"The production of iPS cells from dermal fibroblasts has generated intense interest in the utility of such cells for research purposes and clinical applications. iPS cell production currently requires the use of transcription factor gene delivery to reprogramme cells into iPS cells. Hence, both gene delivery technology and iPS cell characterization and subsequent cell differentiation are critical aspects of iPS cell biology. This meeting will address both issues" Meeting Chairs: Professor Peter Andrews , University of Sheffield, UK and Dr Chris Denning, University of Nottingham¸UK
This event has CPD accreditation
9:00 – 9:30 Registration
9:30 – 9:40 Introduction by the Chairs: Dr Chris Denning, University of Nottingham¸UK and Professor Peter Andrews University of Sheffield, UK Chair: Professor Peter Andrews University of Sheffield, UK
9:40 – 10:25 iPS cell generationProfessor Hans Schöler, Max Planck Institute, Germany 10:25 – 10:50 Forebrain progenitorsNicholas Allen, Cardiff
10:50 – 11:00 Speakers Photo
11:00 – 11:20 Morning Break
11.20-11.45 Human ESCs into specific endodermal cell types Dr Ludvic Vallier, Laboratory for Regenerative medicine, University of Cambridge, UKGeneration of hepatocytes from human embryonic stem cells (hESCs) could represent an advantageous source of cells for cell therapy approaches as alternative to liver transplantation. However, the generation of hepatocytes from hESCs remains a challenge especially using conditions compatible with clinical applications. Here, we report a novel method to differentiate hESCs and hIPSCs into hepatic cells using defined culture system, which recapitulate essential stages of liver development. Importantly, the cells generated under these conditions exhibited hepatic functions in vitro and in vivo. Therefore, this approach represents toward the generation of hESCs derived hepatic cells for cell based therapy.
11.45-12.10 Stem cell states and the single cell Professor Peter Andrews, University of Sheffield, UK After prolonged culture, ES cells are subject to the selection of genetic variants. Accumulating evidence suggests that the ‘stem cell compartment’ in both ES and other stem cells may be composed of distinct substates. One aspect of culture adaption is that it alters the population dynamics of ES cultures, particularly affecting the behavior of substates within the stem cell compartment. Understanding the nature of these substates may provide insights into the mechanisms that control self renewal, commitment to differentiation and lineage selection of ES and, ultimately iPS cells. Inevitably these same mechanisms may also play a role in cancer progression
12.10-12.35 Derivation and utility of cardiomyocytes from human pluripotent stem cellsDr Chris Denning, University of Nottingham, UK
We have demonstrated that functional cardiomyocytes can be derived from human embryonic stem cells, potentially offering a novel cell source for drug screening, disease modelling and cell replacement. However, before these goals can be realised, several issues must be tackled. We have sought to standardise feeder-free culture methods that function in 14 hESC lines derived in 5 different countries, impacting on the ability to improve downstream technologies. Thus, we have demonstrated industrial scale automation of hESC culture to meet demands of commerce. Standardised culture also provides a platform from which differentiation to the cardiac lineage can be improved and directed. Moreover, high efficiency genetic modification has been demonstrated in 11 hESC lines, potentially providing new routes to RNAi library screening for genome analysis. We have also generated transgenic hESC lines that express puromycin N-acetyltransferase from the cardiac specific MYH6 promoter, allowing enrichment of cardiomyocytes to close to 100% purity by incubation with the antibiotic puromycin. This set of technologies is now being applied to proof of principle studies in drug screening and engineering in vitro disease models produced either by genetic modification or by exploitation of induced pluripotency (iPS) technology.
12.35-13.35 Lunch and poster viewing Chair: Dr Chris Denning, University of Nottingham¸UK
13.35-14.00 Humps and Bumps on the road to pluripotency Dr Majlinda Lako, Newcastle University, UK The generation of induced pluripotent stem cells (iPSC) has enormous potential for the development of patient specific regenerative medicine. Human embryonic stem cells (hESC) are able to defend their genomic integrity by maintaining low levels of reactive oxygen species (ROS) through a combination of enhanced removal capacity and limited production of these molecules. Such limited ROS production stems partly from the small numbers of mitochondria present in hESC, thus it was important to determine that human iPSC (hiPSC) generation is able to eliminate the extra mitochondria present in the parental fibroblasts (reminiscent of "bottleneck" situation after fertilisation) and to show that hiPSC have similar antioxidant defences to hESC. We were able to generate seven hiPSC lines from adult human dermal fibroblasts and have fully characterised two of those clones. Both hiPSC clones express pluripotency markers and are able to differentiate in vitro into cells belonging to all three germ layers. One of these clones is able to produce fully differentiated teratoma, whilst the other hiPSC clone is unable to silence the viral expression of OCT4 and c-MYC, produce fully differentiated teratoma and unable to downregulate the expression of some of the pluripotency genes during the differentiation process. In spite of these differences, both clones show similar ROS stress defence mechanisms and mitochondrial biogenesis to hESC. Together our data suggest that during the reprogramming process, certain cellular mechanisms are in place to ensure that hiPSC are provided with the same defence mechanisms against accumulation of ROS as the hESC.
14.00-14.25 Derivation of induced pluripotent cells from adult dermal fibroblasts in patients with CVD and controls.Dr Nicole Kane, University of Glasgow¸Scotland 14.25-14.50 Reprogramming: from Technology to Biology Dr Keisuke Kaji, Edinburgh University, Scotland We have developed a non-viral reprogramming system with multiprotein expression vector and Piggybac transposon/transposase in 2009. In addition to improving the strategy, currently we are using the system to understand the mechanism of the reprogramming process, how the cells go back to the pluripotent state by expression of Oct4, Sox2, Klf4 and c-Myc.
14.50-15.20 Afternoon Tea/Coffee 15.20-15.45 Lenti-vector based generation of human IPS cells from HDFs;similarities and differences form ES cells Dr Sue Kimber and Dr Tristan McKay, University of Manchester, UK
15.45-16.10 Application of induced pluripotent stem cells in modelling human immunodeficiency disordersDr Sayandip Mukherjee, UCL , London Induced pluripotent stem (iPS) cells generated from patient samples can potentially provide a platform for dissecting the molecular mechanisms of inherited disorders, design of drug screening protocols, and also for testing the efficacy and safety profiles of gene replacement therapies. We have focussed on chronic granulomatous disorder (CGD) which is a rare inherited neutrophil disorder and affects four in a million. iPS cells generated from skin biopsies of CGD patients will be employed for studying the efficacy of lentiviral vectors delivering a codon optimized gp91phox transgene, and as a proof of principal study to establish their application in bone marrow reconstitution experiments in murine models of CGD.
16.10-16.20 Chairmans’s summing up
