| dc.description.abstract | The hereditary retinal disease Retinitis pigmentosa (RP) leads to loss of photoreceptor cells and is a significant cause of blindness, affecting over 2 million worldwide. This project focus on Retinitis pigmentosa type 11 (RP11), which occurs due to mutations in one copy of the PRPF31 gene. RP11 generally debuts in early adulthood with impaired night vision, narrowing of the visual field, and in the final stage, loss of central vision. The consequence of a genetic defect in the PRPF31 gene is progressive cell death in the retinal layers, primarily affecting the retina in the retinal pigment epithelial layer and secondarily in the photoreceptor cell layer. There is currently no established treatment for RP11, and it remains a medically challenging disease. Understanding the pathology at cellular and molecular levels is fundamental for developing new treatment modalities, and the use of appropriated research models to study human diseases is therefore crucial. Importantly, human organoids are robust models that can reveal new mechanistic insight into the pathology of diseases and can be used as experimental platforms for drug screening and gene therapy. Thus, the primary goal of this project is to establish a reliable and robust protocol for the generation of human retinal organoids from healthy controls- and RP11 patient-derived induced Pluripotent Stem Cells (iPSC). Prior to this project, fibroblasts were obtained from skin biopsies of four RP11 patients, of which fibroblasts from three patients (one, three, and four) were reprogrammed into induced pluripotent stem cells. Here, the skin fibroblasts of patient two (RP11-2) and the unrelated healthy control (HC) were successfully reprogrammed into iPSC. In addition, previously generated iPSC clones from two patients, namely, one (RP11-1) and three (RP11-3) and healthy controls (ATc1 and ATc2), were thoroughly and successfully characterized as high-quality iPSC, confirming both the quality of fibroblast reprogramming and iPSC cultivation methods. The characterized iPSC were then successfully differentiated into middle-stage retinal organoids harboring visually observable Retinal Pigment Epithelium (RPE) and Neural Retina (NR) structures with significant expression of their respective retinal markers. A retinal organoid protocol from Nakano et al. [1] was applied, modified, and slightly improved during this study. Due to the time constraint of this thesis, organoids were collected on day 96, prior to their required maturation time of 150 days. Experiments with preconditioning of iPSC were shown to alter the iPSC's "naive" state into an active form, more suitable for differentiation into 3D organoids. Moreover, we show that the Notch inhibitor molecule DAPT can fast-track differentiation into photoreceptor progenitor cells (PRP) and RPE cells. The protocol presented here is a work in progress and could be further modified into a more effective and robust protocol with the implementation of preconditioning, DAPT treatment, identified differentiation enhancers, and substitution to defined serum-free media. Notably, the latter enables the exciting possibility of using resulting clinical-grade patient-derived organoids as donors for potential cell therapy. | |