ORCID Profile
0000-0002-9843-1559
Current Organisation
University of Nottingham
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Publisher: Springer Science and Business Media LLC
Date: 12-10-2011
DOI: 10.1038/NATURE10424
Publisher: Oxford University Press (OUP)
Date: 26-07-2011
DOI: 10.1002/STEM.666
Abstract: Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control ergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate ergent cell fate decisions.
Publisher: MyJove Corporation
Date: 07-09-2017
DOI: 10.3791/56318
Publisher: Elsevier BV
Date: 10-2018
Publisher: Springer Science and Business Media LLC
Date: 13-07-2015
DOI: 10.1038/NBT.3275
Publisher: Elsevier BV
Date: 2015
Publisher: Elsevier BV
Date: 10-2013
Publisher: Mary Ann Liebert Inc
Date: 09-2009
Abstract: BMP-11/GDF-11 and Myostatin/GDF-8 are both members of the TGF-beta superfamily that can activate SMAD2/3 phosphorylation via the type I receptors ALK4, ALK5, or ALK7. We tested the ability of BMP-11 and Myostatin to promote self-renewal of human embryonic stem cells (hESC) under feeder-free and serum-free culture conditions in short term (1 week) and medium term cultures (10 weeks). We show that hESC cultured in serum-free medium supplemented with either 20 ng/mL Myostatin or 20 ng/mL BMP-11 maintain the colony and cellular morphology of undifferentiated hESC, maintain POU5f1, NANOG, TRA-1-60, and SSEA4 expression, and display increased SMAD2/3 phosphorylation, similar to hESC cultured in mouse embryonic fibroblast feeder-CM or 20 ng/mL Activin-A. The type I TGF-beta receptor inhibitor SB431542 totally inhibited the maintenance activity of both Myostatin or BMP-11 supplemented medium. Our data show that members of the TGF-beta superfamily, other than Activin-A and GDF3, are able to maintain hES cells in an undifferentiated state under feeder free conditions.
Publisher: American Society for Clinical Investigation
Date: 09-2010
DOI: 10.1172/JCI43122
Publisher: Public Library of Science (PLoS)
Date: 30-06-2009
Publisher: CRC Press
Date: 12-04-2012
DOI: 10.1201/B11937
Publisher: Springer New York
Date: 03-11-2014
DOI: 10.1007/978-1-4939-2074-7_9
Abstract: Hepatocytes produced from the differentiation of human pluripotent stem cells can be used to study human development and liver disease, to investigate the toxicological response of novel drug candidates, and as an alternative source of primary cells for transplantation therapies. Here, we describe a method to produce hepatocytes by differentiating human pluripotent stem cells into definitive endoderm, patterning definitive endoderm into anterior definitive endoderm, specifying anterior definitive endoderm into hepatic endoderm, and differentiating hepatic endoderm into immature hepatocytes. These cells are further matured in either two-dimensional or three-dimensional culture conditions to produce cells capable of metabolizing xenobiotics and generating liver-specific proteins, such as albumin and alpha 1 antitrypsin.
Publisher: The Company of Biologists
Date: 12-2016
DOI: 10.1242/DEV.138081
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 in idual 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.
Publisher: Springer Science and Business Media LLC
Date: 26-09-2012
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 23-12-2009
DOI: 10.1002/HEP.23506
Abstract: Generation of hepatocytes from human embryonic stem cells (hESCs) could represent an advantageous source of cells for cell therapy approaches as an alternative to orthotopic liver transplantation. However, the generation of differentiated hepatocytes from hESCs remains a major challenge, especially using a method compatible with clinical applications. We report a novel approach to differentiate hESCs into functional hepatic cells using fully defined culture conditions, which recapitulate essential stages of liver development. hESCs were first differentiated into a homogenous population of endoderm cells using a combination of activin, fibroblast growth factor 2, and bone morphogenetic protein 4 together with phosphoinositide 3-kinase inhibition. The endoderm cells were then induced to differentiate further into hepatic progenitors using fibroblast growth factor 10, retinoic acid, and an inhibitor of activin/nodal receptor. After further maturation, these cells expressed markers of mature hepatocytes, including asialoglycoprotein receptor, tyrosine aminotransferase, alpha1-antitrypsin, Cyp7A1, and hepatic transcription factors such as hepatocyte nuclear factors 4alpha and 6. Furthermore, the cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, cytochrome activity, and low-density lipoprotein uptake. After transduction with a green fluorescent protein-expressing lentivector and transplantation into immunodeficient uPA transgenic mice, differentiated cells engrafted into the liver, grew, and expressed human albumin and alpha1-antitrypsin as well as green fluorescent protein for at least 8 weeks. In addition, we showed that hepatic cells could be generated from human-induced pluripotent cells derived from reprogrammed fibroblasts, demonstrating the efficacy of this approach with pluripotent stem cells of erse origins. We have developed a robust and efficient method to differentiate pluripotent stem cells into hepatic cells, which exhibit characteristics of human hepatocytes. Our approach should facilitate the development of clinical grade hepatocytes for transplantation and for research on drug discovery.
Publisher: Springer Science and Business Media LLC
Date: 23-03-2017
Publisher: Informa UK Limited
Date: 30-03-2017
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 25-06-2010
DOI: 10.1002/HEP.23761
Publisher: Springer New York
Date: 2016
DOI: 10.1007/7651_2016_7
Abstract: Intestinal human organoids (iHOs) provide an effective system for studying the intestinal epithelium and its interaction with various stimuli. By using combinations of different signaling factors, human induced pluripotent stem cells (hIPSCs) can be driven to differentiate down the intestinal lineage. Here, we describe the process for this differentiation, including the derivation of hindgut from hIPSCs, embedding hindgut into a pro-intestinal culture system and passaging the resulting iHOs. We then describe how to carry out microinjections to introduce bacteria to the apical side of the intestinal epithelial cells (IECs).
Publisher: Springer Science and Business Media LLC
Date: 31-01-2013
Publisher: Elsevier BV
Date: 03-2006
Publisher: Elsevier
Date: 2012
Publisher: Mary Ann Liebert Inc
Date: 05-2009
Abstract: Oct4 is one of the master pluripotency genes that controls differentiation of human embryonic stem cells (hESCs). We generated HES2 and HES3 hESC lines stably transduced with lentivirus carrying Oct4 short hairpin RNA (shRNA) that display 80-90% reduction of Oct4 expression. Analysis of pluripotency marker expression shows that these Oct4 shRNA-transduced hESCs display normal wild-type expression levels of the pluripotency marker CD9 but an absence of GCTM2 expression. These hESC-derived adipocyte precursor cells display a characteristic morphology and can be propagated and cryopreserved as a standard stem cell line. Interestingly, Oct4 shRNA-transduced hESCs display a remarkably high lineage-specific spontaneous differentiation toward adipocytes. After two weeks of spontaneous differentiation under feeder-free conditions, 60-70% of cells display a mature adipocyte morphology as well as the expression of multiple adipocyte-specific mRNAs as assessed by RT-PCR. The upregulation of trophoblast, mesoderm, and endoderm transcripts is, however, also detected in these spontaneously differentiating cultures. These Oct4 shRNA hESCs will be an interesting model system to study human fetal adipogenesis and constitutes a renewable resource for obesity drug screening purposes.
Publisher: Mary Ann Liebert Inc
Date: 15-07-2015
Publisher: Springer Science and Business Media LLC
Date: 03-07-2017
DOI: 10.1038/NM.4360
Abstract: 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.
Publisher: Public Library of Science (PLoS)
Date: 22-01-2014
Publisher: Elsevier BV
Date: 12-2013
Publisher: American Society for Microbiology
Date: 07-2015
DOI: 10.1128/IAI.00161-15
Abstract: The intestinal mucosa forms the first line of defense against infections mediated by enteric pathogens such as salmonellae. Here we exploited intestinal “organoids” (iHOs) generated from human induced pluripotent stem cells (hIPSCs) to explore the interaction of Salmonella enterica serovar Typhimurium with iHOs. Imaging and RNA sequencing were used to analyze these interactions, and clear changes in transcriptional signatures were detected, including altered patterns of cytokine expression after the exposure of iHOs to bacteria. S . Typhimurium microinjected into the lumen of iHOs was able to invade the epithelial barrier, with many bacteria residing within Salmonella -containing vacuoles. An S . Typhimurium invA mutant defective in the Salmonella pathogenicity island 1 invasion apparatus was less capable of invading the iHO epithelium. Hence, we provide evidence that hIPSC-derived organoids are a promising model of the intestinal epithelium for assessing interactions with enteric pathogens.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 2021
DOI: 10.1002/HEP.31252
Abstract: Organoids provide a powerful system to study epithelia in vitro . Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts EHBDs) or inside the liver (intrahepatic bile ducts IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem rogenitor markers leucine‐rich repeat–containing G‐protein‐coupled receptor 5 rominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional‐specific markers corresponding to their location of origin. Of particular interest, down‐regulation of biliary markers and up‐regulation of cell‐cycle genes were observed in organoids. IHBD and EHBD organoids erged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.
Publisher: The Royal Society
Date: 21-05-2018
Abstract: Gastrointestinal diseases are becoming increasingly prevalent in developed countries. Immortalized cells and animal models have delivered important but limited insight into the mechanisms that initiate and propagate these diseases. Human-specific models of intestinal development and disease are desperately needed that can recapitulate structure and function of the gut in vitro . Advances in pluripotent stem cells and primary tissue culture techniques have made it possible to culture intestinal epithelial cells in three dimensions that self-assemble to form ‘intestinal organoids'. These organoids allow for new, human-specific models that can be used to gain insight into gastrointestinal disease and potentially deliver new therapies to treat them. Here we review current in vitro models of intestinal development and disease, considering where improvements could be made and potential future applications in the fields of developmental modelling, drug/toxicity testing and therapeutic uses. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you'.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: Australia
No related grants have been discovered for Nicholas Hannan.