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Carmona S.J.,University of Lausanne | Carmona S.J.,Swiss Institute of Bioinformatics | Teichmann S.A.,European Bioinformatics Institute | Teichmann S.A.,Wellcome Trust Sanger Institute | And 10 more authors.
Genome Research | Year: 2017

The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of trans-membrane proteins in NK cells compared with other immune cell types, and especially T cells, but similar conservation of nuclear and cytoplasmic protein coding genes. To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immunecell- specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates. © 2017 Carmona et al.


Dawson M.A.,University of Cambridge | Dawson M.A.,Cambridge Stem Cell Institute | Gudgin E.J.,University of Cambridge | Horton S.J.,University of Cambridge | And 22 more authors.
Leukemia | Year: 2014

Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common 'core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain 'super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML. © 2014 Macmillan Publishers Limited.


News Article | February 15, 2017
Site: www.eurekalert.org

A $6.1 million, five-year grant from the National Institute of Diabetes, Digestive and Kidney Diseases at the National Institutes of Health may help researchers leverage massive amounts of genomic data to develop medical treatments and pharmaceuticals, according to an international team of researchers. The project -- called VISION or Validated Systematic Integration of Hematopoietic Epigenomes -- will integrate and functionally validate large amounts of emerging genomic and epigenetic data, according to Ross Hardison, T. Ming Chu Professor of Biochemistry and Molecular Biology, Penn State and a member of the Genome Sciences Institute of the Huck Institutes of the Life Sciences. Hardison, who will lead the international multidisciplinary team, added that the group will try to develop new tools for using data to facilitate advances both in basic research as well as medical applications, such as precision medicine. The project will focus on blood cell development as a model system for gene regulation in mammals. Blood cell development is vitally important to health because humans must continually replace old and damaged cells, and because many diseases, like leukemias and anemias, result from mis-regulation of gene expression during blood formation. "We are excited about this project because the methods we are developing can be applied not only to diseases that affect blood, but others as well," Hardison said. "A person's genetic profile can have a significant impact on disease susceptibility and response to specific treatments. However, the critical genetic variants that make up that genetic profile most often do not code for protein, but rather they are located in the much larger noncoding genome. We are studying these noncoding regions and finding new ways to extract valuable information about functional elements within them, which in turn informs us about how genetic variants play a role in disease." The results of the VISION project are being provided to the research community in readily accessible, web-based platforms and online tools that will allow researchers to extract meaningful, experimentally validated interpretations from the data and produce a guide for investigators to translate insights from mouse models to human clinical studies. Hardison is working with Cheryl Keller, project manager, Yu Zhang, associate professor of statistics, and Feng Yue, assistant professor of biochemistry and molecular biology, College of Medicine, all at Penn State; Mitchell Weiss, chair of the department of hematology, St. Jude Children's Research Hospital; Gerd Blobel, Professor of Pediactrics, University of Pennsylvania abd Children's Hospital of Philadelphia; James Taylor, associate professor of biology and associate professor of computer science, Johns Hopkins University; David Bodine, chief and senior investigator, National Human Genome Research Institute, NIH; Berthold Göttgens, principal investigator and professor of haematology, Cambridge Stem Cell Institute, University of Cambridge; Douglas Higgs, group head and principal investigator, and Jim Hughes, associate professor of genome biology, both of the Weatherall Institute of Molecular Medicine, Oxford University.


News Article | November 29, 2016
Site: www.eurekalert.org

Wellcome Trust Sanger Institute and University of Cambridge researchers have created sOPTiKO, a more efficient and controllable CRISPR genome editing platform. Today, in the journal Development, they describe how the freely available single-step system works in every cell in the body and at every stage of development. This new approach will aid researchers in developmental biology, tissue regeneration and cancer. Two complementary methods were developed. sOPiTKO is a knock-out system that turns off genes by disrupting the DNA. sOPTiKD is a knock-down system that silences the action of genes by disrupting the RNA. Using these two methods, scientists can inducibly turn off or silence genes, in any cell type, at any stage of a cell's development from stem cell to fully differentiated adult cell. These systems will allow researchers world wide to rapidly and accurately explore the changing role of genes as the cells develop into tissues such as liver, skin or heart, and discover how this contributes to health and disease. The body contains approximately 37 trillion cells, yet the human genome only contains roughly 20,000 genes. So, to produce every tissue and cell type in the body, different combinations of genes must operate at different moments in the development of an organ or tissue. Being able to turn off genes at specific moments in a cell's development allows their changing roles to be investigated. Professor Ludovic Vallier, one of the senior authors of the study from the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute at the University of Cambridge and the Sanger Institute, said: "As a cell develops from being stem cell to being a fully differentiated adult cell the genes within it take on different roles. Before, if we knocked out a gene, we could only see what effect this had at the very first step. By allowing the gene to operate during the cell's development and then knocking it out with sOPTiKO at a later developmental step, we can investigate exactly what it is doing at that stage." The sOPTiKO and sOPTIKD methods allow scientists to silence the activity of more than one gene at a time, so researchers have the possibility to now investigate the role of whole families of related genes by knocking down the activity of all of them at once. In addition, the freely available system allows experiments to be carried out far more rapidly and cheaply. sOPTiKO is highly flexible so that it can be used in every tissue in the body without needing to create a new system each time. sOPiTKD allows vast improvements in efficiency: it can be used to knock down more than one gene at a time. Before, to silence the activity of three genes, researchers had to knock down one gene, grow the cell line, and repeat for the next gene, and again for the next. Now it can do it all in one step, cutting a nine-month process down to just one to two months. Dr Alessandro Bertero, one of the first authors of the study from the Cambridge Stem Cell Institute, said: "Two key advantages of using sOPTiKO/sOPTIKD over other CRISPR editing systems are that it is truly inducible and can work in almost any cell type. In the past we have been hampered by the fact we could study a gene's function only in a specific tissue. Now you can knock out the same gene in parallel in a diversity of cell type with different functions." For a video of the methods in action: please see: https:/ Bertero A et al. (2016) Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs. Development 143: 4405-4418. doi:10.1242/dev.138081 This work was supported by a European Research Council starting grant Relieve IMDs (L.V., D.O., N.R.F.H., M.C.F.Z., E.G.); the Cambridge University Hospitals National Institute for Health Research Biomedical Research Center (L.V., N.R.F.H., F.Sa.); the EU Seventh Framework Programme TISSUGEN (M.C.d.B.); theWellcome Trust PhD program (L.Y.); a British Heart Foundation PhD Studentship (A.B.); a research fellowship fromthe Deutsche Forschungsgemeinschaft (M.P.) [PA 2369/1-1]; and a core support grant from theWellcome Trust and Medical Research Council to the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute. Coronary heart disease is the UK's single biggest killer. For over 50 years we've pioneered research that's transformed the lives of people living with heart and circulatory conditions. Our work has been central to the discoveries of vital treatments that are changing the fight against heart disease. But so many people still need our help. From babies born with life-threatening heart problems to the many Mums, Dads and Grandparents who survive a heart attack and endure the daily battles of heart failure. Join our fight for every heartbeat in the UK. Every pound raised, minute of your time and donation to our shops will help make a difference to people's lives. For more information visit https:/ About the University of Cambridge The mission of the University of Cambridge is to contribute to society through the pursuit of education, learning and research at the highest international levels of excellence. To date, 96 affiliates of the University have won the Nobel Prize. Founded in 1209, the University comprises 31 autonomous Colleges, which admit undergraduates and provide small-group tuition, and 150 departments, faculties and institutions. Cambridge is a global university. Its 19,000 student body includes 3,700 international students from 120 countries. Cambridge researchers collaborate with colleagues worldwide, and the University has established larger-scale partnerships in Asia, Africa and America. The University sits at the heart of one of the world's largest technology clusters. The 'Cambridge Phenomenon' has created 1,500 hi-tech companies, 14 of them valued at over US$1 billion and two at over US$10 billion. Cambridge promotes the interface between academia and business, and has a global reputation for innovation. http://www. The Wellcome Trust Sanger Institute is one of the world's leading genome centres. Through its ability to conduct research at scale, it is able to engage in bold and long-term exploratory projects that are designed to influence and empower medical science globally. Institute research findings, generated through its own research programmes and through its leading role in international consortia, are being used to develop new diagnostics and treatments for human disease. http://www. Wellcome exists to improve health for everyone by helping great ideas to thrive. We're a global charitable foundation, both politically and financially independent. We support scientists and researchers, take on big problems, fuel imaginations and spark debate. http://www.


Two complementary methods were developed. sOPiTKO is a knock-out system that turns off genes by disrupting the DNA. sOPTiKD is a knock-down system that silences the action of genes by disrupting the RNA. Using these two methods, scientists can inducibly turn off or silence genes, in any cell type, at any stage of a cell's development from stem cell to fully differentiated adult cell. These systems will allow researchers world wide to rapidly and accurately explore the changing role of genes as the cells develop into tissues such as liver, skin or heart, and discover how this contributes to health and disease. The body contains approximately 37 trillion cells, yet the human genome only contains roughly 20,000 genes. So, to produce every tissue and cell type in the body, different combinations of genes must operate at different moments in the development of an organ or tissue. Being able to turn off genes at specific moments in a cell's development allows their changing roles to be investigated. Professor Ludovic Vallier, one of the senior authors of the study from the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute at the University of Cambridge and the Sanger Institute, said: "As a cell develops from being stem cell to being a fully differentiated adult cell the genes within it take on different roles. Before, if we knocked out a gene, we could only see what effect this had at the very first step. By allowing the gene to operate during the cell's development and then knocking it out with sOPTiKO at a later developmental step, we can investigate exactly what it is doing at that stage." The sOPTiKO and sOPTIKD methods allow scientists to silence the activity of more than one gene at a time, so researchers have the possibility to now investigate the role of whole families of related genes by knocking down the activity of all of them at once. In addition, the freely available system allows experiments to be carried out far more rapidly and cheaply. sOPTiKO is highly flexible so that it can be used in every tissue in the body without needing to create a new system each time. sOPiTKD allows vast improvements in efficiency: it can be used to knock down more than one gene at a time. Before, to silence the activity of three genes, researchers had to knock down one gene, grow the cell line, and repeat for the next gene, and again for the next. Now it can do it all in one step, cutting a nine-month process down to just one to two months. Dr Alessandro Bertero, one of the first authors of the study from the Cambridge Stem Cell Institute, said: "Two key advantages of using sOPTiKO/sOPTIKD over other CRISPR editing systems are that it is truly inducible and can work in almost any cell type. In the past we have been hampered by the fact we could study a gene's function only in a specific tissue. Now you can knock out the same gene in parallel in a diversity of cell type with different functions." More information: Bertero A et al. (2016) Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs. Development 143: 4405-4418. DOI: 10.1242/dev.138081


News Article | November 30, 2016
Site: www.biosciencetechnology.com

Wellcome Trust Sanger Institute and University of Cambridge researchers have created sOPTiKO, a more efficient and controllable CRISPR genome editing platform. In the journal Development, they describe how the freely available single-step system works in every cell in the body and at every stage of development. This new approach will aid researchers in developmental biology, tissue regeneration and cancer. Two complementary methods were developed. sOPiTKO is a knock-out system that turns off genes by disrupting the DNA. sOPTiKD is a knock-down system that silences the action of genes by disrupting the RNA. Using these two methods, scientists can inducibly turn off or silence genes, in any cell type, at any stage of a cell's development from stem cell to fully differentiated adult cell. These systems will allow researchers world wide to rapidly and accurately explore the changing role of genes as the cells develop into tissues such as liver, skin or heart, and discover how this contributes to health and disease. The body contains approximately 37 trillion cells, yet the human genome only contains roughly 20,000 genes. So, to produce every tissue and cell type in the body, different combinations of genes must operate at different moments in the development of an organ or tissue. Being able to turn off genes at specific moments in a cell's development allows their changing roles to be investigated. Professor Ludovic Vallier, one of the senior authors of the study from the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute at the University of Cambridge and the Sanger Institute, said: "As a cell develops from being stem cell to being a fully differentiated adult cell the genes within it take on different roles. Before, if we knocked out a gene, we could only see what effect this had at the very first step. By allowing the gene to operate during the cell's development and then knocking it out with sOPTiKO at a later developmental step, we can investigate exactly what it is doing at that stage." The sOPTiKO and sOPTIKD methods allow scientists to silence the activity of more than one gene at a time, so researchers have the possibility to now investigate the role of whole families of related genes by knocking down the activity of all of them at once. In addition, the freely available system allows experiments to be carried out far more rapidly and cheaply. sOPTiKO is highly flexible so that it can be used in every tissue in the body without needing to create a new system each time. sOPiTKD allows vast improvements in efficiency: it can be used to knock down more than one gene at a time. Before, to silence the activity of three genes, researchers had to knock down one gene, grow the cell line, and repeat for the next gene, and again for the next. Now it can do it all in one step, cutting a nine-month process down to just one to two months. Dr. Alessandro Bertero, one of the first authors of the study from the Cambridge Stem Cell Institute, said: "Two key advantages of using sOPTiKO/sOPTIKD over other CRISPR editing systems are that it is truly inducible and can work in almost any cell type. In the past we have been hampered by the fact we could study a gene's function only in a specific tissue. Now you can knock out the same gene in parallel in a diversity of cell type with different functions." For a video of the methods in action see below:


Riddell A.,Flow Cytometry Core Facility | Riddell A.,Cambridge Stem Cell Institute | Gardner R.,Instituto Gulbenkian Of Ciencia | Perez-Gonzalez A.,Flow Cytometry Core Facility | And 3 more authors.
Methods | Year: 2015

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instrument's sort count. Yield and Recovery are often neglected, mostly due to difficulties in their measurement. Purity of the sort product is often cited alone but is not sufficient to evaluate sorting performance. All of these three performance metrics require re-sampling of the sorted fraction. But, unlike Purity, calculating Yield and/or Recovery calls for the absolute counting of particles in the sorted fraction, which may not be feasible, particularly when dealing with rare populations and precious samples. In addition, the counting process itself involves large errors.Here we describe a new metric for evaluating instrument sort Recovery, defined as the number of particles sorted relative to the number of original particles to be sorted. This calculation requires only measuring the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch (CSC), avoiding re-sampling the sorted fraction and absolute counting. We called this new metric Rmax, since it corresponds to the maximum expected Recovery for a particular set of instrument parameters. Rmax is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter, or any instrument related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument performance before single-cell sorting experiments. Because we do not perturb the sort fraction we can calculate Rmax during the sort process, being especially valuable to check instrument performance during rare population sorts. © 2015 The Authors.


PubMed | Flow Cytometry Core Facility, Instituto Gulbenkian Of Ciencia, ETH Zurich, Flow Cytometry Unit and Cambridge Stem Cell Institute
Type: | Journal: Methods (San Diego, Calif.) | Year: 2015

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instruments sort count. Yield and Recovery are often neglected, mostly due to difficulties in their measurement. Purity of the sort product is often cited alone but is not sufficient to evaluate sorting performance. All of these three performance metrics require re-sampling of the sorted fraction. But, unlike Purity, calculating Yield and/or Recovery calls for the absolute counting of particles in the sorted fraction, which may not be feasible, particularly when dealing with rare populations and precious samples. In addition, the counting process itself involves large errors. Here we describe a new metric for evaluating instrument sort Recovery, defined as the number of particles sorted relative to the number of original particles to be sorted. This calculation requires only measuring the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch (CSC), avoiding re-sampling the sorted fraction and absolute counting. We called this new metric Rmax, since it corresponds to the maximum expected Recovery for a particular set of instrument parameters. Rmax is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter, or any instrument related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument performance before single-cell sorting experiments. Because we do not perturb the sort fraction we can calculate Rmax during the sort process, being especially valuable to check instrument performance during rare population sorts.


Lorber B.,University of Cambridge | Lorber B.,Paracelsus Medical University | Hsiao W.-K.,Research Center Pharmaceutical Engineering GmbH | Martin K.R.,University of Cambridge | And 3 more authors.
Current Opinion in Ophthalmology | Year: 2016

Purpose of review: Biological three-dimensional printing has received a lot of media attention over recent years with advances made in printing cellular structures, including skin and heart tissue for transplantation. Although limitations exist in creating functioning organs with this method, the hope has been raised that creating a functional retina to cure blindness is within reach. The present review provides an update on the advances made toward this goal. Recent findings It has recently been shown that two types of retinal cells, retinal ganglion cells and glial cells, can be successfully printed using a piezoelectric inkjet printer. Importantly, the cells remained viable and did not change certain phenotypic features as a result of the printing process. In addition, recent advances in the creation of complex and viable three-dimensional cellular structures have been made. Summary Some first promising steps toward the creation of a functional retina have been taken. It now needs to be investigated whether recent findings can be extended to other cells of the retina, including those derived from human tissue, and if a complex and viable retinal structure can be created through three-dimensional printing. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.


McDowell G.S.,Tufts University | McDowell G.S.,The Future of Research | Philpott A.,University of Cambridge | Philpott A.,Cambridge Stem Cell Institute
International Journal of Developmental Biology | Year: 2016

The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile “cell-in-a-test-tube” system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis. © 2016 UPV/EHU Press.

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