Retinoic acid delays initial photoreceptor differentiation and results in a highly structured mature retinal organoid.

BACKGROUND: Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Most protocols use common media supplements; however, protocol-dependent variability impacts data interpretation. To date, the lack of a systematic comparison of a given protocol with or without supplements makes it difficult to determine how they influence the differentiation process and morphology of the retinal organoids. METHODS: A 2D-3D differentiation method was used to generate retinal organoids, which were cultured with or without the most commonly used media supplements, notably retinoic acid. Gene expression was assayed using qPCR analysis, protein expression using immunofluorescence studies, ultrastructure using electron microscopy and 3D morphology using confocal and biphoton microscopy of whole organoids. RESULTS: Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. At later stages, the presence of retinoic acid led to the generation of mature retinal organoids with a well-structured stratified photoreceptor layer containing a predominant rod population. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer. CONCLUSIONS: This study proves the importance of supplemented media for culturing retinal organoids. More importantly, we demonstrate for the first time that the role of retinoic acid goes beyond inducing a rod cell fate to enhancing the organisation of the photoreceptor layer of the mature organoid.

Novel roles for voltage-gated T-type Ca2+ and ClC-2 channels in phagocytosis and angiogenic factor balance identified in human iPSC-derived RPE.

Human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) is a powerful tool for pathophysiological studies and preclinical therapeutic screening, as well as a source for clinical cell transplantation. Thus, it must be validated for maturity and functionality to ensure correct data readouts and clinical safety. Previous studies have validated hiPSC-derived RPE as morphologically characteristic of the tissue in the human eye. However, information concerning the expression and functionality of ion channels is still limited. We screened hiPSC-derived RPE for the polarized expression of a panel of L-type (CaV 1.1, CaV 1.3) and T-type (CaV 3.1, CaV 3.3) Ca2+ channels, K+ channels (Maxi-K, Kir4.1, Kir7.1), and the Cl- channel ClC-2 known to be expressed in native RPE. We also tested the roles of these channels in key RPE functions using specific inhibitors. In addition to confirming the native expression profiles and function of certain channels, such as L-type Ca2+ channels, we show for the first time that T-type Ca2+ channels play a role in both phagocytosis and vascular endothelial growth factor (VEGF) secretion. Moreover, we demonstrate that Maxi-K and Kir7.1 channels are involved in the polarized secretion of VEGF and pigment epithelium-derived factor (PEDF). Furthermore, we show a novel localization for ClC-2 channel on the apical side of hiPSC-derived RPE, with an overexpression at the level of fluid-filled domes, and demonstrate that it plays an important role in phagocytosis, as well as VEGF and PEDF secretion. Taken together, hiPSC-derived RPE is a powerful model for advancing fundamental knowledge of RPE functions.