Using cell surface antigens for the isolation of photoreceptor precursor cells for retinal stem cell therapy

Lead Research Organisation: University College London
Department Name: Institute of Child Health

Abstract

Diseases of the retina resulting in the death of the light-sensing photoreceptor cells are the leading cause of blindness in the developed world. These conditions affect 1 in 3000 people from birth and more than 10% of the ageing population. They are currently untreatable and irreversible. We recently demonstrated in proof-of-concept experiments in mice that replacing lost photoreceptors by cell transplantation is a feasible clinical treatment strategy. We discovered that by transplanting immature photoreceptor cells, these cells can make new functional photoreceptors in the diseased retina, and restore some visual function. To translate our findings to a human therapy we need to isolate equivalent human photoreceptor precursors from stem cell cultures grown in the laboratory. Our hypothesis is that retinal cells generated from stem cell cultures will be effective and safe for human therapy provided they are treated so that they reach exactly the right stage in a cell culture dish and provided only these correctly-staged cells are transplanted. One critical challenge therefore is developing methods to identify and collect correctly-staged donor cells to transplant into the diseased retina. We will develop methods for isolating live cells by using antibodies that bind to special markers on the surface of the immature photoreceptor cells. The efficiency of the newly isolated cells for retinal repair will be tested in transplantation experiments. This research will develop cell isolation protocols for human photoreceptor precursor cells, which is an essential step towards development of clinical trials for incurable retinal diseases causing blindness.

Technical Summary

Retinal degenerative conditions result in the irrevocable loss of photoreceptor cells and are the leading cause of untreatable blindness in the Western world. We recently demonstrated in proof-of-concept experiments in mice that replacing lost photoreceptors by transplantation is a feasible clinical treatment strategy. We showed that post-mitotic, yet immature, rod photoreceptor precursor cells can efficiently integrate into the diseased murine retina after transplantation, differentiate into functional photoreceptors, and restore some visual function. Our data indicate that the ontogenetic stage of the donor cell is of paramount importance for transplantation success and that optimal integration is achieved only by transplanting immature precursors already committed to the photoreceptor lineage. To translate our findings in mice directly to a human therapy we need to isolate equivalent human photoreceptor precursors. Several stem cell sources have been identified for the generation of new photoreceptors. Our hypothesis is that retinal cells generated from stem cell cultures will be effective and safe for human therapy provided they are differentiated in vitro into post-mitotic photoreceptor precursors, and provided only these correctly-staged cells are transplanted. One critical challenge therefore facing clinical application is the ability to isolate homogeneous correctly-staged donor populations.

We propose to develop live cell sorting methods using antibodies that bind to surface antigens to isolate pure populations of rod and cone photoreceptor precursor cells, without genetic modification, initially from mouse and then from human stem cell cultures. Transcriptome and proteome analysis of precursors expressing the photoreceptor-specific transgenes Nrl-gfp and Crx-gfp will be used to define a panel of cell surface markers that identify the ontogenetic stages of photoreceptor development. From preliminary transcriptome analyses we have already identified several rod precursor cell surface markers. New cell surface antigen signatures will be tested using flow cytometry for isolation of donor precursor cells from the developing retina and from in vitro differentiated stem cell cultures. Isolation protocols will be tested on mouse and human cells and the efficacy of isolated cells for retinal repair will be tested in transplantation experiments using our established protocols and retinal disease models. The goal of this project is to demonstrate that cell surface markers can be used to isolate pure populations of mouse and human photoreceptor precursor cells from in vitro stem cell cultures that are effective for retinal repair. This study will develop cell isolation protocols for human photoreceptor precursor cells, which is a requisite step towards development of clinical trials.

Publications

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Ali RR (2011) Regenerative medicine: DIY eye. in Nature

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Barber AC (2013) Repair of the degenerate retina by photoreceptor transplantation. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Cell and Gene Therapy MSc, PG Diploma; PG Certificate: Module co-organiser 2011-2016, Project Examiner (2015-8), Board of Examiners (2016-8),
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description ARVO Travel Grant program
Amount £700 (GBP)
Organisation Association for Research in Vision and Ophthalmology 
Sector Charity/Non Profit
Country United States
Start 05/2011 
End 05/2011
 
Description Bogue Research Fellowship
Amount £3,000 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 02/2014 
End 04/2014
 
Description Central Research Fund, University of London
Amount £5,000 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 07/2010 
End 11/2010
 
Description Fight for Sight Project grant - Using Crx-positive precursors for cone photoreceptor replacement
Amount £169,800 (GBP)
Funding ID 1351/52 
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description Great Ormond Street Hospital Children's Charity Research Leadership Award
Amount £253,945 (GBP)
Funding ID V1257 
Organisation Great Ormond Street Hospital Children's Charity (GOSHCC) 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2012 
End 03/2015
 
Description Great Ormond Street Hospital Special Trustees PhD Studentship
Amount £67,500 (GBP)
Organisation Great Ormond Street Hospital Children's Charity (GOSHCC) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2009 
End 10/2012
 
Description Newlife Project Grant 2013 Cell therapy for congenital retinal dystrophy
Amount £120,000 (GBP)
Funding ID 2AAAB 
Organisation Newlife 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2015 
End 12/2016
 
Description PhD Studentship
Amount £71,000 (GBP)
Organisation Great Ormond Street Hospital Children's Charity (GOSHCC) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 10/2016
 
Description PhD Studentship
Amount £71,000 (GBP)
Organisation Great Ormond Street Hospital (GOSH) 
Department NIHR Great Ormond Street Biomedical Research Centre
Sector Academic/University
Country United Kingdom
Start 09/2013 
End 09/2016
 
Title Biomarkers for photoreceptor isolation 
Description Combination of cell surface biomarkers for the non-genetic isolation of photoreceptor cells for retinal cell therapy 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This tool enables the non-genetic isolation of human donor cells from human pluripitent stem cell culture systems for retinal cell replacement therapy. 
 
Title Cell sorting using unique antibody sets to isolate photoreceptor precursors 
Description New approach for the isolation of specific cell types from tissues/stem cell cultures. Development of cell sorting protocols for isolation of photoreceptor precursors from mixed cell populations using antibodies recognising cell surface markers. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact These published new protocols enable the isolation of photoreceptor precursors for photoreceptor transplantation experiments in our laboratory and by other groups which will impact research into the development of retinal stem cell therapies. 
 
Title Photoreceptor precursor transcriptome 
Description Microarray data providing information on the transcriptome of transplantable photoreceptor precursor cells 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2011 
Provided To Others? Yes  
Impact Identification of cell surface markers useful for selection of photoreceptor precursor cells that can be effectively transplanted - published in Stem cells 2011 Lakowski et al 
 
Title THRB2-EGFP 
Description THRB2-GFP H9 human embryonic stem cell reporter line for isolation of cone photoreceptors from retinal differentiation culture systems (organoids) 
Type Of Material Cell line 
Provided To Others? No  
Impact This research tool enables for the first time the isolation and characterization of human cone photoreceptors from hPSC derived differentiation cultures. Cone photoreceptors are crucial for human vision and central to the development of a cell therapy for retinal dystrophies. This cell line will facilitate studies into how cone photoreceptors develop and are maintained as well as make possible small molecule/drug screening approaches for reagents modulating cone cell metablism and physiology, 
 
Description Merck 
Organisation Merck
Country Germany 
Sector Private 
PI Contribution Test and use of novel gene editing tool for generation of human pluripotent stem cell fluorescent reporter lines specific for cone and rod photoreceptors
Collaborator Contribution Merck is providing a newly generated human pluripotent stem cell line engineered to facilitate the generation of fluorescent reporter lines.
Impact This collaboration has recently started. The first outcome is expect within 6month.
Start Year 2017
 
Description Professor David Gamm, Department of Ophthalmology and Visual Sciences University of Wisconsin 
Organisation University of Wisconsin-Madison
Department Department of Ophthalmology and Visual Sciences
Country United States 
Sector Academic/University 
PI Contribution New funding for laboratory visit to Department of Ophthalmology and Visual Sciences, University of Wisconsin USA (Bogue fellowship) to increase our expertise in human pluripotent stem cell differentiation for retinal therapy and application of cell surface markers for cell sorting.
Collaborator Contribution Prof Gamm's laboratory at the University of Wisconsin, Department of Ophthalmology and Visual Sciences, is a focal point of innovation in the area of stem cell therapy, benefiting from interaction with Prof. James Thompson, a key figure in the development in current human ES-cell technology. By modelling retinal development using both human ES-cell/iPSC-cell systems the Gamm group has, over the last 6 years, provided the basis for the development of effective differentiation strategies of donor cells for future clinical applications.
Impact This collaboration will enhance progress towards isolation of human photoreceptor cells for transplantation.
Start Year 2013
 
Description The Lasker Foundation/International Retinal Research Foundation (IRRF) Initiative for Innovation in Vision Science 
Organisation Lasker Foundation
Country United States 
Sector Charity/Non Profit 
PI Contribution Invited speaker, discussant, session chair, author of summary document. The Lasker/IRRF Initiative is currently examining rapidly developing cutting-edge technologies that can be applied to re-establish light sensitivity and visual perception to the blind. Leaders in retinal degeneration, ocular genetics, electrophysiology and sensorimotor research, molecular biology, neuro-ophthalmology, nanotechnology and regenerative medicine, ophthalmic imaging and other disciplines relevant to the project met at two workshops held during the summer of 2013 and in 2014. The collaborative nature of this multi-disciplinary approach is designed to bridge the gaps between scientific disciplines and encourage the best minds to define the most creative solutions to daunting scientific problems; to design streamlined approaches that use all available tools; to accelerate discovery, and to facilitate translational research leading to new treatments to reverse retinal degenerative vision loss.
Collaborator Contribution As above
Impact Invited participant in The Lasker Foundation/International Retinal Research Foundation (IRRF) Initiative for Innovation in Vision Science 2013-14 Chaired by John Dowling of Harvard University. Invited speaker at Woods Hole, Massachusetts, USA July 8-9. Session Leader and Speaker "Stem Cells and Transplantation" at Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburne Virginia, US on March 23-26, 2014. Prepared Chapter 4 "Restoring Vision to the Blind: Stem Cells and Transplantation" and presented at Special Interest Group Symposium Association for Research in Vision and Ophthalmology (ARVO) Meeting, Denver, USA May 2015. ARVO published the Lasker report as a special issue of its online journal Translational Vision Science & Technology. The journal is listed in PubMed (Transl Vis Sci Technol. 2014 Dec; 3(7): 1.) and is open access, so the report is readily accessible to a global audience. Published online 2014 Dec 30. doi: 10.1167/tvst.3.7.1
Start Year 2013
 
Description Fund raising activities for GOSHCC 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We host 3-50 invited lay visitors to the lab, or to research showcasing events, several times each year.

Interest and engagement from lay visitors/donors in translation research towards development of new treatments for childhood blindness.
Year(s) Of Engagement Activity 2011,2012,2013
 
Description GOSH Research Review 2014 . A Vision for Treating Blindness 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Increased profile of ongoing research

Invitations to give talks
Year(s) Of Engagement Activity 2014
URL https://www.ucl.ac.uk/ich/research/research-review2013-14
 
Description Inaugural lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Inaugural symposium attended by wide range of scientific, clinical staff, members of the public and representatives of fundraising bodies.

Speaker Jane Sowden, Professor of Development Biology and Genetics, University College London
Inaugural lecture - "Frontiers in the genetics of eye development and retinal repair" given following promotion to Professor at UCL in 2011.

Dissemination of research aims and achievements.
Year(s) Of Engagement Activity 2013
 
Description Invited participant and speaker in Velux Foundation Ophthalmology workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited participant and speaker in Velux Foundation Ophthalmology workshop; Detecting hotspots of research in ophthalmology; 9-11 April 2015 Collegium Helveticum, Zurich Switzerland




Policy document advising areas for future funding in vision and ophthalmological research
Year(s) Of Engagement Activity 2015
URL http://www.efc.be/organisation/the-velux-foundations/
 
Description Pediatric Ophthalmology Subspecialty Day Faculty, Invited speaker, Annual Meeting: American Academy of Ophthalmology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Health professionals
Results and Impact The American Academy of Ophthalmology
is the largest national membership association of Eye M.D.s with >500 attendees at the Pediatric Subspeciality Day. The meeting provides insight into new therapeutic options for treatment of pediatric eye disorders.

My platform presentation and participation as a member of the session discussion panel provided the opportunity to showcase our research to US Ophthalmologists. It stimulated discussion and received an exceptional vote of thanks by the Program Director.
Year(s) Of Engagement Activity 2011
 
Description Press for Human Molecular Genetics cone transplantation paper 2010 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Our paper published in Human Molecular Genetics reporting the first transplantation of cone photoreceptors into the retina featured in New Scientist and was covered by BioNews published by the Progress Education Trust, which provides accurate, news and comment on genetics and stem cell research, as well as several other international online news services.

Increased public awareness of the MRC research programme
Year(s) Of Engagement Activity 2010
 
Description Press for Nature Biotechnology paper 2013 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Our paper published in Nature Biotechnology (online 21 July 2013) received widespread media coverage on the BBC and national newspapers as well as a wide range of other international online news services.
This study showed for the first time that photoreceptor precursors grown from embryonic stem cell lines can be transplanted into the retina of blind mice. This work follows from our discovery in 2006 that transplanting immature rod photoreceptor cells from the retinas of very young mice can restore light sensitivity. We showed that it is possible to grow equivalent cells in a dish rather than taking the cells from young mice.
http://www.guardian.co.uk/science/2013/jul/21/embryonic-stem-cells-sight-blind-retinas

This paper is the culmination of more than a decade of investigations funded by the MRC, Fight for Sight and a number of other charities and opens the way for development of similar approaches to transplant human photoreceptor cells.

Increased publicity about the funded research and the feasibility of future treatments for blindness.
Year(s) Of Engagement Activity 2013
URL http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.2643.html
 
Description Press for Nature paper 2012 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Our paper published in Nature reporting restoration of night blindness in mice was widely reported online on the BBC and national newspapers as well as several other international online news services.

http://www.bbc.co.uk/news/health-17748165

Increased publicity and interest in our funded research work and the potential for future treatments for blindness

http://www.gosh.nhs.uk/news/press-releases/2012-press-release-archive/photoreceptor-transplant-restores-vision-in-mice/

http://www.nature.com/nature/journal/v485/n7396/full/nature10997.html
Year(s) Of Engagement Activity 2012
 
Description Press- photoreceptor transplantation 2010 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact • The Jackson laboratory, a leading genetics research laboratory, USA reported our research findings on cone transplantation as a news item and the study was reported on the MRC, GOSH and UCL news websites.
http://jaxmice.jax.org/news/2010/Blind.html
http://www.mrc.ac.uk/Newspublications/News/MRC007246


Increased publicity
Year(s) Of Engagement Activity 2010
 
Description Radio interview The Naked Scientist April 2011 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact We wrote a research commentary for Nature "DIY eye" on a new breakthrough and then gave a radio interview/podcast on this topic on "The Naked Scientists" Radio science and Science Podcasts.

This award winning BBC weekly radio programme, The Naked Scientists, reaches a potential audience of 6 million listeners across the east of England, and also has an international following on the web.


Increased the profile of MRC funded work and retinal stem cell therapy
Year(s) Of Engagement Activity 2011