39 results on '"Hannan, Nicholas R.F."'
Search Results
2. Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications
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Durczak, Paulina M., primary, Fair, Kathryn L., additional, Jinks, Nicholas, additional, Cuevas Ocaña, Sara, additional, Sainz Zuñiga, Carlos B., additional, and Hannan, Nicholas R.F., additional
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- 2024
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3. A rapid method for generating transplantable and biologically responsive colonic tissue from human induced pluripotent stem cells.
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Dalleywater, William, primary, Predeus, Alexander V, additional, Cakir, Batuhan, additional, Mazin, Pavel, additional, Vadakekolathu, Jayakumar, additional, Rutella, Sergio, additional, Meakin, Marian L, additional, Ritchie, Alison A, additional, Montazid, Shamir, additional, Cuevas Ocaña, Sara, additional, Holmes, Nadine, additional, Wright, Victoria, additional, Seng, Fei, additional, Bills, Adam, additional, Sculthorpe, Declan, additional, Elmentaite, Rasa, additional, Teichmann, Sarah A, additional, Irshad, Shazia, additional, Tomlinson, Ian, additional, Silver, Andrew, additional, Wildman, Ricky D, additional, Hannan, Nicholas R.F., additional, Rose, Felicity RAJ, additional, and Ilyas, Mohammad, additional
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- 2023
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4. hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency
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Segeritz, Charis-Patricia, Rashid, Sheikh Tamir, de Brito, Miguel Cardoso, Serra, Maria Paola, Ordonez, Adriana, Morell, Carola Maria, Kaserman, Joseph E., Madrigal, Pedro, Hannan, Nicholas R.F., Gatto, Laurent, Tan, Lu, Wilson, Andrew A., Lilley, Kathryn, Marciniak, Stefan J., Gooptu, Bibek, Lomas, David A., and Vallier, Ludovic
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- 2018
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5. Identification and functional characterisation of a rare MTTP variant underlying hereditary non-alcoholic fatty liver disease
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Grove, Jane I., primary, Kiu Lo, Peggy Cho, additional, Shrine, Nick, additional, Barwell, Julian, additional, Wain, Louise V., additional, Tobin, Martin D., additional, Salter, Andrew M., additional, Bennett, Neil, additional, John, Catherine, additional, Ntalla, Ioanna, additional, Jones, Gabriela E., additional, Neal, Christopher P., additional, Thomas, Mervyn G., additional, Kuht, Helen, additional, Gupta, Pankaj, additional, Vemala, Vishwaraj M., additional, Grant, Allister, additional, Adewoye, Adeolu B., additional, Shenoy, Kotacherry T., additional, Balakumaran, Leena K., additional, Hollox, Edward J., additional, Hannan, Nicholas R.F., additional, and Aithal, Guruprasad P., additional
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- 2021
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6. Targeted gene correction of [alpha].sub.1-antitrypsin deficiency in induced pluripotent stem cells
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Yusa, Kosuke, Rashid, S. Tamir, Strick-Marchand, Helene, Varela, Ignacio, Liu, Pei-Qi, Paschon, David E., Miranda, Elena, Ordonez, Adriana, Hannan, Nicholas R.F., Rouhani, Foad J., Darche, Sylvie, Alexander, Graeme, Marciniak, Stefan J., Fusaki, Noemi, Hasegawa, Mamoru, Holmes, Michael C., and Di Santo, James P.
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Stem cells -- Genetic aspects -- Health aspects ,Mutation (Biology) -- Health aspects ,Gene therapy -- Methods ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
Fixing the genes in iPS cells Before human induced pluripotent stem (iPS) cells can be used to treat genetically inherited human disease, it will be necessary to develop methods of correcting disease-causing mutations that are compatible with clinical applications, combining efficiency with efficacy and leaving no residual sequences in the targeted genome. Yusa et al. present a proof-of-principle experiment demonstrating the complete genetic correction of a disease-causing mutation in patient-specific iPS cells. They use zinc finger nucleases and piggyBac technology to correction a point mutation in the [alpha].sub.1-antitrypsin gene, which is responsible for [alpha].sub.1-antitrypsin deficiency (A1ATD). The corrected iPS cells could efficiently differentiate to form hepatocyte-like cells and engraft into an animal model for liver injury without tumour formation. Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders.sup.1,2,3,4. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications.sup.3,5. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome.sup.6. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs).sup.7 and piggyBac.sup.8,9 technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the [alpha].sub.1-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for [alpha].sub.1-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies., Author(s): Kosuke Yusa [sup.1] , S. Tamir Rashid [sup.2] [sup.3] , Helene Strick-Marchand [sup.4] [sup.5] , Ignacio Varela [sup.6] , Pei-Qi Liu [sup.7] , David E. Paschon [sup.7] , Elena [...]
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- 2011
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7. Regional Differences in Human Biliary Tissues and Corresponding In Vitro–Derived Organoids
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Rimland, Casey A., primary, Tilson, Samantha G., additional, Morell, Carola M., additional, Tomaz, Rute A., additional, Lu, Wei‐Yu, additional, Adams, Simone E., additional, Georgakopoulos, Nikitas, additional, Otaizo‐Carrasquero, Francisco, additional, Myers, Timothy G., additional, Ferdinand, John R., additional, Gieseck, Richard L., additional, Sampaziotis, Fotios, additional, Tysoe, Olivia C., additional, Ross, Alexander, additional, Kraiczy, Judith M., additional, Wesley, Brandon, additional, Muraro, Daniele, additional, Zilbauer, Matthias, additional, Oniscu, Gabriel C., additional, Hannan, Nicholas R.F., additional, Forbes, Stuart J., additional, Saeb‐Parsy, Kourosh, additional, Wynn, Thomas A., additional, and Vallier, Ludovic, additional
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- 2021
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8. Activation of the selenoprotein SEPS1 gene expression by pro-inflammatory cytokines in HepG2 cells
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Gao, Yuan, Hannan, Nicholas R.F., Wanyonyi, Stephen, Konstantopolous, Nicky, Pagnon, Joanne, Feng, Helen C., Jowett, Jeremy B.M., Kim, Kee-Hong, Walder, Ken, and Collier, Greg R.
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- 2006
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9. hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in ?1-antitrypsin deficiency
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Segeritz, Charis-Patricia, Rashid, Sheikh Tamir, de Brito, Miguel Cardoso, Serra, Maria Paola, Ordonez, Adriana, Morell, Carola Maria, Kaserman, Joseph E., Madrigal, Pedro, Hannan, Nicholas R.F., Gatto, Laurent, Tan, Lu, Wilson, Andrew A., Lilley, Kathryn, Marciniak, Stefan J., Gooptu, Bibek, Lomas, David A., and Vallier, Ludovic
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Inflammation ,Human-induced pluripotent stem cell ,Hepatocyte ,Inherited liver disease ,?1-Antitrypsin deficiency - Abstract
© 2018 The Authors Background & Aims: α1-Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies. Methods: Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and “omics” comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures. Results: Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line. Conclusions: Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD. Lay summary: This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.
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- 2018
10. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids
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Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, and Vallier, Ludovic
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Cholangiocytes, Bile duct, Bio-engineering, Tissue engineering, Organoids, Regenerative medicine, Cell-based therapy, Biliary atresia, PGA scaffold, Collagen scaffold, Densified collagen - Abstract
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 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.
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- 2017
11. Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs
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Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Sinha, Sanjay, Kotter, Mark, and Vallier, Ludovic
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Human pluripotent stem cells, shRNA, CRISPR/Cas9,OCT4, POU5F1, T, brachyury, DPY30 - Abstract
© 2016. 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.
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- 2016
12. Immunostaining for DNA Modifications: Computational Analysis of Confocal Images
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Ramsawhook, Ashley H., primary, Lewis, Lara C., primary, Eleftheriou, Maria, primary, Abakir, Abdulkadir, primary, Durczak, Paulina, primary, Markus, Robert, primary, Rajani, Seema, primary, Hannan, Nicholas R.F., primary, Coyle, Beth, primary, and Ruzov, Alexey, primary
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- 2017
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13. Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells
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Hannan, Nicholas R.F., Sampaziotis, Fotios, Segeritz, Charis-Patricia, Hanley, Neil A., Vallier, Ludovic, Sampaziotis, Fotios [0000-0003-0812-7586], Vallier, Ludovic [0000-0002-3848-2602], and Apollo - University of Cambridge Repository
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Cystic Fibrosis ,Endoderm ,Induced Pluripotent Stem Cells ,Thyroid Nuclear Factor 1 ,Cell- and Tissue-Based Therapy ,Cystic Fibrosis Transmembrane Conductance Regulator ,Nuclear Proteins ,Cell Differentiation ,Forkhead Transcription Factors ,Respiratory Mucosa ,respiratory system ,Pulmonary Surfactant-Associated Protein C ,Epithelium ,Cell Line ,Original Research Reports ,GATA6 Transcription Factor ,Humans ,Fibroblast Growth Factor 10 ,Lung ,Transcription Factors - Abstract
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.
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- 2015
14. Disease modeling using human induced pluripotent stem cells: Lessons from the liver
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Gieseck, Richard L., primary, Colquhoun, Jennifer, additional, and Hannan, Nicholas R.F., additional
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- 2015
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15. Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after Injury
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Fordham, Robert P., primary, Yui, Shiro, additional, Hannan, Nicholas R.F., additional, Soendergaard, Christoffer, additional, Madgwick, Alison, additional, Schweiger, Pawel J., additional, Nielsen, Ole H., additional, Vallier, Ludovic, additional, Pedersen, Roger A., additional, Nakamura, Tetsuya, additional, Watanabe, Mamoru, additional, and Jensen, Kim B., additional
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- 2013
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16. Generation of Multipotent Foregut Stem Cells from Human Pluripotent Stem Cells
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Hannan, Nicholas R.F., primary, Fordham, Robert P., additional, Syed, Yasir A., additional, Moignard, Victoria, additional, Berry, Andrew, additional, Bautista, Ruben, additional, Hanley, Neil A., additional, Jensen, Kim B., additional, and Vallier, Ludovic, additional
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- 2013
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17. BMP-11 and Myostatin Support Undifferentiated Growth of Human Embryonic Stem Cells in Feeder-Free Cultures
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Hannan, Nicholas R.F., primary, Jamshidi, Pegah, additional, Pera, Martin F., additional, and Wolvetang, Ernst J., additional
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- 2009
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18. Adipocyte Differentiation in Human Embryonic Stem Cells Transduced With Oct4 shRNA Lentivirus
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Hannan, Nicholas R.F., primary and Wolvetang, Ernst J., additional
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- 2009
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19. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids
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Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Gómez-Vázquez, María J., Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Zonneveld, Mariëlle C.F., Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, Vallier, Ludovic, Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Gómez-Vázquez, María J., Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Zonneveld, Mariëlle C.F., Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, and Vallier, Ludovic
- Abstract
Treatment of common bile duct disorders such as biliary atresia or ischaemic strictures is limited to liver transplantation or hepatojejunostomy due to the lack of suitable tissue for surgical reconstruction. Here, we report a novel method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree and we explore the potential of bioengineered biliary tissue consisting of these extrahepatic cholangiocyte organoids (ECOs) and biodegradable scaffolds for transplantation and biliary reconstruction in vivo. ECOs closely correlate with primary cholangiocytes in terms of transcriptomic profile and functional properties (ALP, GGT). Following transplantation in immunocompromised mice ECOs self-organize into tubular structures expressing biliary markers (CK7). When seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary marker expression (CK7) and function (ALP, GGT). This bioengineered tissue can reconstruct the wall of the biliary tree (gallbladder) and rescue and extrahepatic biliary injury mouse model following transplantation. Furthermore, it can be fashioned into bioengineered ducts and replace the native common bile duct of immunocompromised mice, with no evidence of cholestasis or lumen occlusion up to one month after reconstruction. In conclusion, ECOs can successfully reconstruct the biliary tree following transplantation, providing proof-of-principle for organ regeneration using human primary cells expanded in vitro.
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20. Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs
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Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark, Vallier, Ludovic, Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark, and Vallier, Ludovic
- Abstract
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.
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21. Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells
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Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas R.F., Vallier, Ludovic, Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas R.F., and Vallier, Ludovic
- Abstract
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.
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22. Immunostaining for DNA modifications: computational analysis of confocal images
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Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth, Ruzov, Alexey, Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth, and Ruzov, Alexey
- Abstract
For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.
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23. Dynamics of 5-carboxylcytosine during hepatic differentiation: potential general role for active demethylation by DNA repair in lineage specification
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Lewis, Lara C., Lo, Peggy Cho Kiu, Foster, Jeremy M., Dai, Nan, Correa, Ivan R., Durczak, Paulina M., Duncan, Gary, Ramsawhook, Ashley, Aithal, Guruprasad P., Denning, Chris, Hannan, Nicholas R.F., Ruzov, Alexey, Lewis, Lara C., Lo, Peggy Cho Kiu, Foster, Jeremy M., Dai, Nan, Correa, Ivan R., Durczak, Paulina M., Duncan, Gary, Ramsawhook, Ashley, Aithal, Guruprasad P., Denning, Chris, Hannan, Nicholas R.F., and Ruzov, Alexey
- Abstract
Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognised and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operative in this system, however it is still unknown if TDG/BER-dependent demethylation is utilised during other types of cellular differentiation. Here we analyse dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells (hPSCs) towards hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment and drop in differentiating cells concurrently with the onset of expression of Alpha Fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the commencement of their expression. Our data indicate that transient 5caC accumulation is a common feature of two different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.
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24. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids
- Author
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Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Gómez-Vázquez, María J., Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Zonneveld, Mariëlle C.F., Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, Vallier, Ludovic, Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Gómez-Vázquez, María J., Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Zonneveld, Mariëlle C.F., Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, and Vallier, Ludovic
- Abstract
Treatment of common bile duct disorders such as biliary atresia or ischaemic strictures is limited to liver transplantation or hepatojejunostomy due to the lack of suitable tissue for surgical reconstruction. Here, we report a novel method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree and we explore the potential of bioengineered biliary tissue consisting of these extrahepatic cholangiocyte organoids (ECOs) and biodegradable scaffolds for transplantation and biliary reconstruction in vivo. ECOs closely correlate with primary cholangiocytes in terms of transcriptomic profile and functional properties (ALP, GGT). Following transplantation in immunocompromised mice ECOs self-organize into tubular structures expressing biliary markers (CK7). When seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary marker expression (CK7) and function (ALP, GGT). This bioengineered tissue can reconstruct the wall of the biliary tree (gallbladder) and rescue and extrahepatic biliary injury mouse model following transplantation. Furthermore, it can be fashioned into bioengineered ducts and replace the native common bile duct of immunocompromised mice, with no evidence of cholestasis or lumen occlusion up to one month after reconstruction. In conclusion, ECOs can successfully reconstruct the biliary tree following transplantation, providing proof-of-principle for organ regeneration using human primary cells expanded in vitro.
- Full Text
- View/download PDF
25. Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells
- Author
-
Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas R.F., Vallier, Ludovic, Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas R.F., and Vallier, Ludovic
- Abstract
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.
- Full Text
- View/download PDF
26. Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs
- Author
-
Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark, Vallier, Ludovic, Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark, and Vallier, Ludovic
- Abstract
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.
- Full Text
- View/download PDF
27. Immunostaining for DNA modifications: computational analysis of confocal images
- Author
-
Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth, Ruzov, Alexey, Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth, and Ruzov, Alexey
- Abstract
For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.
- Full Text
- View/download PDF
28. Dynamics of 5-carboxylcytosine during hepatic differentiation: potential general role for active demethylation by DNA repair in lineage specification
- Author
-
Lewis, Lara C., Lo, Peggy Cho Kiu, Foster, Jeremy M., Dai, Nan, Correa, Ivan R., Durczak, Paulina M., Duncan, Gary, Ramsawhook, Ashley, Aithal, Guruprasad P., Denning, Chris, Hannan, Nicholas R.F., Ruzov, Alexey, Lewis, Lara C., Lo, Peggy Cho Kiu, Foster, Jeremy M., Dai, Nan, Correa, Ivan R., Durczak, Paulina M., Duncan, Gary, Ramsawhook, Ashley, Aithal, Guruprasad P., Denning, Chris, Hannan, Nicholas R.F., and Ruzov, Alexey
- Abstract
Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognised and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operative in this system, however it is still unknown if TDG/BER-dependent demethylation is utilised during other types of cellular differentiation. Here we analyse dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells (hPSCs) towards hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment and drop in differentiating cells concurrently with the onset of expression of Alpha Fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the commencement of their expression. Our data indicate that transient 5caC accumulation is a common feature of two different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.
- Full Text
- View/download PDF
29. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids
- Author
-
Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Gómez-Vázquez, María J., Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Zonneveld, Mariëlle C.F., Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, Vallier, Ludovic, Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Gómez-Vázquez, María J., Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Zonneveld, Mariëlle C.F., Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, and Vallier, Ludovic
- Abstract
Treatment of common bile duct disorders such as biliary atresia or ischaemic strictures is limited to liver transplantation or hepatojejunostomy due to the lack of suitable tissue for surgical reconstruction. Here, we report a novel method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree and we explore the potential of bioengineered biliary tissue consisting of these extrahepatic cholangiocyte organoids (ECOs) and biodegradable scaffolds for transplantation and biliary reconstruction in vivo. ECOs closely correlate with primary cholangiocytes in terms of transcriptomic profile and functional properties (ALP, GGT). Following transplantation in immunocompromised mice ECOs self-organize into tubular structures expressing biliary markers (CK7). When seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary marker expression (CK7) and function (ALP, GGT). This bioengineered tissue can reconstruct the wall of the biliary tree (gallbladder) and rescue and extrahepatic biliary injury mouse model following transplantation. Furthermore, it can be fashioned into bioengineered ducts and replace the native common bile duct of immunocompromised mice, with no evidence of cholestasis or lumen occlusion up to one month after reconstruction. In conclusion, ECOs can successfully reconstruct the biliary tree following transplantation, providing proof-of-principle for organ regeneration using human primary cells expanded in vitro.
- Full Text
- View/download PDF
30. Immunostaining for DNA modifications: computational analysis of confocal images
- Author
-
Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth, Ruzov, Alexey, Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth, and Ruzov, Alexey
- Abstract
For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.
- Full Text
- View/download PDF
31. Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs
- Author
-
Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark, Vallier, Ludovic, Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark, and Vallier, Ludovic
- Abstract
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.
- Full Text
- View/download PDF
32. Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells
- Author
-
Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas R.F., Vallier, Ludovic, Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas R.F., and Vallier, Ludovic
- Abstract
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.
- Full Text
- View/download PDF
33. Dynamics of 5-carboxylcytosine during hepatic differentiation: potential general role for active demethylation by DNA repair in lineage specification
- Author
-
Lewis, Lara C., Lo, Peggy Cho Kiu, Foster, Jeremy M., Dai, Nan, Correa, Ivan R., Durczak, Paulina M., Duncan, Gary, Ramsawhook, Ashley, Aithal, Guruprasad P., Denning, Chris, Hannan, Nicholas R.F., Ruzov, Alexey, Lewis, Lara C., Lo, Peggy Cho Kiu, Foster, Jeremy M., Dai, Nan, Correa, Ivan R., Durczak, Paulina M., Duncan, Gary, Ramsawhook, Ashley, Aithal, Guruprasad P., Denning, Chris, Hannan, Nicholas R.F., and Ruzov, Alexey
- Abstract
Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognised and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operative in this system, however it is still unknown if TDG/BER-dependent demethylation is utilised during other types of cellular differentiation. Here we analyse dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells (hPSCs) towards hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment and drop in differentiating cells concurrently with the onset of expression of Alpha Fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the commencement of their expression. Our data indicate that transient 5caC accumulation is a common feature of two different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.
- Full Text
- View/download PDF
34. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids
- Author
-
Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Gómez-Vázquez, María J., Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Zonneveld, Mariëlle C.F., Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, Vallier, Ludovic, Sampaziotis, Fotios, Justin, Alexander W., Tysoe, Olivia C., Sawiak, Stephen, Godfrey, Edmund M., Upponi, Sara S., Gieseck, Richard L., de Brito, Miguel Cardoso, Berntsen, Natalie Lie, Gómez-Vázquez, María J., Ortmann, Daniel, Yiangou, Loukia, Ross, Alexander, Bargehr, Johannes, Bertero, Alessandro, Zonneveld, Mariëlle C.F., Pedersen, Marianne T., Pawlowski, Matthias, Valestrand, Laura, Madrigal, Pedro, Georgakopoulos, Nikitas, Pirmadjid, Negar, Skeldon, Gregor M., Casey, John, Shu, Wenmiao, Materek, Paulina M., Snijders, Kirsten, Brown, Stephanie, Rimland, Casey A., Simonic, Ingrid, Davies, Susan E., Jensen, Kim B., Zilbauer, Matthias, Gelson, William T.H., Alexander, Graeme J., Sinha, Sanjay, Hannan, Nicholas R.F., Wynn, Thomas A., Karlsen, Tom H., Melum, Espen, Markaki, Athina E., Saeb-Parsy, Kourosh, and Vallier, Ludovic
- Abstract
Treatment of common bile duct disorders such as biliary atresia or ischaemic strictures is limited to liver transplantation or hepatojejunostomy due to the lack of suitable tissue for surgical reconstruction. Here, we report a novel method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree and we explore the potential of bioengineered biliary tissue consisting of these extrahepatic cholangiocyte organoids (ECOs) and biodegradable scaffolds for transplantation and biliary reconstruction in vivo. ECOs closely correlate with primary cholangiocytes in terms of transcriptomic profile and functional properties (ALP, GGT). Following transplantation in immunocompromised mice ECOs self-organize into tubular structures expressing biliary markers (CK7). When seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary marker expression (CK7) and function (ALP, GGT). This bioengineered tissue can reconstruct the wall of the biliary tree (gallbladder) and rescue and extrahepatic biliary injury mouse model following transplantation. Furthermore, it can be fashioned into bioengineered ducts and replace the native common bile duct of immunocompromised mice, with no evidence of cholestasis or lumen occlusion up to one month after reconstruction. In conclusion, ECOs can successfully reconstruct the biliary tree following transplantation, providing proof-of-principle for organ regeneration using human primary cells expanded in vitro.
- Full Text
- View/download PDF
35. Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs
- Author
-
Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark, Vallier, Ludovic, Bertero, Alessandro, Pawlowski, Matthias, Ortmann, Daniel, Snijders, Kirsten, Yiangou, Loukia, Cardoso de Brito, Miguel, Brown, Stephanie, Bernard, William G., Cooper, James D., Giacomelli, Elisa, Gambardella, Laure, Hannan, Nicholas R.F., Iyer, Dharini, Sampaziotis, Fotios, Serrano, Felipe, Zonneveld, Mariëlle C.F., Sinha, Sanjay, Kotter, Mark, and Vallier, Ludovic
- Abstract
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.
- Full Text
- View/download PDF
36. Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells
- Author
-
Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas R.F., Vallier, Ludovic, Sampaziotis, Fotios, de Brito, Miguel Cardoso, Geti, Imbisaat, Bertero, Alessandro, Hannan, Nicholas R.F., and Vallier, Ludovic
- Abstract
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.
- Full Text
- View/download PDF
37. Immunostaining for DNA modifications: computational analysis of confocal images
- Author
-
Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth, Ruzov, Alexey, Ramsawhook, Ashley, Lewis, Lara C., Eleftheriou, Maria, Abakir, Abdulkadir, Durczak, Paulina M., Markus, Robert, Rajini, Seema, Hannan, Nicholas R.F., Coyle, Beth, and Ruzov, Alexey
- Abstract
For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.
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38. Dynamics of 5-carboxylcytosine during hepatic differentiation: potential general role for active demethylation by DNA repair in lineage specification
- Author
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Lewis, Lara C., Lo, Peggy Cho Kiu, Foster, Jeremy M., Dai, Nan, Correa, Ivan R., Durczak, Paulina M., Duncan, Gary, Ramsawhook, Ashley, Aithal, Guruprasad P., Denning, Chris, Hannan, Nicholas R.F., Ruzov, Alexey, Lewis, Lara C., Lo, Peggy Cho Kiu, Foster, Jeremy M., Dai, Nan, Correa, Ivan R., Durczak, Paulina M., Duncan, Gary, Ramsawhook, Ashley, Aithal, Guruprasad P., Denning, Chris, Hannan, Nicholas R.F., and Ruzov, Alexey
- Abstract
Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognised and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operative in this system, however it is still unknown if TDG/BER-dependent demethylation is utilised during other types of cellular differentiation. Here we analyse dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells (hPSCs) towards hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment and drop in differentiating cells concurrently with the onset of expression of Alpha Fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the commencement of their expression. Our data indicate that transient 5caC accumulation is a common feature of two different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.
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39. Disease modeling using human induced pluripotent stem cells: Lessons from the liver.
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IIIGieseck, Richard L., Colquhoun, Jennifer, and Hannan, Nicholas R.F.
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INDUCED pluripotent stem cells , *HEPATOLOGY , *LIVER diseases , *HUMAN body , *REGENERATIVE medicine , *DRUG use testing , *ANIMAL disease models , *MEDICAL transcription - Abstract
Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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