A single cell sequencing approach to determine the heterogeneity, dynamics and cell fate decisions of retinal progenitor cells in vivo and in vitro

Being told that you have a visual impairment that can't be treated can be difficult to accept but this is the burden that 285 million people worldwide must bear. 26% of global blindness is caused by dysfunction of the retina, which is the innermost, light sensitive tissue that lines the back of the eye and is vital for light sensing and image processing. Dysfunction of retina and subsequent vision loss can occur through the effect of faulty genes we inherit from our parents as well as the accumulation of damage and the effect of various diseases throughout our lives. Our ability to prevent and treat vision loss is closely linked to our knowledge of "how our retinas form" and when and what is likely to go wrong. Our retinas develop mostly before birth; hence the availability of tissue to study from this time period is very limited. My group is in a unique position to bridge this gap, having access to human retinas through close and well established collaborations with the Human Developmental Biology Resource, which collects samples from aborted embryos and fetuses with the mother's consent. We also have the advantage of creating in the lab three-dimensional structures called "retinal organoids", which resemble the formation of human retina during development and contain the key retinal cell types. Our aim is to use both of these unique resources to understand how and when the retina forms and the role of genes that cause loss of vision when faulty.
Retina is a complex tissue and is composed of seven cell types: these emerge at different points during our development from a pool of progenitor cells, which in itself is heterogeneous with various subsets suggested to give rise to the different cell types in a concise progression through time. For this reason, it has been difficult to pinpoint the progenitor cells which give rise to all the cell types that make up the human retina with the traditional research methods that rely on studies of cell mixtures. Here we propose to use an important new technology called single cell analysis which allows us to look at which genes are turned on in each cell in the population. Gene expression at the single cell level is a very reliable tool for the precise categorisation of cells and allows us to identify types of cells that are not noticeable when looking under the microscope at their shape or position. We will use this as a first step to explore the molecular differences of individual retinal cells in both developing retinas and the retinal organoids generated in our lab. The use of advanced data analysis techniques will then allow us to build a catalogue of cell types and the genes that characterise them, to match the progenitors to the various cell types across development, to predict their ultimate fate and to assess how closely the lab generated retinal organoids mimic the development of human retina. Second, we will use the single cell sequencing data to reconstruct a lineage tree using bioinformatics tools. This approach organises cells in 'pseudo-time', predicting the order and mode in which cell fate decisions are made, enabling us to predict genes that occupy special positions around branch points of the tree. Third, we will apply a new approach, which allows us to correlate the gene expression profile of individual cells with their location in the retina, thus creating a spatial map of our retinas as they develop. This spatial map will allow us to validate the expression of key genes that are found near the branch points which may be important to understand the decision that progenitor cells make towards their final trip to become retinal cells. Finally, we will assess whether genes expressed around branch points play an active role in controlling cell fate decisions by manipulating their expression. The information will be available to all scientists and clinicians to help their understanding of retinal development and disease.

Retina is the innermost, light-sensitive layer of the tissue that lines the back of the eye and is vital for light sensing and image processing. Retina is comprised of six neuronal and one glial cell type, which are derived from heterogeneous and dynamic multipotent retinal progenitor cells (RPCs) in an orderly manner. A significant part of retinal development in humans occurs in utero, which poses logistical issues for systematic studies of human retinogenesis. My group has established close collaborations with the Human Developmental Biology Resource, which has enabled us to perform immunohistochemical and bulk transcriptional studies of human retinal development up to 20 post conception weeks. Utilizing our expertise in pluripotent stem cell biology, we are able to generate light-responsive retinal organoids, which contain all the key retinal cell types within a laminated structure that resembles the human retina. Notwithstanding these achievements, to date we have very little molecular information about human RPCs competency and/or heterogeneity during human retinal development both in vivo and in vitro. This application aims to utilize the most recent advances in single cell sequencing (RNA-Seq, ATAC-Seq and spatial transcriptomics) to determine the spectrum of transcriptional and chromatin accessibility profiles of human RPCs in time and space, and to identify key genes, whose function is essential for RPCs determination and differentiation. Using inducible lentiviral vectors proven to work in retinal organoids, we will manipulate the expression of these key genes and assess their function in RPCs maintenance and differentiation ability. Data generated from this project will be deposited into Data Coordination Platform contributing to the Human Cell Atlas and providing the scientific and clinical community with comprehensive datasets that can be further mined and exploited in the context of normal development, stem cell differentiation and retinal disease.

Visual impairment is a significant healthcare challenge affecting 285 million people worldwide with 26% of these suffering from diseases of the retina. Retinal diseases impose a substantial burden, both in economic and personal terms upon our society; however to develop better ways of treating such diseases, we need a greater understanding of human retinal development and function. To date, most of our knowledge in this field has been inferred from animal models, which are unable to fully replicate human disease phenotypes due to anatomical, genetic and functional species-specific differences; thus there is a pressing need to improve our understanding of human retinogenesis in vivo, to identify the mechanisms that underlie human retinal disease and to improve tissue engineering of cells suitable for human transplantation in vitro. In this proposal we address the fundamental question of how retinal progenitor cells acquire their unique fate to build the whole retina during development. The project is multidisciplinary, combining biology, development, molecular and computational approaches and cuts across several BBSRC priority areas e.g. data driven biology, systems approaches to biosciences and technology development in biosciences and replacement, refinement and reduction in research using animals.
The proposed project will benefit various academic beneficiaries in the field of neuroscience, developmental, stem cell and systems biology and tissue engineering. Importantly, the project will benefit clinical researchers involved in cell replacement therapies and developmental disease. Our data will be published in open access journals, disseminated at national and international meetings and deposited in open access resources, enabling other users to mine the data in the context of their work; hence the benefits will occur during the course of the project as well as after the project has ended. The project will train highly skilled researchers in multi- disciplinary research, allowing them to acquire transferable skills such as organisation, critical thinking, analysis of large data sets, problem solving, modelling complex scenarios, cross-disciplinary interactions and collaboration. This will therefore contribute to strengthening the UK science and medicine by providing highly skilled personnel for the academic or private sector. Through our outreach activities, we will encourage young people to take up a career in science, thus supporting the UKs ambition for strong science underpinning growth of the economy, entrepreneurial activities and industrial development. The focus on computational skills will attract and train new researchers in the bioinformatics arena, which in the short term should alleviate the current shortage of skilled staff. In longer term, the data generated from this project will help to identify key genes, which may be used to mobilise progenitor cell or reprogram cell types from one to another in vivo as well as developing small molecule treatments. The work is innovative, cross disciplinary and at the forefront of biomedical science, so it will help to enhance the UKs scientific reputation.