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

Ellie L Wagstaff,Camiel J F Boon,Anneloor L M A Ten Asbroek,Arthur A Bergen,Andrea Heredero Berzal,Peter M J Quinn

doi:10.3390/ijms22137081

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.

Highlights

Once we established which methods were previously used to generate retinal organoids, we systematically reviewed the steps of each protocol and indexed the external factors added to help the early development of the retinal lineage (Table 1), alongside other essentials, such as starting material and medium composition (Figure 8)
We found that the majority of methods that did not start with a Dulbecco’s Modified Eagle Medium (DMEM)-based medium during the early differentiation stages often opted to switch to a DMEM base during later culture stages
Since the introduction of retinal organoid technology, there has been a need to emulate in vivo retinal development as closely as possible in vitro

Summary

Introduction

Human eye development can be separated into four main stages: the development of the neural tube, the formation of the optic vesicle, the invagination of the double layered optic cup, and the development of the fully differentiated retina. Neural tube formation is induced by the developing notochord, a long rod that forms along the anteroposterior axis of the embryo (Figure 1A). The notochord secretes growth factors that prompts the differentiation of the overlying ectoderm into the neural ectoderm via hedgehog, BMP and Wnt signaling [1]. This structure thickens into the neural plate (Figure 1Ai). Neural tube formation is known as primary neurulation and occurs by the end of the fourth week of embryonic development

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