Targeting retinal cell types by synthetic promoters in 3D stem cell derived-retinal organoids

Abstract

The retina is a light-sensitive and about 200 µm thin tissue located at the posterior of the eye, crucial for converting photons into electrical signals that are delivered to the brain. Functionally, the retina acts like a camera; structurally, it consists of an intricate architecture and complex intercellular interactions.<br /> For decades, research has focused on the cellular and molecular mechanisms underlying diseases such as Glaucoma, AMD, and Retinitis Pigmentosa. This is critical because mammalian retinal cells cannot self-repair. Therefore, their degeneration leads to irreversible vision loss.<br /> Our understanding of retinal biology has been greatly enhanced by animal models and 2D cell cultures. These stem cells are typically generated using either Yamanaka factors or, more recently, small molecule chemical cocktails.<br /> However, these traditional models often fail to fully recapitulate human physiology. This is why we utilize human iPSC-derived 3D Retinal Organoids (ROs). These organoids are highly advanced: they closely resemble the morphology of the human retina and, most importantly, they mimic in vivo human retinal development.<br /> Despite their advantages, a major limitation remains: In most preliminary studies, retinal cells within organoids could only be analyzed be fixed or lysed.<br /> Therefore, the primary aim of my thesis was to use synthetic promoters to establish a reporter cell line. This allows us to specifically target and visualize retinal cell types—specifically RGCs and Cones—within living organoids. This establishes a powerful, accessible tool for observing cell development in real-time, which we then validated using standard downstream analyses.