CRB1 in the human retina in vitro: A functional, pathomechanistical and developmental study using Human Induced Pluripotent Stem Cell-derived retinal organoids

• Applicant: Professor Dr. Stefan Liebau
• Subject Area: Ophthalmology ; Molecular Biology and Physiology of Neurons and Glial Cells ; Molecular and Cellular Neurology and Neuropathology
• Term: from 2017 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 382162936

The presented study is designed to gain further mechanistical insights of the protein CRB1 during human retinal development and explicitly its role in the pathomechanism of hereditary retinal diseases. For that scientific approach, we aim to make use of human retinal organoids derived from induced pluripotent stem cells (hiPSCs). Consequently, we plan to 1) to generate patient specific retinal organoids from hiPSCs with determined homozygous mutations in the CRB1 gene as well as from control hiPSCs, 2) we will investigate distinct roles of CRB1 during retinal cell development and maturation. Here, we will one the one hand investigate transcript and protein expression levels of the CRB gene family, with a focus on CRB1, during retinal development and on the other hand investigate patient specific deregulations of expression and/or localization of mutated CRB1. Moreover, we aim to elucidate functional differences in patient specific retinal organoids via electrophysiology and structural differences in e.g. the morphology of the outer limiting membrane, where CRB1 seems to be localized. Here, the available developmental time-window starts with early retinal progenitor differentiation in neuroectodermal structures, leads over early states of retinal layering and differentiation until a final development of functional, light-sensitive photoreceptor cells. The various cells at different maturation stages will be determined and investigated either in fixed cells via immunocytochemistry and FACS and live-FACS in living cells with the help of cell surface markers and fluorescent promoter reporter constructs stably inserted into the genome already at the stage of the hiPS cell. Subsequently we will investigate the functional and pathomechanistical role of CRB1 in cellular mechanisms, such as signaling pathways. (Outlook) These pathways may then be pharmacologically modulated in the last steps of the study, to gain insights into possible rescue-approaches of CRB1-mediated pathomechanisms. Combined, it is possible to elucidate the developmental role of CRB1 during human retinal development and on the other hand, investigate the role of mutated CRB1 in retinal pathomechanisms. Their characterization may help to gain knowledge about the role of CRB1 in the disease cause and progression.

• DFG Programme: Research Grants