Towards establishing clinical-grade human embryonic stem cell lines

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

Abstract

Human embryonic stem cells (hESC) are derived from surplus embryos made available through in vitro fertilization programmes. These cells are very much like the cells of the early embryo and they retain the ability to make all the different specialized types of cells in our organs and tissues e.g. blood cells, brain cells, skin cells etc.  By culturing hESC in laboratory conditions we can study how these cells develop into the specialised cells of our organs and tissues. There is also the future prospect of using hESC to generate specialised cells that can be used to treat degenerative diseases that would benefit from stem cell transplantation (e.g. in diabetes, heart disease, Parkinson disease etc) or accidental injuries (e.g. skin burns). We have been able to derive hES-NCL1 embryonic stem cell line in 2004. However, the derivation and culture process for this cell line was done in the presence of foreign (mouse) cells and/or chemicals derived from animals. Although this cell line provides an excellent tool for basic research, it is unsuitable for clinical applications. 

The object of this application is to develop specialised laboratory and culture techniques so that new lines of hESC can be isolated and expanded under conditions whereby they are not exposed to animal cells or to animal products. Such cells would then have the potential to be used in treating patients (stem cell therapy).

Technical Summary

Human embryonic stem cells are pluripotent cells derived from the inner cell mass of blastocysts. They can be differentiated into numerous cell types and as such they offer new opportunities for cell replacement therapies, drug discovery and understanding of human embryonic development.

We have been successful in deriving a new fully characterised human embryonic stem cell line (hES-NCL1). We are culturing this cell line on novel human feeder cells which we have recently derived, and extracellular matrix (ECM) protein supplemented with the conditioned medium from these feeders. All the available human ES cell lines have been derived in the presence of animal ingredients, but our novel human feeders offer great advantages for deriving clinical grade human ES cell lines without the risk of pathogen transfer from animal based ingredients. We intend to improve xeno-free and feeder-free culture conditions by using human feeders and new human components of the ECM with the conditioned medium derived from our novel feeders.

Publications

10 25 50

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Athanasopoulos T (2017) Nonintegrating Gene Therapy Vectors. in Hematology/oncology clinics of North America

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

 
Description Evidence session for the select committee for the Science and Technology at the House of Commons. This served to gather evidence for proposed changes to the HFEA bill.
Geographic Reach Multiple continents/international 
Policy Influence Type Gave evidence to a government review
Impact The impact of this and other evidence sessions was to formulate the changes to the HFEA bill that resulted in this becoming law in November 2008; The most notable achievement was that our group was awarded a licence from the HFEA to permit interspecies somatic cell nuclear transfer (human into animal) following the evidence and our group´s work with human embryonic stem cells.
 
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 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 ERC CONSOLIDATOR FELLOWSHIP
Amount € 1,300,000 (EUR)
Funding ID 614620 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
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 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 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 Integrated Mres/PhD studentship
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2012 
End 08/2016
 
Description MRC UK project grant
Amount £617,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 07/2018 
End 07/2021
 
Description MRC/BBSRC Human iPS cell research project grant
Amount £62,500 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 07/2008 
End 03/2009
 
Description MRC/BBSRC Human iPS cell research project grant
Amount £625,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 07/2008 
End 03/2009
 
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 01/2011 
End 01/2012
 
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 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 Sabbatical fellowship/Conselleria de Sanidad (Generalitat Valenciana), and the Instituto de Salud Carlos III (Ministry of Science and
Amount £750,000 (GBP)
Organisation Spanish Ministry of Science and Innovation 
Sector Public
Country Spain
Start 09/2009 
End 12/2010
 
Description Single Cell Genomics Unit
Amount £2,000,000 (GBP)
Funding ID MR/M008886/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
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
 
Title hES-NCL1 - 9 and NCL hiPSC 1 -50 
Description Nine new human ESC lines were derived during the G0301182 MRC funded study and these have been deposited at the UK Stem Cell Bank. More than 50 hiPSC lines have also been derived with the supplementary funding received in 2009 as extension to this project. 
Type Of Material Cell line 
Year Produced 2008 
Provided To Others? Yes  
Impact The nine new cell lines and their characteristics have been registered in the EU hESC registry which will promote interactions between european stem cells scientists. In addition, two of published iPSC lines have been sent to our collaborators in Madison, USA and Oxford UK for further studies. 
 
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 transcriptional data 
Description Affymetrix transcriptional data has been generated for the hES-NCL1, H9 and H1 human ESC lines and embryoid bodies (day6, day10, day 14, day 21) derived from them. 
Type Of Material Biological samples 
Year Produced 2008 
Provided To Others? Yes  
Impact We are using the "know how" from this exercise to analyse transcriptional data generated from hESC derived haematopoietic stem cells. The set of transcriptional data has been sent to our collaborators in Sweden, Australia and Spain to enable large scale comparisons with other human ESC lines. 
 
Description International Stem Cell Initiative 
Organisation University of Sheffield
Department Department of Biomedical Science
Country United Kingdom 
Sector Academic/University 
PI Contribution We used the first human embryonic stem cell derived by our group, hES-NCL1 to be included in ICSI 1 and 2. Samples of RNA and DNA were used for expression, methylation and epigenetic analyses. In addition, in house characterisation of cell surface markers by flow cytometry and immunocytochemistry was submitted to ISCI 1 for data comparison with other human ESC lines.
Collaborator Contribution This is part of the International Stem Cell Initiative (ISCI) spearheaded by Prof. Peter Andrews at Sheffield University. My group has been part of ISCI 1 and 2 and I have participated in the annual conferences organised at Bar Harbor, USA. The results of these initiatives have helped the characterisation of the nine human embryonic stem cell lines derived by our group (and already reported in Stem Cells 2006; 24: 2669-2776 and Stem Cells 22: 790-797) as well as characterisation of newly derived iPSC lines (reported in Stem cells Armstrong et al. 2009, under revision).
Impact This has resulted in 4 publication as follows: 17572666, 16990582, 15342943 and the manuscript that is under revision by Stem Cells shown in publication section.
Start Year 2006
 
Description Testing of functionality of lab made retina using MEA 
Organisation Newcastle University
Department Institute of Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution We are improving methods for generating hESC and hiPSC lab made retina.
Collaborator Contribution Dr. Sernagor's group is testing the functionality of lab made retina using MEA.
Impact collaboration, manuscript and further funding
Start Year 2009
 
Description collaboration between Tufts Medical Centre and my group 
Organisation Tufts Medical Center
Country United States 
Sector Academic/University 
PI Contribution All our gene and protein expression data were made available to our collaborator to enable his comparative cell cycle regulation in neural stem cells to embryonic stem cells.
Collaborator Contribution We over-expressed human NANOG in human ESC with the aim of identifying new secreted factors that could perhaps be used to further define human ESC media to be applied on GMP laboratories. On the course of this investigation we discovered an important role for NANOG in cell cycle regulation of human ESC. This was a new area of investigation for my group and Prof. Hind´s group expertise at Tufts Medical Centre was essential in establishment of all cell cycle related techniques and the transfer of such expertise in my group.transfer of skills and expertise in the cell cycle field
Impact Notable outputs comprise the following publications: 19305136, 19139263,18806832, 18521083, 1863806
Start Year 2006
 
Description collaboration between Tufts Medical Centre and my group 
Organisation Tufts Medical Center
Country United States 
Sector Academic/University 
PI Contribution All our gene and protein expression data were made available to our collaborator to enable his comparative cell cycle regulation in neural stem cells to embryonic stem cells.
Collaborator Contribution We over-expressed human NANOG in human ESC with the aim of identifying new secreted factors that could perhaps be used to further define human ESC media to be applied on GMP laboratories. On the course of this investigation we discovered an important role for NANOG in cell cycle regulation of human ESC. This was a new area of investigation for my group and Prof. Hind´s group expertise at Tufts Medical Centre was essential in establishment of all cell cycle related techniques and the transfer of such expertise in my group.transfer of skills and expertise in the cell cycle field
Impact Notable outputs comprise the following publications: 19305136, 19139263,18806832, 18521083, 1863806
Start Year 2006
 
Description collaboration with CIPF, Valencia, Spain 
Organisation Regional Government of Valencia
Department Príncipe Felipe Research Centre (CIPF)
Country Spain 
Sector Charity/Non Profit 
PI Contribution We have a strong collaboration with Prof. Ximo Dopazo's group for bioinformatics analysis of RNA seq data.
Collaborator Contribution Centro de Investigacion Principe Felipe provided laboratory space, reagents and staff costs to enable myself and some members of my group to carry out a one year sabbatical vocation during which we concentrated on studies of signalling pathways in hESC and methods to increase the efficiency of iPSC derivation. At this time we forged strong links with Prof. Dopazo's group and initiated a number of bioinformatics expression studies which are still ongoing.
Impact Several publications resulted from this collaboration and the PMID numbers are outlined below: PMID: 20882529 PMID: 20235273 PMID: 2007308
Start Year 2009
 
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 collaboration with Dr. Sophie Hambleton's group 
Organisation Newcastle University
Department Institute of Cellular Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution our groups have been deriving iPSC lines from patients with hematopoietic deficiencies
Collaborator Contribution provision of primary fibroblasts samples from patients and acquisition of ethical permission
Impact Dr. Hambleton has applied for an MRC senior fellowship
Start Year 2011
 
Description collaboration with MRC Centre for Regenerative Medicine Centre Edinburgh 
Organisation University of Edinburgh
Department MRC Centre for Regenerative Medicine
Country United Kingdom 
Sector Public 
PI Contribution We are transferring our retinal differentiation skills and protcols to the group led by Prof. Charles Ffrench Constant
Collaborator Contribution provision of antibodies
Impact award of a MRC clinical fellowship to Dr. Rolan Megan
Start Year 2012
 
Description collaboration with Prof. Chinnery's group 
Organisation Newcastle University
Department Institute of Genetic Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution our groups have been deriving iPSC lines from patients with mitochondrial disorders
Collaborator Contribution our groups have been deriving iPSC lines from patients with mitochondrial disorders
Impact award of a three year project grant by BBSRC
Start Year 2010
 
Description collaboration with Prof. Penny Jeggo's group 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution We have derived the iPSC lines from samples sent by Jeggo group and have fully characterised those in close collaboration.
Collaborator Contribution provision of patient specific samples and data analysis
Impact Publications: 23818183, 23722522
Start Year 2009
 
Description collaboration with Prof. Robin Ali 
Organisation University College London
Department Institute of Ophthalmology UCL
Country United Kingdom 
Sector Academic/University 
PI Contribution Our research groups are testing in collaboration the engraftment of hESC/hiPSC derived photoreceptor precursors
Collaborator Contribution provision of skils, murine models and transplantation techniques
Impact succesful award from Fight for Sight UK (two year project grant)
Start Year 2010
 
Description collaboration with Prof. Stefan Pryzborski 
Organisation Durham University
Country United Kingdom 
Sector Academic/University 
PI Contribution We rely on Prof. Pryzboski's team to do the teratoma analysis for our group. We provide assistance and advice with respect to iPSC culture.
Collaborator Contribution Teratoma analysis and ICC is performed by Przyborski's group.
Impact 18 in total here are some selected recent one: 23818183 23722522 22311747
 
Description non degradable substrates for RPE cell culture 
Organisation University Hospital Southampton NHS Foundation Trust
Country United Kingdom 
Sector Hospitals 
PI Contribution we provide the pluripotent stem cell derived RPE and photoreceptors
Collaborator Contribution 1. Dr Carl Sheridan and Dr. Andrew Lotery have provided for us FDA approved non degradable substrates that have been tested by his group for retinal pigmented epithelial (RPE) cell culture and transplantation in a rabbit model of retinal degeneration. We have started to test these substrates for combined expansion of pluripotent- stem cell-derived photoreceptors and RPE with the aim of optimising culture conditions and delivering human cells suitable for clinical transplantation.
Impact multi-disciplinary
Start Year 2010
 
Description non degradable substrates for RPE cell culture 
Organisation University of Liverpool
Department Institute of Ageing and Chronic Disease
Country United Kingdom 
Sector Academic/University 
PI Contribution we provide the pluripotent stem cell derived RPE and photoreceptors
Collaborator Contribution 1. Dr Carl Sheridan and Dr. Andrew Lotery have provided for us FDA approved non degradable substrates that have been tested by his group for retinal pigmented epithelial (RPE) cell culture and transplantation in a rabbit model of retinal degeneration. We have started to test these substrates for combined expansion of pluripotent- stem cell-derived photoreceptors and RPE with the aim of optimising culture conditions and delivering human cells suitable for clinical transplantation.
Impact multi-disciplinary
Start Year 2010
 
Title WO 2005/080551A2 (Stem Cells) 
Description The patent describes the methods used for the derivation of human ESC lines from arrested and normal embryos as well as derivation of autologous feeder cells. 
IP Reference WO2005080551 
Protection Patent granted
Year Protection Granted
Licensed No
Impact Autologous feeder cells are routinely being used for derivation of human ESC and more recently promoted to be the best feeder layer for efficient derivation of hiPSC. Derivation of hESC from arrested embryos has also been validates as way forward in many catholic countries like Switzerland since it does not involve usage of good quality embryos that are a priority for reproductive purposes.
 
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 Cheltenham Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presented data about human embryonic stem cells and their potential use for biological studies, drug discovery and regenerative medicine.

education of public about stem cell research in UK and worldwide
Year(s) Of Engagement Activity 2008
 
Description Foreign Office travel visit to Israel 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Health professionals
Results and Impact To explain how legislative changes in UK helped the stem cell research to policymakers in Israel

televised debate in Jerusalem followed the talk
Year(s) Of Engagement Activity 2008
 
Description Invited speaker at the annual research day organised by Macular Disease society UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Around 80 patients and their families attended this event. Prof. Lako and Dr. Collin delivered two talks on the impact of stem cell developments and technologies on disease modelling and understanding.

We have been asked to participate in the next's year annual event.
Year(s) Of Engagement Activity 2014
 
Description Invited speaker at the annual research day organised by RP Fighting Blindness UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact more than 200 RP patients and their families attended the events as well as health professionals and researchers working in this area

we were asked to participate in the 2014 patient day event organised by RPFB
Year(s) Of Engagement Activity 2014
 
Description New tools for treating human inherited diseases 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker: LivES symposium: Embryonic Stem Cells: Paris, 12-13th March 2012

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Scientic visit to Suzhou Nanotechnology Institute China 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Health professionals
Results and Impact 100 research fellow and group leaders attended the talk

collaboration has been established between Dr Dai's group and Lako's group
Year(s) Of Engagement Activity 2011
 
Description UK-Korea symposium on Guidelines, Ethics and Regulation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Talk about UK regulation on human ESC derivation and nuclear transfer

large audience (500 people)
Year(s) Of Engagement Activity 2008
 
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 inaugural meeting of Stem Cell Therapy Interest Group, Glasgow 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Lako: keynote speaker

This was an excellent event bringing together stem cell biologists from Glasgow and surrounding regions and great opportunity for networking.
Year(s) Of Engagement Activity 2013
 
Description talk at the science and technology select commitee, House of Commons 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact to lobby in favour of the changes proposed for the new HFEA BILL (now the act)

press conference followed the talk to select commitee
Year(s) Of Engagement Activity 2008