Quantification (lower panel) of PDE6B expression normalised to β-tubulin expression of two independent experiments. S Representative western blot (upper panel) of PDE6B expression in Protocol 2 and Protocol 3 organoids collected at D150, D180 and D225 of differentiation. Quantification of rods (green bars) and cones (red bars), as determined by areas of normalised rhodopsin and arrestin fluorescence, respectively, and expressed as a percentage of the total photoreceptors at D150 ( Q) and D225 ( R) in Protocol 2 and Protocol 3 organoids. Data are represented as mean ± SEM * p < 0.05, n = 3 Mann and Whitney test. Quantification was performed on 6–13 images per organoid and 3 organoids were analysed per condition. Quantification analysis of the relative areas of NR2E3 ( M), RG opsin ( N), arrestin ( O) and rhodopsin ( P) fluorescence within the ONL normalised to the area of Hoechst fluorescence in the ONL (see Additional file 1: Fig. Representative images of rod-specific rhodopsin (in green) and cone-specific arrestin (in red) expression in Protocol 2 ( I, K) and Protocol 3 ( J, L) organoids. Representative images of the expression of the rod transcription factor NR2E3 (in green) and the cone-specific red–green opsins (RG opsin in red) in Protocol 2 ( E, G) and Protocol 3 ( F, H) organoids. Representative images of the expression of the nuclear cone–rod homeobox protein (CRX in green) and the calcium binding protein recoverin (RCVRN in red) in Protocol 2 ( A, C) and Protocol 3 ( B, D) organoids. Retinal organoids were analysed at D150 ( A, B, E, F, I, J) and D225 ( C, D, G, H, K, L) of differentiation. Qualitative and quantitative analysis of photoreceptor markers in mid-stage and mature 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.Ĭones Induced pluripotent stem cells Photoreceptors Retinal organoids Retinoic acid Rods. This study proves the importance of supplemented media for culturing retinal organoids. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer. 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. Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. 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. 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.Ī 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. Most protocols use common media supplements however, protocol-dependent variability impacts data interpretation. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |