Three-dimensional automated reporter quantification (3D-ARQ) technology enables quantitative screening in retinal organoids

Abstract

The advent of stem cell-derived retinal organoids has brought forth unprecedented opportunities for developmental and physiological studies, while presenting new therapeutic promise for retinal degenerative diseases. From a translational perspective, organoid systems provide exciting new prospects for drug discovery, offering the possibility to perform compound screening in a three-dimensional (3D) human tissue context that resembles the native histoarchitecture and to some extent recapitulates cellular interactions. However, inherent variability issues and a general lack of robust quantitative technologies for analyzing organoids on a large scale pose severe limitations for their use in translational applications. To address this need, we have developed a screening platform that enables accurate quantification of fluorescent reporters in complex human iPSC-derived retinal organoids. This platform incorporates a fluorescence microplate reader that allows xyz-dimensional detection and fine-tuned wavelength selection. We have established optimal parameters for fluorescent reporter signal detection, devised methods to compensate for organoid size variability, evaluated performance and sensitivity parameters, and validated this technology for functional applications.

Keywords: 3D-ARQ; Fluorescence reporter quantification; Human; Retinal organoids; Screening.

Fig. 1.

Sensitivity and reproducibility of signal detection. (A-F) Signal-to-background (S:B) ratios of live retinal organoids at 5 weeks of differentiation, stained with different fluorescent dyes or expressing fluorescent reporters as indicated. Each bar corresponds to an individual organoid. Six independent measurements per organoid per condition were performed within a 2 h period to assess technical reproducibility. Error bars represent s.e.m. of technical replicates. (A′-F′) Confocal images of whole-mount organoids representative of each condition, showing the subcellular distribution of fluorophores. Scale bars: 100 µm.

Fig. 2.

Normalization of fluorescence intensity using a globally expressed fluorophore. (A-D) Live retinal organoids at 5 weeks of differentiation were double stained with Bodipy TR and Calcein AM. Fluorescence intensity readouts are shown for 12 individual organoids for each fluorophore (A,B), and their Bodipy TR values after Calcein AM normalization (C). The correlation between the fluorescence values for both fluorophores is shown in D. (E) The correlation between fluorescence intensity of Bodipy TR-stained retinal organoids (RO) and their volume (n=16). (F,G) Five-week retinal organoids were treated with serial dilutions of Bodipy TR and counterstained with a fixed concentration of Calcein AM. Mean fluorescence is shown for Bodipy TR staining before (F) and after (G) normalization. Error bars represent s.e.m. for three biological replicates per dilution. RFU, relative fluorescence units.

Fig. 3.

Quantification of transgene expression levels. (A-C) Confocal images of live retinal organoids at 5 weeks of differentiation comparing (A) wild-type (non-transgenic; WT), (B) chimeric (wild-type/m-YFP) and (C) global m-YFP transgenic organoids. Scale bars: 100 µm. (D) Fold change in m-YFP fluorescence intensity measured for each condition and normalized using Bodipy TR counterstaining. Chimeric organoids generated at a 45% YFP ratio were used in this experiment. Error bars represent s.e.m. of five biological replicates per condition. **P<0.01, *P<0.05, by two-tailed Student's t-test with unequal variance. (E) The YFP fluorescence intensity of randomly generated chimeric organoids was evaluated using 3D-ARQ normalized to Bodipy TR counterstaining, and compared with the percentage of YFP⁺ cells in individual organoids by flow cytometry analysis after dissociation (n=20).

Fig. 4.

Assessment of developmental progression of gene expression. (A-D) Confocal images of fixed whole-mount retinal organoids immunostained for Pou4f2 at various differentiation time points [weeks (W) 5-9]. (E) Mean S:B ratios for Pou4f2 immunofluorescence at each time point, normalized to Sytox Green staining. (F-I) Confocal images of fixed whole-mount retinal organoids immunostained for Otx2 at weeks 5-11 of differentiation. (J) Mean S:B ratios for Otx2 immunofluorescence at each time point, normalized to Bodipy TR staining. Error bars represent s.e.m. of five biological replicates per time point; *P<0.05, **P<0.01, by two-tailed Student's t-test with unequal variance; n.s., not significant. Scale bars: 100 µm.

Fig. 5.

Assessment of the physiological status of retinal organoids. (A) Live retinal organoids at 5 weeks of differentiation were treated with 0 (Cont.), 2 and 4 mM H₂O₂, and stained with DHE to measure ROS production. Shown is the mean fluorescence intensity fold change among conditions. n=5 biological replicates per condition. Error bars represent s.e.m. *P<0.05, by two-tailed Student's t-test with equal variance. (B-C′) Confocal imaging of live retinal organoids at 87 weeks of differentiation stained with JC-1 dye. Red fluorescence, representing JC-1 aggregates (B,B′), and green fluorescence, representing the monomeric form of the dye (C,C′), were concentrated in cells in the outer layer of the organoids. B′ and C′ are confocal z-stack reconstructions corresponding to the organoids shown in B and C. Scale bars: 100 µm. Inset in B shows recoverin immunolabeling (magenta) in the outer surface of the neural retina, with DAPI-stained nuclei in blue. Scale bar: 50 µm. (D) Mitochondrial membrane depolarization in live retinal organoids at 87 weeks of differentiation was assessed by JC-1 staining. After measuring initial JC-1 aggregate/monomer ratios, organoids were treated with 10 µM CCCP or DMSO, and re-evaluated at 6, 24 and 48 h, and once again after a 3-day recovery period. n=4 organoids per group. Error bars represent s.e.m.