Neuroprotection with oxcarbazepine and caspase-2 siRNA in MOG^TCRxThy1CFP mice.

<p>(A) MOG^TCRxThy1CFP transgenic mice received the following: optic neuritis control (n=7), 100µl daily dose of vehicle (1:1 PBS and DMSO) (n=7) or 100μl 10mg/kg OXC (dissolved in 1:1 PBS and DMSO) (n=7). Mice were sacrificed on day 21. Eyes were flatmounted and RGC were counted using stereology software. Results represent mean ± SEM of RGC density. (B) Cross sections of the retina stained with total caspase-2 and (C) active caspase-2 and both were secondary labelled with Alex Fluor 568 (Red). RGC are shown in green (arrow), but degenerating RGC with lower CFP expression could be detected (dashed arrow). (D) RNFL thickness after ON induction and treatment with either nonsense or caspase-2 siRNA. (E) Quantification of RGC density after ON induction and treatment with either a nonsense (n=7) or caspase-2 siRNA (n=8). Plots show mean ± SEM of RGC density and RNFL thickness.</p

OCT imaging during ON in MOG^TCRxThy1CFP mice

<p>(A) Histological changes in the thickness of RGC layer in control mice (n=5) and MOG^TCRxThy1CFP mice (n=5) at day 21. Results represent the mean ± SEM of RGC layer thickness. **P<0.01 and ***P<0.001 between control mice and MOG-specific TCR mice at the same distance from optic nerve head. (B) Multiline OCT, a Spectralis® HRA + OCT modified by Heidelberg Engineering Inc to allow imaging of CFP-expressing RGC and adapted to comply with mouse optics with a +25D lens and fitted animal mount. Mouse is ABH strain. (C) OCT images of retina were acquired using a circular OCT scan surrounding the optic nerve head on (D) day 0 and (E) day 21 of disease induction and RNFL thickness was calculated. (F) Decrease in RNFL thickness after immunisation of MOG^TCRxThy1CFP mice to develop ON (n=10). (G) Correlation between RGC density (measured using retinal flatmounts) and RNFL thickness (measured using OCT). Results represent the mean ± SEM of RNFL thickness. * P<0.05, **P<0.01, ***P<0.001 compared to control.</p

RGC loss during ON in MOG^TCRxThy1CFP mice.

<p>(A) Decrease in mean RGC density after immunisation of MOG^TCRxThy1CFP mice (n=7) compared to control mice (n=7) at day 21. (B) Retinal flatmount of control mouse at 20x magnification showing a high density of CFP expressing RGC and (C) retinal flatmount of retina after immunisation of MOG^TCRxThy1CFP mice showing a low density of CFP expressing RGC.</p

Visual sensitivity loss and electrophysiological changes during ON in MOG^TCRxThy1CFP mice.

<p>(A) Decrease in visual sensitivity after immunisation of MOG^TCRxThy1CFP to develop ON (n=10). Results represent the mean ± SEM of net positive head movements. **P<0.01 compared to control. (B) Typical waveform VEP showing P1 (first positive peak), N1 (first negative peak), P2 (second positive peak). (C) Decrease in mean VEP amplitude after immunisation of MOG^TCRxThy1CFP mice to develop ON (n=5). (D) Increase in mean N1-P2 latency of VEP amplitude after immunisation of MOG^TCRxThy1CFP mice to develop ON (n=5). Results represent the mean ± SEM of amplitude. *P<0.05 compared to control.</p

cSLO imaging during ON in MOG^TCRxThy1CFP mice.

<p>(A) Example of retina before disease induction on day 0 and (B) after disease induction on day 21. (C) Quantification of mean RNFL thickness by cSLO on day 0 and after ON disease induction at day 21 and comparison with conventional histology at day 21 (n=8). Images of individual RGC taken over a 30° field of view and a contrast sensitivity of 80% at (D) day 0 and (E) day 21. RGC survival (yellow) and RGC loss (red) are identifiable. Results represent the mean ± SEM of RGC density.</p

Optic and retinal nerve loss during ON in MOG^TCRxThy1CFP mice.

<p>(A) Semi-thin toluidine blue stained optic nerve cross section and (B) ultra-thin lead citrate stained sections from wildtype mice showing uniformly myelinated axons (M). (C) Semi-thin toluidine blue stained section and (D) ultra-thin lead citrate stained section of optic nerves from immunized MOG^TCRxThy1CFP developing ON on day 21, showing evidence of demyelination (D) and areas of axonal loss (AL). (E) Semi-thin toluidine blue stained cross section of retina from wildtype mouse, showing healthy RGC (RGC) and (F) semi-thin toluidine blue stained cross section of retina from immunised MOG^TCRxThy1CFP mice developing ON on day 21 showing irregular RGC.</p

Additional file 1: of Use of bioreactors for culturing human retinal organoids improves photoreceptor yields

Figure S1. Flow cytometric analysis and quantification of proportion of RECOVERIN/CD73 and CD133/CD73 double-positive cells within RECOVERIN and CD133 photoreceptor-positive populations. Representative FC plots of control vs bioreactor retinal organoids. A FC quantification of CD133/CD73 double-positive developing rods within CD133-positive population. B Quantification of RECOVERIN/CD73 double-positive mature photoreceptor cells by gating only in RECOVERIN-positive live cell population. Error bars, mean ± SEM; n = 50 retinal organoids, N = 3–4 independent differentiation experiments carried out per control or bioreactor condition; *P < 0.05, **P < 0.01, two-tail unpaired t test with Welch’s correction. Figure S2. Flow cytometry gating strategy employed for all flow cytometric analysis for each individual sample. A Dead cells excluded by using DRAQ7 vs FSC-A (or SYTOX Blue vs FSC-A; data not shown). Cellular aggregates gated out (FSC-A vs FSC-H) to ensure only single live cells (SSC-A vs FSC-A) used for subsequent analysis. B Representative plots of control vs bioreactor for RECOVERIN staining. Gates drawn using only secondary control samples for both control and bioreactor samples. C Representative plots of gating strategy used for CD73 staining in combination with CD133 antibody staining for both control and bioreactor samples. Unstained and fluorescence minus one (FMO) controls for CD73 and CD133 used to define positive fraction of cells for both control and bioreactor samples. D Representative plots for RECOVERIN and CD73 staining. Unstained and FMO gating controls used to determine RECOVERIN and CD73-positive cells for both control and bioreactor samples. Figure S3. Immunofluorescence analysis showing Müller glia (CRALBP-positive) and photoreceptor (RECOVERIN-positive) cells of week 15 retinal organoids in control (A) and bioreactor (B) conditions. Scale Bars: 200 μM. Figure S4. SEM and TEM images of hPSC-derived retinal organoid OLM regions. A, B SEM image showing photoreceptors of bioreactor-generated retinal organoid. C, D TEM illustrating photoreceptor outer limiting membrane (OLM), inner segments, CC and developing outer segments of control (C) and bioreactor (D) retinal organoids. Scale bars: 2 μm (B–D). Figure S5. SEM images of whole retinal organoid. Topographic features of neuroepithelia showing photoreceptor cell density and morphology from control (A–C) vs bioreactor (E–G) at ascending magnifications. Scale bars: 10 μM. Table S1. Antibody catalogue numbers and dilutions (DOCX 8526 kb