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

This protocol describes how to recapitulate biliary development by differentiation of hPSCs into endoderm, foregut progenitor cells, hepatoblasts, cholangiocyte progenitors and mature 3D cholangiocyte-like cell organoids. The difficulty in isolating and propagating functional primary cholangiocytes...

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Published in:	Nature protocols Vol. 12; no. 4; pp. 814 - 827
Main Authors:	Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas RF, Vallier, Ludovic
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 01-04-2017 Nature Publishing Group
Subjects:	631/136/1425 631/136/532/1360 631/1647/1407/651 631/532/2064 Analytical Chemistry Bile ducts Bile Ducts - cytology Biliary tract diseases Biological Techniques Cell culture Cell Culture Techniques - methods Cell Differentiation Chemical compounds Computational Biology/Bioinformatics Differentiation Disease Endoderm Epidermal growth factor Epithelial Cells - cytology Extracellular matrix Fibroblasts Foregut Growth Humans Induced Pluripotent Stem Cells - cytology Kinases Life Sciences Maturation Microarrays Organic Chemistry Organoids Pharmacology Pluripotency Progenitor cells Protocol Stem cells United Kingdom > UK
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Abstract	This protocol describes how to recapitulate biliary development by differentiation of hPSCs into endoderm, foregut progenitor cells, hepatoblasts, cholangiocyte progenitors and mature 3D cholangiocyte-like cell organoids. 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.
AbstractList	This protocol describes how to recapitulate biliary development by differentiation of hPSCs into endoderm, foregut progenitor cells, hepatoblasts, cholangiocyte progenitors and mature 3D cholangiocyte-like cell organoids. 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. 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. This protocol describes how to recapitulate biliary development by differentiation of hPSCs into endoderm, foregut progenitor cells, hepatoblasts, cholangiocyte progenitors and mature 3D cholangiocyte-like cell organoids.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. 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. Keywords: Cholangiocyte, stem cell, cholangiopathy, disease modeling, Alagille Syndrome, Cystic Fibrosis, VX809, human pluripotent stem cell, PSC, hPSC, biliary, foregut progenitor cell, hepatoblast, cholangiocyte progenitor, 3D cell culture, organoid, differentiation, biliary disorder, cholangiocyte-like cell, CLC, endoderm, bile duct, biliary epithelial cell, gall bladder The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in studying biliary disorders and testing 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-day protocol closely recapitulates key stages of biliary development starting with the differentiation of hPSCs into endoderm and subsequently foregut progenitor cells, followed by the generation of hepatoblasts, cholangiocyte progenitors expressing early biliary markers and mature CLCs displaying cholangiocyte functionality. Compared to alternative protocols for biliary differentiation of hPSCs, our system does not require co-culture with other cell types and relies on chemically defined conditions up to and including the generation of cholangiocyte progenitors. 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.
Audience	Academic
Author	Geti, Imbisaat Bertero, Alessandro de Brito, Miguel Cardoso Hannan, Nicholas RF Vallier, Ludovic Sampaziotis, Fotios
AuthorAffiliation	4 Center for Biomolecular Sciences, University of Nottingham, UK 2 Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, UK 1 Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, Anne McLaren Laboratory, Department of Surgery, University of Cambridge, Cambridge, UK 3 Department of Hepatology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK 5 Wellcome Trust Sanger Institute, Hinxton, UK
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Notes	Ludovic Vallier and Nicholas RF Hannan share senior authorship for this manuscript.
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Snippet	This protocol describes how to recapitulate biliary development by differentiation of hPSCs into endoderm, foregut progenitor cells, hepatoblasts,... The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in the study of biliary disorders and the testing of novel... The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in studying biliary disorders and testing novel therapeutic...
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SubjectTerms	631/136/1425 631/136/532/1360 631/1647/1407/651 631/532/2064 Analytical Chemistry Bile ducts Bile Ducts - cytology Biliary tract diseases Biological Techniques Cell culture Cell Culture Techniques - methods Cell Differentiation Chemical compounds Computational Biology/Bioinformatics Differentiation Disease Endoderm Epidermal growth factor Epithelial Cells - cytology Extracellular matrix Fibroblasts Foregut Growth Humans Induced Pluripotent Stem Cells - cytology Kinases Life Sciences Maturation Microarrays Organic Chemistry Organoids Pharmacology Pluripotency Progenitor cells Protocol Stem cells
Title	Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells
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