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

ABSTRACT: 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, ?-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.

SUBMITTER: Sampaziotis F

PROVIDER: S-EPMC4768345 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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

Sampaziotis Fotios F de Brito Miguel Cardoso MC Madrigal Pedro P Bertero Alessandro A Saeb-Parsy Kourosh K Soares Filipa A C FAC Schrumpf Elisabeth E Melum Espen E Karlsen Tom H TH Bradley J Andrew JA Gelson William Th WT Davies Susan S Baker Alastair A Kaser Arthur A Alexander Graeme J GJ Hannan Nicholas R F NRF Vallier Ludovic L

Nature biotechnology 20150713 8

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 ...[more]

PMID: 26167629

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Project description:Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC.

Project description:BackgroundIn diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for modeling of diabetic nephropathy and genetic podocytopathies and to complement animal studies. Recently, several methods have been published to derive podocytes from human-induced pluripotent stem cells (iPSCs) by directed differentiation. However, these methods have major variations in media composition and have not been compared.MethodsWe characterized our accelerated protocol by guiding the cells through differentiation with four different medias into MIXL1+ primitive streak cells with Activin A and CHIR for Wnt activation, intermediate mesoderm PAX8+ cells via increasing the CHIR concentration, nephron progenitors with FGF9 and Heparin for stabilization, and finally into differentiated podocytes with Activin A, BMP-7, VEGF, reduced CHIR, and retinoic acid. The podocyte morphology was characterized by scanning and transmission electron microscopy and by flow cytometry analysis for podocyte markers. To confirm cellular identity and niche localization, we performed cell recombination assays combining iPSC-podocytes with dissociated mouse embryonic kidney cells. Finally, to test iPSC-derived podocytes for the modeling of diabetic kidney disease, human podocytes were exposed to high glucose.ResultsPodocyte markers were expressed at similar or higher levels for our accelerated protocol as compared to previously published protocols that require longer periods of tissue culture. We confirmed that the human podocytes derived from induced pluripotent stem cells in twelve days integrated into murine glomerular structures formed following seven days of culture of cellular recombinations. We found that the high glucose-treated human podocytes displayed actin rearrangement, increased cytotoxicity, and decreased viability.ConclusionsWe found that our accelerated 12-day method for the differentiation of podocytes from human-induced pluripotent stem cells yields podocytes with comparable marker expression to longer podocytes. We also demonstrated that podocytes created with this protocol have typical morphology by electron microscopy. The podocytes have utility for diabetes modeling as evidenced by lower viability and increased cytotoxicity when treated with high glucose. We found that multiple, diverse methods may be utilized to create iPSC-podocytes, but closely mimicking developmental cues shortened the time frame required for differentiation.

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

Publications

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

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