Defensa de Tesis Doctoral – Iván Aldavero Muñoz

Visual dystrophies leading to vision impairment are becoming a global problem as life spans increase and the global population rises exponentially. Age-related macular degeneration (AMD) is the most common cause of blindness in people over 50 years old in industrialized countries. There are currently no effective, targeted, and efficient treatment options for this neurodegenerative disease. We need accessible and reproducible in vitro human models to continue making progress in understanding these retinal diseases and in identifying potential therapeutic targets for intervention. Human induced pluripotent stem cells (iPS) offer new opportunities for modeling retinal diseases, especially thanks to their ability to differentiate to retinal pigment epithelium (RPE), which plays a critical role in maintaining visual function and retinal homeostasis¹.
                We aimed, first, to develop a protocol for generating pure RPE sheets from iPS with high efficiency and reproducibility able to recapitulate in vitro innate retinal functions². We also explored developing a human post-mortem retinal tissue model which retains similar characteristics³. And secondly, we aimed to study the effects of the deletion of CRB2, a protein involved in the physiological maturation and polarization of the RPE, which are essential to establish a functional RPE cell layer⁴; and the deletion of VEGFA, a protein whose polarized secretion is vital for the proper functioning of the vessels that innervate the RPE and is strongly involved in the neovascular subtype of AMD⁵.
                For this, PGP1 line, an iPS commercial line sourced from primary human fibroblasts from a control male donor with a well-defined genetic and clinical background was employed. Then, RPE cells were generated and differentiated from a CRB2 CRISPR-deleted pool of iPS cells, another cell pool with VEGFA deleted, and from control cells, electroporated with RNP complexes without sgRNA guide. This directed differentiation protocol via manipulation of the insulin growth factors (IGF), basic fibroblast growth factor (FGF-2; FGF-basic), transforming growth factor beta (TGF-β), and WNT pathways leads to increased RPE fate differentiation efficiency and enables RPE sheet generation at high purity without manual selection. This method⁶ produced a homogeneous, pigmented, polarized⁷, phagocytic, post-mitotic, and characteristically polygonal monolayer of RPE cells. The results presented in this PhD thesis strengthen the evidence supporting the validity of iPS-derived RPE models as a robust platform for the mechanistic study of AMD. These models can be also utilized to assess potential drug treatments and to develop novel regenerative cell therapies for AMD and other retinal diseases. However, more data is needed to determine how key proteins like CRB2 or VEGFA impact both healthy and pathological RPE.

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