Fig. 1: STGD1 ROs develop typically with expression of all key retinal neurons and demonstrate expression of ABCA4 on transcript and protein level. | Cell Death & Disease

Fig. 1: STGD1 ROs develop typically with expression of all key retinal neurons and demonstrate expression of ABCA4 on transcript and protein level.

From: Unravelling genotype-phenotype correlations in Stargardt disease using patient-derived retinal organoids

Fig. 1: STGD1 ROs develop typically with expression of all key retinal neurons and demonstrate expression of ABCA4 on transcript and protein level.The alternative text for this image may have been generated using AI.

A Representative ROs derived using the BMP4-activated method of RO differentiation are displayed in the purple shaded box. PT2 and WT2 are displayed at various timepoints of differentiation spanning Day 60 to Day 220. Similarly, representative ROs derived from the IGF1-dependent method of differentiation are displayed in the green shaded box. Across all lines and protocols, clearly defined RO structures are developed by Day 120 of differentiation. By Day 180, the characteristic inner and outer segment brush border is apparent on the organoid’s apical edge and continues to develop longer structures by the final timepoint of Day 220 as evidenced by the red arrows in the final column. B All iPSC-derived ROs display expression of markers for REC (photoreceptor cells), SNCG (RGCs, yellow arrows), PROX1 (HCs), AP2α (ACs), PKCα (bipolar cells, white arrows) and CRALBP (Müller glia). REC+ cells appear to mislocalise to the central part of the ROs in a patient-specific manner. C ABCA4 (green) and cone-specific GT335 (red) protein expression in post-mortem retina tissue sample. The ABCA4 protein localises specifically to the POS tips of cone photoreceptor cells. ABCA4 (yellow) protein expression in nascent photoreceptor inner segments (PIS) and POS in Day 220 ROs. Staining patterns appear more diffuse throughout the PIS and POS likely due to immature disc stacking in this developmental model. The degree of observable ABCA4 fluorescence appears to correlate with the estimated residual protein levels determined via the severity of patient line genotype. AC displays the lowest intensity of fluorescence, followed by PT1, PT2 and the unaffected control WT2. D Representative western blotting revealed an abundance of protein at the expected size of ~250 kDa. The intensity of the bands correlated with the severity of genotype possessed by each patient case. The intensity of the ABCA4 protein bands was normalised against ACTIN (ACTB) and quantified. One-way ANOVA test revealed a significant reduction in ABCA4 expression in AC and PT1 samples. Whilst PT2 ABCA4 protein levels remained relatively close to WT2 samples. N = 16 ROs per sample. **** = p-value < 0.0001.

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