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

Abstract

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. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-03146-x.

TBC1D32 variants disrupt retinal ciliogenesis and cause retinitis pigmentosa

“…The TEM analysis of iPSC-derived RPE grown on Transwell inserts was performed as described ( 53 ). Retinal organoids were differentiated from adherent iPSCs and cultured as free-floating structures in DMEM/F12+GlutaMAX (Gibco) supplemented with taurine and retinoic acid, as described ( 27 ).…”

USH2A variants causing retinitis pigmentosa or Usher syndrome provoke differential retinal phenotypes in disease-specific organoids

“…We first looked at brush border formation, which represents the inner segment (IS)- and outer segment (OS)-like structures of rod and cone photoreceptors, 28 in mid-stage organoids on day 150 of differentiation ( Figures 4 A, 4B, and 4E). We observed a shorter or sometimes absent brush border in USH2A-RP-1 organoids ( Figure 4 A), which was significantly (3-fold) shorter (7.2 ± 1 μm) compared with control (22.1 ± 1 μm) organoids ( Figure 4 E).…”

“…Retinal organoids were differentiated from adherent iPSC cultures and, following excision, cultured as free-floating structures in DMEM/F12+GlutaMAX (Gibco) supplemented with taurine and retinoic acid, as described previously. 28 A minimum of 5 differentiations were performed per line, and for each differentiation, retinal organoids were monitored using an Olympus CKX53 microscope. For analysis of brush border length, 5 measurements were performed and averaged per organoid.…”

“…The iPSC-derived retinal organoids were processed and embedded as described previously without modification. 28 Counterstained 70-nm sections were observed using a Tecnai F20 transmission electron microscope at 200 kV in the in-house CoMET facility.…”

“…Over recent years, induced pluripotent stem cell (iPSC)-derived retinal organoids containing features of mature photoreceptors 26 , 27 , 28 have proven their worth for disease modeling of diverse IRDs and for testing the efficacy of potential therapies. 27 , 29 , 30 , 31 , 32 , 33 , 34 We previously reported generation of iPSC lines from two individuals carrying the most prevalent exon 13 USH2A mutations: a non-syndromic RP individual compound heterozygous for c.2299delG and c.2276G>T alleles 35 and an USH individual homozygous for the c.2299delG allele.…”