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

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. New three-dimensional retinal culture methods can produce large numbers of new retinal cells. Our hypothesis is that photoreceptor cells generated from these 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 human cells to transplant into the diseased retina. We will develop live cell sorting methods using antibodies that bind to special markers on the surface of the immature photoreceptor cells providing a tag to isolate them with. The efficacy of isolated cells for retinal repair will be tested in transplantation experiments in animal models of retinal disease. This study will develop cell isolation protocols for human photoreceptor precursor cells, which is a requisite step towards development of clinical trials for incurable retinal diseases causing blindness.

Retinal degenerations with loss of photoreceptors are the leading cause of untreatable blindness in Europe. In 2006 we showed that photoreceptors can be transplanted into the adult retina and recently we have been able to provide the first definitive evidence of restoration of rod-mediated vision, following transplantation of rods into a model of stationary night blindness. Importantly we have also recently proved in the mouse model the concept of developing and utilising a cell surface biomarker panel to isolate transplantation-competent photoreceptor precursor cells from 3D embryonic stem cell retinal differentiation cultures. Whilst we have demonstrated that retinal repair by photoreceptor transplantation is feasible, for clinical application we need to develop protocols for purifying human photoreceptors from a renewable source. In this project we aim to identify a human cell surface biomarker panel and to develop robust methodologies for identification and purification of human rod and cone photoreceptor precursors from clinical grade human pluripotent stem cell cultures that will support the development of a clinical trial in retinal dystrophy patients.
To achieve this goal we will:
(1) Test the biomarkers we have identified in the mouse for labelling and isolation of human fetal photoreceptor precursor cells and immunophenotype human fetal photoreceptor cells;
(2) Define the biomarker profiles of human ESC-derived rod and cone photoreceptors by transcriptome analysis at sequential stages of maturation in human ESC-derived retinal cultures, and compare these profiles to transplantation competent mouse photoreceptor precursor cells;
(3) Evaluate combinations of identified biomarkers for purification of hESC-derived rod and cone photoreceptors;
(4) Evaluate biomarker selected hESC-derived cells in transplantation experiments;
(5) Develop robust biomarker panel-based isolation protocols and quality control assays for human photoreceptor precursors.

Hereditary retinal disease and age related macular degeneration (AMD) leading to photoreceptor loss are the leading causes of untreatable blindness in the UK. There is currently no cure for these conditions, and available therapies at best, can provide a delay in disease progression to a minority of patients. Inherited retinal degenerations affect 1 in 3000 of the population whilst AMD affects 1 in 10 people over the age of 60 and its prevalence is increasing. In children more than 15% of blind registrations are due to inherited retina disease. These include children who are born without functional photoreceptors and who may have mutations in 1 of more than 100 different retinal disease genes, or have defects in unknown genes. Of these early onset conditions, those not amenable to gene therapy approaches, present a target group for whom photoreceptor cell transplantation may be first offered. There is currently no treatment option and these individuals face a lifetime of blindness. Beyond this patient group photoreceptor transplantation could be effective for a wide range of conditions resulting in photoreceptor cell death, although it may be necessary for transplants to be part of combined therapies in which abnormalities in additional tissues such as the retinal vasculature are also controlled. We have discovered that transplantation of immature photoreceptors improves vision in mouse models of retinal degeneration.The proposed project aims to builds on our discovery and to develop methods for isolation of equivalent human cells that could be used for stem cell therapy for the treatment of retinal diseases.