A state of the art multiparametric flow cytometry analysis system for multidisciplinary stem cell research

Lead Research Organisation: Newcastle University
Department Name: Institute of Human Genetics

Abstract

Stem cells are capable of essentially extensive growth whilst retaining the ability to differentiate into most of the cell types found in the adult but unless we can find ways to grow them in large numbers while directing them to differentiate into therapeutically useful cells much of this potential will not be realised. A major obstacle impacting the derivation of differentiated cell types from stem cells is that current in vitro differentiation strategies often only produced the desired cells in small numbers. This means they must be separated from the other cell types present and one of the best techniques we have for doing this is flow cytometry. This technology relies upon the binding of fluorescent labelled antibodies to the surfaces of our desired cells such that when they pass through the flow cytometer they can be separated from the others and retain their viability. If appropriate antibodies are available, up to 12 different colours can be analysed at any one time using the instrumentation detailed in this proposal and this offers us an unparalleled advantage in cell identification and separation. There are a number of active research groups at the Institute of Human Genetics investigating a range of differentiation protocols designed to produce useful cells from a range of stem cells and flow cytometry instrumentation is not only indispensable to this work but is also subject to very heavy demand. In view of this, a successful outcome of this proposal would greatly enhance our ability to develop useful techniques for stem cell differentiation that will ultimately benefit medical science in general.

Technical Summary

Stem cells, both embryonic and adult offer great promise for the development of therapeutic applications but for both of these stem cell types, separation of specific cells will be required. In the case of embryonic stem cells there is a need for culture strategies that can direct their differentiation towards therapeutically useful cells but currently available techniques produce heterogeneous mixtures from which we need to separate the desired cells. Adult stem cells exist in heterogeneous microenvironments or niches and again are often present in only very small numbers underlining the requirement for advanced separation technologies. Fluorescence activated cell sorting (FACS) is the most versatile method for separating such small numbers, provided that specific antibodies are available for the surface proteins of a specific cell type. A great advantage of FACS is that multiple antibodies labelled with different fluorochromes may be used in a single cell sample thereby increasing the total data available from a single experiment. Such multiparametric flow cytometry is cost effective in terms of antisera consumption and permits more detailed identification and isolation of rare cells but this requires the development of additional fluorescent labelled antibodies against proteins specific to these cells. This can only be achieved by increasing our understanding of stem cell biology which is a major focus of stem cell research at the Institute of Human Genetics. Stem cells of all types are difficult to grow in vitro hence a better understanding of the signal transduction mechanisms which promote pluripotency in embryonic stem cells and contribute to interaction with the niche for adult stem cells are an essential part of this proposal. Multiparametric flow cytometry will be an important part of this investigation since may use it for parallel examination of multiple intracellular proteins with small numbers of cells.

Publications

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Atkinson SP (2013) Potential for pharmacological manipulation of human embryonic stem cells. in British journal of pharmacology

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Baylis O (2011) 13 years of cultured limbal epithelial cell therapy: a review of the outcomes. in Journal of cellular biochemistry

 
Description The funding from this award has been used to establish the new multicolour state of the art flow cytometry system at the Institute of Genetic Medicine. This euqipment has provided a unique resource for Stem Cell groups in Newcastle and Durham University enabling research in areas of embryonic and somatic stem cell biology as well as developmental and cell biology projects across both campuses. The equipment is now part of the University wide flow cytometry core facility which is now self-funded through FEC based internal charging system.
Exploitation Route Through collaborative links with Guava and BD Biosciences, we test a range of monoclonal antibodies and cell surface markers prior to their commercialisation using funds obtained via the KTP scheme. The equipment purchased via this equipment award is now part of the University wide flow cytometry core facility which is now self-funded through FEC based internal charging system.
Sectors Leisure Activities, including Sports, Recreation and Tourism

 
Description 3D hiPSC derived laminated retina model
Amount £1,000,000 (GBP)
Funding ID NC/C016106/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2017 
End 10/2020
 
Description BBSRC Equipment Grant
Amount £141,849 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2008 
End 05/2009
 
Description BBSRC Equipment Grant (A state of the art multiparametric flow cytometry analysis system for multidisciplinary stem cell research)
Amount £251,513 (GBP)
Funding ID BB/E012841/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2007 
End 03/2008
 
Description BBSRC project grant
Amount £460,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2012 
End 12/2014
 
Description BBSRC strategic tools and objectives scheme
Amount £140,000 (GBP)
Funding ID BB/I02333X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2011 
End 07/2012
 
Description BHF PROJECT GRANT
Amount £140,000 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 02/2014
 
Description BRC project grant
Amount £60,000 (GBP)
Organisation National Institute for Health Research 
Department NIHR Newcastle Biomedical Research Centre
Sector Academic/University
Country United Kingdom
Start 01/2009 
End 12/2009
 
Description Capacity Building in Higher Education
Amount £700,000 (GBP)
Organisation erasmus + 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Cell surface marking of corneal epithelial stem cells: a powerful approach to stem cell enrichment prior to transplantation
Amount £50,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 07/2018
 
Description Chronic Granulomatous Disease project grant
Amount £150,000 (GBP)
Organisation The Chronic Granulomatous Disorder Society 
Department Chronic Granulomatous Disorder (CGD) Research Trust
Sector Charity/Non Profit
Country United Kingdom
Start 08/2010 
End 07/2012
 
Description Corneal epithelial stem cell therapy: enhancing safety and stability of cultured product
Amount £120,000 (GBP)
Organisation UCL Partners 
Department UCL Partners AHSC and AHSN
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 09/2015
 
Description Demonstrating proof of principle for generating a platform technology to direct differentiation of pluripotent stem cell using Sendai Viral vectors
Amount £62,000 (GBP)
Organisation Medical Research Council (MRC) 
Department MRC Confidence in Concept Scheme
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 08/2017
 
Description ERC CONSOLIDATOR FELLOWSHIP
Amount € 1,300,000 (EUR)
Funding ID 614620 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 06/2014 
End 05/2018
 
Description European Bank of induced pluripotent stem cells (EbiSC)
Amount € 232,500 (EUR)
Funding ID EbiSC 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 05/2014 
End 09/2016
 
Description Exploiting the role of exosomes to treat age related macular degeneration and provide biomarkers for early diagnosis of the disease
Amount £64,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 08/2018
 
Description Fanconi Anaemia Fund project grant
Amount £50,000 (GBP)
Organisation Fanconi Anemia Research Fund (FARF) 
Sector Charity/Non Profit
Country United States
Start 12/2009 
End 12/2010
 
Description Fight for Sight UK project grant
Amount £166,000 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2009 
End 03/2012
 
Description Fight for Sight project grant
Amount £170,000 (GBP)
Funding ID 1456/1457 
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 08/2017
 
Description Generation of disease models for Fanconi Anaemia using pluripotent stem cells
Amount £78,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2011 
End 12/2011
 
Description Investigating haematopoietic development in Ligase IV and XLF patients using induced pluripotent stem cells
Amount £166,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2009 
End 04/2012
 
Description Investigation of the haematopoietic differentiation of human ES cells
Amount £146,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2005 
End 07/2008
 
Description MRC Confidence in Concept Scheme
Amount £26,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 09/2016
 
Description MRC Integrated Mres/PhD studentship
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2012 
End 08/2016
 
Description MRC Translational Stem Cell Research Committee (TSCRC) Grant
Amount £1,206,610 (GBP)
Funding ID G0900879 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2010 
End 03/2015
 
Description MRC/BBSRC Human iPS cell research project grant
Amount £625,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2008 
End 03/2009
 
Description MRC/BBSRC Human iPS cell research project grant
Amount £62,500 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2008 
End 03/2009
 
Description Macular Disease Society PROJECT GRANT
Amount £170,000 (GBP)
Organisation Macular Society 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2014 
End 03/2017
 
Description NOVARTIS PROJECT GRANT
Amount £41,400 (GBP)
Organisation Novartis 
Sector Private
Country Global
Start 04/2012 
End 03/2013
 
Description Newcastle Health Charity project grant
Amount £49,000 (GBP)
Organisation Newcastle upon Tyne Hospitals NHS Foundation Trust 
Department Newcastle Healthcare Charity
Sector Charity/Non Profit
Country United Kingdom
Start 01/2007 
End 12/2007
 
Description Newcastle Health Charity project grant
Amount £49,000 (GBP)
Organisation Newcastle upon Tyne Hospitals NHS Foundation Trust 
Department Newcastle Healthcare Charity
Sector Charity/Non Profit
Country United Kingdom
Start 01/2011 
End 01/2012
 
Description PhD studentship
Amount £100,000 (GBP)
Organisation Newcastle University 
Department Dr William Edmund Harker Foundation
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description RP Fighting Blindness project grant
Amount £150,000 (GBP)
Funding ID GR584 
Organisation RP Fighting Blindness 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 12/2015
 
Description Single Cell Genomics Unit
Amount £2,000,000 (GBP)
Funding ID MR/M008886/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2015 
End 12/2015
 
Description Sir James Knott Trust project grant
Amount £600,000 (GBP)
Organisation Sir James Knott Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2008 
End 12/2008
 
Description Stem cells for biological assays of novel drugs and predictive toxicology
Amount € 2,200,000 (EUR)
Funding ID STEMBANCC 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 10/2012 
End 09/2017
 
Description Understanding the role of autophagy in the pathogenesis of AMD using a patient specific iPSC model
Amount £170,000 (GBP)
Organisation Macular Society 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2017 
End 12/2020
 
Title donor contructs for introducing GFP reporter into the 3' UTR of CRX gene 
Description a donor construct was made to introduce GFP into the endogenous CRX locus 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact this will enable other researchers to introduce this reporter into their human ESC and iPSC lines and monitor differentiation process 
 
Title human iPSC lines from diseased individuals 
Description human iPSC lines from patients with Fanconi Anaemia (type C), Ligase IV and XLF deficient patients, CGD patients, Muscular Dystrophy and HLHS patients 
Type Of Material Cell line 
Year Produced 2011 
Provided To Others? Yes  
Impact several publications have risen from this work as follows; 22311747 23818183 23722522 23280624 23582880 
 
Title human iPSC lines from patients with PRPF31 mutations 
Description these are patient specific cell lines generated from reprogramming of adult fibroblasts taken from skin biopsies of PRPF31 patients 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? No  
Impact the availability of this material will enable generation of in vitro disease models 
 
Description Testing of monoclonal antibodies 
Organisation Inova Technology
Country United States 
Sector Private 
PI Contribution We are very active testing monoclonal antibodies for: Becton Dickinson Miltenyi Biotec Biolegend Biostatus Beta testing software for Applied Cytometry Sheffield (Venturi one software) Inivai Technologies, Victoria Australia (Gatelogic & Flow Logic) This allows us to do three things: 1 Acquire free antibodies from the companies allowing us to thoroughly test the suitability of the antibodies and fluorochromes at no cost to ourselves, in addition we have residual antibody left after this test that, if acceptable, we can use and or distribute to users. 2 Develop a symbiotic relationship with the companies whereby we often get free kits to run our internal user training (such as the Apoptosis course) at no cost in terms of reagents. 3 Because of the relationship we develop we often get asked to give our opinion on newly developed kits , and also , recently train users on instrumentation for Becton Dickinson 4 The ability to try and evaluate software inevitably leads to us acquiring free of charge the software in question and so adding to the users choice of free software available in the core facility.
Start Year 2007
 
Description collaboration with Dr. Saretzki and Prof. von Zglinicki 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution We are interested in the role of telomerase, DNA damage and oxidative stress in ESC and iPSC as well as their derivatives for disease modelling studies.
Collaborator Contribution Our collaborators provide advice and assistance on telomerase, DNA damage and oxidative stress measurement assays.
Impact 23818183 23722522 23280624 22311747 20073085 18203676 18055443 15790773 15536187 15511642
Start Year 2006
 
Description Core member of committee C, BBSRC 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact member of committee C: reading and reviewing proposals, helping to shape UK policy
Year(s) Of Engagement Activity 2017,2018
 
Description BIO 2008 conference presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Health professionals
Results and Impact 30 minutes talk to a large audience (25,000 participants)

Radio conference followed the talk
Year(s) Of Engagement Activity 2008
 
Description European Commission, Evaluator of REA-FET-OPEN-2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact panel member: discuss proposals and reach funding decisions
Year(s) Of Engagement Activity 2017
 
Description European Commission, Member of H02020 panel "Clinical Research on regenerative medicine", June 2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact panel member: proposal discussion and selection for funding
Year(s) Of Engagement Activity 2017
 
Description Flow cytometry training course 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Flow cytometry training course: Side population by flow cytometry This course is organised every year: number of participants between 20 and 30. Handbook produced

no actual impacts realised to date
Year(s) Of Engagement Activity 2007
URL http://www.guardian.co.uk/science/video/2010/mar/31/genetics-autism-zebra-finch
 
Description Member of the H2020-JTI-IMI2-panel 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Ho2020 panel member: proposal discussion and funding, helping to share EU research policy and funding.
Year(s) Of Engagement Activity 2017,2018
 
Description Member of the H2020: SC1-BHC-09-2018 panel 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Ho2020 panel member: proposal discussion and funding, helping to share EU research policy and funding.
Year(s) Of Engagement Activity 2006,2017
 
Description Organiser of the Newcastle RP Patient Information Day, 8th June 2016 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact 98 patients and their families attended the event which sparked a lot of questions, discussion and increased interest in application of stem cell therapies for retinal disease.
Year(s) Of Engagement Activity 2016
URL https://campus.recap.ncl.ac.uk/Panopto/Pages/Viewer.aspx?id=d7b01a8a-3dfe-486e-b0ae-bc8d02b3e3f9
 
Description Using stem cells to understand our development and human disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Talk

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description stem cell festival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact about 300 members of the public attended the event

we have been asked to participate every year
Year(s) Of Engagement Activity 2012,2013,2014