The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development

Abstract

Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.

Retinal organoid differentiation methods determine organoid cellular composition

“…Germline deletion of BMP4 is embryologically lethal in mice between 6.5 and 9.5 days post conception [51] . Conditional deletion of BMP4 from the optic vesicle resulted in an optic cup devoid of neural retinal markers, indicating that BMP4 is required for retinal specification [52] . In vitro, BMP4 does not appear to be required for retinal differentiation, since the protocols without BMP4 also produce organoids.…”

Self-Organization of the Retina during Eye Development, Retinal Regeneration In Vivo, and in Retinal 3D Organoids In Vitro

“…In addition to Wnt, BMP, and IGF1, other factors should also be included in the system of regulation of the early retinal formation stages. It was found that FGF, TGFβ, Notch, Vax, retinoids, and Gas1 are responsible for the diversification and stabilization of the two major visual domains (RPE and NR) in the optic vesicles and eye cup [ 61 , 62 ]. The role of FGF and Wnt signaling has been studied in mice both during RPE and NR formation and in maintaining the properties of the retinal growth zone (ciliary margin (CM)).…”

“…Along with studies of regulatory genetic networks, with the full complexity of this problem, attention is paid to epigenetic regulatory mechanisms, including the chromatin landscape, histone modifications, DNA methylation, non-encoded RNAs, etc. Thousands of enhancers are shown to be active in the developing retinae, and many of them have features of cell- and developmental stage-specific activity [ 23 , 62 , 92 , 93 , 94 ]. Attempts to identify the role of miRNAs were made earlier.…”

Erythropoietin in Optic Neuropathies: Current Future Strategies for Optic Nerve Protection and Repair

“…Additionally, EPO binding to the EPOR/βcR heterodimer could activate Wingless (Wnt) signaling (Figure 1) [28], which could regulate cells survival, and differentiation. Wnt signaling is an important pathway responsible for the development of different ocular structure [29]. This heterodimer receptor contributes to the majority of the protective effects of EPO, which potentially underlines a vast quantity of therapeutic approaches.…”