Using cell surface antigens for the isolation of 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. 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.

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.