studies of liver disease

Liver disease is a leading cause of death in the Western world. However, our insight into the underlying disease mechanisms and the development of novel therapeutic agents has been hindered by limited availability of primary tissue, intraspecies variability associated with the use of animal models, and reduced long‐term viability of isolated and diseased liver cells. The emergence of human induced pluripotent stem cells and differentiation protocols to generate hepatocyte‐like cells has opened the possibility of addressing these issues. Here, we discuss the recent progress and potential in the production of various cell types constituting the liver and their applications to model liver diseases and test drug toxicity in vitro. (Hepatology 2015;62:303‐311)

Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells

The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in the study of biliary disorders and the testing of novel therapeutic agents. To overcome this problem, we have developed a platform for the differentiation of human pluripotent stem cells (hPSCs) into functional cholangiocyte-like cells (CLCs). We have previously reported that our 26-d protocol closely recapitulates key stages of biliary development, starting with the differentiation of hPSCs into endoderm and subsequently into foregut progenitor (FP) cells, followed by the generation of hepatoblasts (HBs), cholangiocyte progenitors (CPs) expressing early biliary markers and mature CLCs displaying cholangiocyte functionality. Compared with alternative protocols for biliary differentiation of hPSCs, our system does not require coculture with other cell types and relies on chemically defined conditions up to and including the generation of CPs. A complex extracellular matrix is used for the maturation of CLCs; therefore, experience in hPSC culture and 3D organoid systems may be necessary for optimal results. Finally, the capacity of our platform for generating large amounts of disease-specific functional cholangiocytes will have broad applications for cholangiopathies, in disease modeling and for screening of therapeutic compounds.

Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development. Highlighted article: Novel optimized inducible knockdown and knockout platforms are developed and used to assess gene function in human pluripotent stem cells and their differentiated progeny.

Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells

Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.

Severe coagulopathy caused by rifampicin in patients with primary sclerosing cholangitis and refractory pruritus.

Pruritus is one of the most disabling clinical manifestations of cholestatic liver disease. Antipruritic agents are effective only in a proportion of patients [1], [2]. Rifampicin is an antituberculous agent recommended for relieving pruritus refractory to bile acid sequestrants [3]–[6]. This is the first report of severe, reversible prolongation of prothrombin time in patients with primary sclerosing cholangitis (PSC), following the introduction of rifampicin.

A retrospective study assessing fully covered metal stents as first-line management for malignant biliary strictures.

Objectives Fully covered self-expanding metal stents (FCSEMS) constitute the first type of metal stent that can easily be removed endoscopically and/or intraoperatively, which may be advantageous in the management of distal malignant biliary strictures (DMBS). To assess the efficacy of FCSEMS as first-line treatment for DMBS, we compared patency, survival and complication rates between FCSEMS, uncovered self-expanding metal stents (USEMS) and plastic stents (PS). Methods This was a multicentre retrospective study of 315 consecutive patients with DMBS, who underwent endoscopic retrograde cholangiopancreatography and stenting (FCSEMS, USEMS or PS) at two hospitals between 1 January 2007 and 31 December 2013. Stent patency and patient survival were compared using the Kaplan–Meier method; complication rates were compared using Fisher’s exact test; and Cox regression analysis was used to screen for confounding factors. Results FCSEMS were associated with prolonged stent patency (median=145 days) compared with USEMS (median=110 days, P<0.003) and PS (median=34 days, P<0.001). Biliary sepsis rates were lower for FCSEMS compared with PS (4.7 vs. 17.8%, P=0.02), whereas pancreatitis rates were higher for FCSEMS compared with USEMS (7.8 vs. 1.0%, P=0.04), but not PS (2.6%, P=NS). Conclusion The use of FCSEMS as first-line management for DMBS is associated with longer patency and reduced complication rates compared with the use of PS. However, the higher rate of pancreatitis compared with USEMS requires further evaluation in a large randomized controlled trial.

A case of acute liver failure due to etodolac

Etodolac is a cyclooxygenase 2 (COX-2)-specific non-steroidal anti-inflammatory agent, used predominantly in the management of musculoskeletal disease and arthritis. Dyspepsia, headache, dizziness, rash and pruritus are the commonest side effects. Hepatic impairment is extremely rare, occurring in less than 0.3% of patients [1, 2]. Liver failure has been reported only once, in a patient with underlying alcohol related chronic liver disease and cocaine abuse, contributing to the presentation [3]. We report the first case of etodolac induced acute liver failure in the absence of confounding factors such as chronic liver disease or concomitant use of other hepatotoxic agents.

Building better bile ducts

An organoid-based model promises to improve our understanding of bile duct disorders The bile ducts form a network of tubes within the liver and transfer bile produced in the liver to the bowel. In biliary disorders, this transport system fails, leading to the accumulation of toxic bile in the liver, damage, and permanent scarring (cirrhosis), which can ultimately be treated only through liver transplantation. Indeed, bile duct diseases (cholangiopathies) are the leading disorder treated (70%) by pediatric liver transplantation and account for a third of adult transplanted livers.