Human induced pluripotent stem cells generate light responsive retinal organoids with variable and nutrient dependent efficiency

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format which enhanced generation of retinal organoids with retinal pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. © AlphaMed Press 2018

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<p>P124_04_10_16_HFgrating</p

Light responses of ON-transient ganglion cells are longer and faster in NCBE-deficient mice.

<p><b><i>A–C,</i></b> Representative single PSTH light responses of representative NCBE KO (red) and WT (black) ON-transient ganglion cells responding to stimuli with increasing spot sizes (<b><i>A</i></b>), increasing light intensities (<b><i>B</i></b>) and increasing frequencies (<b><i>C</i></b>). <b><i>D, G,</i></b> The response durations <i>(A1τ2)</i> of NCBE KO ON-transient ganglion cells compared to NCBE WT ON-transient ganglion cells were increased when either the spot size (<b><i>D</i></b>; RM two-way ANOVA: F (1, 59) = 6.68; p = 0.0123) or the intensity (<b><i>G</i></b>; RM two-way ANOVA: F (1, 59) = 7.05; p = 0.0102) was increased. <b><i>E, H,</i></b> The time to peak <i>(L1)</i> of NCBE KO ON-transient ganglion cells was decreased compared to NCBE WT ON-transient ganglion cells for both stimuli (<b><i>E</i></b>; RM two-way ANOVA: F (1, 59) = 7.67; p = 0.0075; <b><i>H</i></b>; RM two-way ANOVA: F (1, 59) = 4.83; p = 0.0319). <b><i>F,</i></b> NCBE KO and WT ON-transient ganglion cell responses <i>(A1)</i> of a spot size series were normalized and KO response amplitudes were larger for stimuli >300 (<b><i>F</i></b>; RM two-way ANOVA: F (1, 59) = 2.81; p = 0.1449). <b><i>I,</i></b> Normalized cell responses <i>(A1)</i> of a flicker series showed no significant differences (<b><i>I</i></b>; RM two-way ANOVA: F (1, 59) = 0.02; p = 0.8866). Vertical lines an <b><i>A</i></b> and <b><i>B</i></b> represent stimulus onset and offset, respectively. Values are presented as mean ± standard error of the mean (SEM). NCBE WT (n = 31), NCBE KO (n = 30).</p

NCBE is colocalized with KCC2.

<p><i>A–C,</i> Projections (3 µm) of NCBE WT retinal sections stained for NCBE (<b><i>A, C</i></b><i>,</i> green) and the chloride extruder KCC2 (<b><i>B</i></b>, <b><i>C</i></b>, magenta). Higher magnification of single scans (0.5 µm) of NCBE WT retinal sections double-stained for NCBE (<b><i>D, F</i></b>, green) and KCC2 (<b><i>E,</i></b><i> </i><b><i>F</i></b><i>,</i> magenta) revealed that KCC2 and NCBE are expressed on the same cellular compartments in the retina (arrows). Scale bar = 10 µm.</p

Primary antibodies used in this study.

<p>Primary antibodies used in this study.</p

NCBE is strongly expressed in the mouse retina.

<p><i>A, B,</i> H/E stainings of NCBE WT (A) and KO (B) retinal sections. Gross morphology of the retina did not differ between both genotypes. <b><i>C, D,</i></b> Projections (5 µm) of NCBE WT (<b><i>C</i></b>) and KO (<b><i>D</i></b>) retinal sections stained for NCBE (green). Bipolar (arrows) and amacrine (arrowheads) cells express NCBE, with a strong expression in both plexiform layers (<b><i>C</i></b>). No NCBE expression was found in the retina of NCBE KO mice (<b><i>D</i></b>), confirming the specificity of the antibody. Numbers 1–5 are labeling the IPL strata, scale bars = 10 µm.</p

Summary of the NCBE expression in the mouse retina.

<p>NCBE is expressed (grey) on the axon terminals of rod ON, cone ON and OFF bipolar cells and on the dendrites of OFF bipolar cells. NCBE is not expressed in dendrites of ON bipolar cells and OFF bipolar cell type 3B. NCBE is also expressed in starburst amacrine cells (SAC), but not in dendrites of ganglion cells (GC). NCBE is also not expressed in photoreceptors (not shown) and horizontal cells (HC).</p

Light responses of ON-OFF ganglion cells are slower in NCBE-deficient mice.

<p><b>A–C,</b> Single PSTH of representative NCBE KO (red) and WT (black) ON-OFF ganglion cell light responses, evoked with increasing spot sizes (<b><i>A</i></b>), light intensities (<b><i>B</i></b>) and stimulus frequencies (<b><i>C</i></b>). <b><i>D, G,</i></b> No significant differences were found between the ON <i>(A1τ2)</i> and OFF(<i>A2τ2)</i> response durations of WT and KO cells when spot size was increased [<b><i>D</i></b>; top (ON): F(1, 29) = 0.06; p = 0.8141; bottom (OFF): F(1, 29) = 0.01; p = 0.9425]. However, when light intensity was increased, response durations were longer in NCBE KO mice [<b><i>G</i></b>; top (ON): F(1, 29) = 5.98; p = 0.0207; bottom (OFF): F(1, 29) = 4.50; p = 0.0426]. <b><i>E, H</i></b><b>,</b> Time to peak of the ON response <i>(L1</i>) but not of the OFF response (<i>L2</i>) was significantly increased in NCBE KO when either the spot size [<b><i>E</i></b>; Top (ON): F(1, 29) = 6.13; p = 0.0194; bottom (OFF): F(1, 29) = 1.94; p = 0.1741] or the intensity were increased [<b><i>H</i></b>; top (ON): F(1, 29) = 12.56; p = 0.0014; bottom (OFF): F(1, 29) = 0.53; p = 0.4736]. <b><i>F,</i></b> No significant differences were found between the normalized ON (<i>A1</i>) and OFF response amplitudes (<i>A2</i>) with increasing spot size (<b><i>F</i></b>; top (ON): F(1, 29) = 0.03; p = 0.8565; bottom (OFF): F(1, 29) = 0.28; p = 0.5978). <b><i>I,</i></b> Normalized cell responses <i>(A1)</i> of a flicker series. NCBE KO cells showed decreased responses and were not able to follow higher frequencies comparably to NCBE WT cells (<b><i>I</i></b>; F(1, 29) = 2.94; p = 0.0969). Vertical lines an <b><i>A</i></b> and <b><i>B</i></b> represent stimulus onset and offset, respectively. Values are presented as mean ± SEM. Statistical values were obtained from repeated measurement ANOVA. NCBE WT (n = 16), NCBE KO (n = 15).</p

Electroretinography in NCBE-deficient mice indicates ON bipolar cell dysfunction.

<p><i>A, D,</i> Representative single flash ERG recordings from NCBE WT (black) and KO (red) mice (age 12 months) for increasing intensities under dark-adapted (<b><i>A</i></b>, scotopic) and light-adapted (<b><i>D</i></b>, photopic) conditions. <b><i>B, C, E, F,</i></b> Box-and-whisker plots of single flash ERG b-wave amplitudes (<b><i>B, E</i></b>) and latencies (<b><i>C, F</i></b>), plotted against flash intensity. Scotopic b-wave but not a-wave amplitudes in NCBE KO mice were reduced (<b><i>A,</i></b><i> </i><b><i>B</i></b>), and b-wave latencies (<b><i>C</i></b>) were increased compared to NCBE WT mice. <i>Inset</i> in <b><i>C</i></b>: Overlay of scotopic single flash ERG response traces of NCBE WT (black) and NCBE KO (red) mice at −2.0 log cd*s/m² intensity (arrow head). Scale bar: horizontal 50 ms, vertical 100 µV. Under photopic conditions, b-wave amplitudes (<b><i>D, E</i></b>) and b-wave latencies (<b><i>F</i></b>) of NCBE KO mice were similarly affected. <b><i>G,</i></b><i> </i><b><i>I,</i></b> Representative ERG recordings of a flicker frequency series (flash intensity <b><i>G</i></b>: −2 log cd*s/m²; <b><i>I</i></b>: 0.5 log cd*s/m²) under scotopic conditions. Flicker amplitudes (<b><i>H, J</i></b>) of NCBE KO mice decreased at much lower flash frequencies than that of WT controls. Vertical lines an <b><i>A</i></b>, <b><i>D</i></b>, <b><i>G</i></b>, <b><i>I</i></b> represent stimulus onset. In all quantitative plots (<b><i>B, C, E, F, H, J</i></b>), boxes indicate the 25% and 75% quantile range, whiskers indicate the 5% and 95% quantiles, and solid lines connect the medians of the data. NCBE WT (n = 2), NCBE KO (n = 3).</p

NCBE is expressed in bipolar cells and amacrine cells.

<p><b><i>A,</i></b> NCBE (green) is expressed in axon terminals (arrowheads) and somata (arrows) of type 1 and/or 2 OFF bipolar cells, labeled with NK3R (magenta). <b><i>B,</i></b> HCN4-labeled type 3A OFF bipolar cells (magenta) showed NCBE (green) distributed on dendrites (arrows) and axon terminals (arrowheads). <b><i>C, D,</i></b> PKARIIβ-labeled type 3B OFF bipolar cells (<b><i>C</i></b>, magenta) and CSEN-labeled type 4 OFF bipolar cells (<b><i>D</i></b>, magenta) with NCBE (green). Only proximal (arrows) but not distal dendrites (arrowheads) of type 3B OFF bipolar cells showed NCBE expression (<b><i>C</i></b>). Dendrites (arrows) and somata (arrowheads) of CSEN-labeled type 4 OFF bipolar cells were also positive for NCBE (<b><i>D</i></b>). <b><i>E,</i></b> Gα0-labeled dendrites (magenta, arrows) of ON bipolar cells did not show NCBE expression (green). However, some ON bipolar cell somata (arrowheads) were NCBE-positive. <b><i>F, G,</i></b> PKCα-labeled rod bipolar cells dendrites (magenta) showed no NCBE expression (<b>F</b>, green, arrows), whereas axon terminals strongly express NCBE (<b><i>G</i></b>, arrows). <b><i>H,</i></b> ZNP-1-labeled axon terminals (magenta) of type 6 ON bipolar cells showed NCBE expression (green, arrows). <b><i>I, J,</i></b> Retinal sections of NCBE WT mice were stained for NCBE (<b><i>I, J</i></b>, green) and ChAT (<b><i>J</i></b><i>,</i> magenta). Merging the NCBE channel (<b><i>I</i></b>) with the ChAT channel (<b><i>J</i></b>) revealed that somata (arrows) and dendrites (arrowheads) of ChAT-labeled starburst amacrine cells express NCBE. <b><i>K, L,</i></b> NCBE- (<b><i>K, L</i></b><i>,</i> green) and calretinin-labeled retinal sections (<b><i>L</i></b><i>,</i> magenta). NCBE expression was found on calretinin-labeled dendrites (arrowheads) and somata (arrows) of amacrine cells in the retina. <b><i>M, N,</i></b> Calbindin-labeled horizontal cells (arrows) do not express NCBE, but calbindin-labeled somata (arrowheads) of amacrine cells do. All images represent projections (3 µm) of confocal stacks. Numbers 1–5 are labeling the IPL strata, scale bars (<i>A–H</i>) = 5 µm; (<i>I–M</i>) = 10 µm.</p