Fig 1.
Schematic diagram describing a novel method for human retinal organoid generation and representative images of their morphological features.
(A) Culture conditions and the timing of treatment with differentiation-promoting agents are indicated. The time after the initiation of the differentiation of retinal organoids differentiation was indicated as the DD. The timing and combinations of pharmacological treatments were carefully designed to accelerate retinal organoid differentiation and maturation. (B) Representative phase contrast images of retinal organoids differentiated at DD15, DD20, and DD40. Scale bar: 500 μm. (C) Higher magnification of a phase contrast image of retinal organoids fully differentiated at DD90. White and black arrowheads indicate the hair-like structure and the definitive border (outer nuclear layer/outer plexiform layer border), respectively. Scale bar: 200 μm. The representative images were arbitrarily chosen from eight independent experiments.
Fig 2.
Neural retinal progenitor differentiation in the modified SEAM method.
Human iPSC colonies were cultured using the SEAM method with specific pharmacological treatments. These colonies were initially exposed to dual SMAD inhibition and then switched to BMP4 treatment. (A) RAX (green)- and VSX2 (red)-double-positive neural retinal progenitor cells emerged at DD10, as revealed by immunocytochemistry of developing retinal clusters. Scale bars: 200 μm (left) and 100 μm (right). (B) MITF- (green) and PAX6-positive (red) retinal progenitor/RPE precursors. Scale bar: 200 μm. Nuclei were stained with DAPI (blue). Differentiating cells were examined in six wells in three independent experiments. Representative images were chosen arbitrarily.
Fig 3.
Accelerated differentiation of human retinal organoids into photoreceptors, RGCs, and Müller glia in floating culture with optimized pharmacological treatments.
Human retinal organoids were generated in the presence of three differentiation-promoting agents (SAG, activin A, and RA) and harvested at each DD throughout the 90-day study period. Retinal organoids were cryosectioned and immunostained with antibodies against various retinal cell marker proteins. CRX (green): Photoreceptor precursor/photoreceptor, BRN3 (red): RGC, CRALBP (white): Müller glia. White arrowheads indicate the intermediate layer between two consecutive nuclear layers, presumably representing the OPL-like structure. Nuclei (blue) were stained with DAPI. Scale bar: 100 μm. Ten sections prepared from ten retinal organoids were examined in three independent experiments. Representative images were chosen arbitrarily.
Fig 4.
Rapid maturation of photoreceptors and bipolar cells in human retinal organoids derived from two different iPSC lines.
(A) Retinal organoids derived from the human iPSC line 1231A3 at a late DD were cryosectioned and immunostained with antibodies against various retinal cell marker proteins. DD70 to DD90: Rhodopsin (green) and L/M opsin (red) for mature rod and cone photoreceptors, respectively; DD80: PKCα (green, bipolar cell marker), NRL (green, rod photoreceptor marker), recoverin (red, photoreceptor marker), and RXRγ (red, cone photoreceptor marker). Scale bar: 100 μm. (B) Mature photoreceptors in retinal organoids derived from the human iPSC line M8. rhodopsin (green), L/M opsin (red), and S opsin (white, cone photoreceptor marker) at DD80. Scale bar: 100 μm. Nuclei (blue) were stained with DAPI for all experiments. Ten sections prepared from ten retinal organoids were examined in three independent experiments. Representative images were arbitrarily chosen from three independent experiments.
Fig 5.
Promoting effects of concurrent pharmacological modulation of multiple signal transduction on photoreceptor differentiation in the outermost layer of human retinal organoids.
Retinal organoids were cultured in maturation medium supplemented with SAG, activin A, and RA, alone or in combination from DD10 through DD40. At DD40, retinal organoids were cryosectioned and immunostained with antibodies against the photoreceptor precursor/photoreceptor marker CRX (green) and RGC marker BRN3 (red). Nuclei (blue) were stained with DAPI for all experiments. Scale bar: 100 μm. Ten sections prepared from ten retinal organoids were examined in three independent experiments. Representative images were chosen arbitrarily.
Fig 6.
Determinant effect of SAG on mature photoreceptor lamination in human retinal organoids.
Retinal organoids were cultured in maturation medium supplemented with SAG and RA, alone or in combination from DD40 through DD90. At DD90, retinal organoids were cryosectioned and immunostained with antibodies against the photoreceptor marker rhodopsin (green) and L/M opsin (red). White arrowheads indicate ectopic cone photoreceptors that developed in the inner region of retinal organoids. Nuclei (blue) were stained with DAPI for all the experiments. Scale bar: 100 μm. Ten sections prepared from ten retinal organoids were examined in three independent experiments. Representative images were chosen arbitrarily.
Fig 7.
Altered gene expression of various retinal progenitor cell makers in human retinal organoids treated with differentiation-promoting agents.
Retinal organoids were cultured in maturation medium supplemented with SAG, activin A, and RA, alone or in combination, from DD10 through DD20. Retinal organoids were harvested and subjected to gene expression analysis for CRX, MITF, RAX, and PAX6 by qPCR. Each expression level was normalized to that of GAPDH and is indicated as a fold-change from the respective basal expression levels observed at DD0. Data are representative from three independent experiments with three biological replicates in each treatment group. Error bars represent standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Dunnett’s multiple comparison test.