Immunohistochemistry of the injected eyes and control at day 70.

<p>Confocal microscopy at day 70 of the hWJ-MSCs and saline injected eye. Positive staining of the retinal antibody markers was detected in the hWJ-MSCs injected eyes but not in the saline injected eye. The antibodies used were Stem 121(green) for mesenchymal stem cells, Rhodopsin (red) for rod photoreceptors, MITF (green) for RPE specific markers, β tubulin (green) for immature neurons, anti-cone arrestin (red) for cone photoreceptors, PKC-α (red) for bipolar cells and recoverin (red) for cone bipolar. All slides were counterstained with DAPI (blue) to label the nucleus. Scale bar (white) represents 1000 μm.</p

Transmission electron microscopy.

<p>Transmission electron microscopy demonstrated the uptake of gold nanoparticles (arrow heads) by the hWJ-MSCs in vitro (A). At day 5 after injection into the cell, the gold nanoparticles laden cell was located in the subretinal space (B) and some of the gold nanoparticles were seen taken up by the retinal pigment (arrow heads), (C) showed a magnified version of the gold laden cell <i>in vivo</i>.</p

Retinal function.

<p>The dark adapted ERG a- and b-wave response amplitudes (10 cd.s/m²) and the isolated cone response at days -3, 15 and 30 for each experimental group. Although there was a trend that the ERG amplitude of the injected group was higher than that of the non-injected and control groups at days 15 and 30, the differences in ERG responses between the studied groups were not statistically significant at any time point post injection (p>0.05). All groups showed undetectable ERG at days 70. Error bars indicate standard error of the mean.</p

Retinal histology of the injection and non-injection sites at day 70.

<p>In eye injected with hWJ-MSCs, there was a generalised preservation of outer nuclear layer (double-headed arrows) over the whole retina as shown at the site of injection (A), middle region (B) and at the site furthest away from the site of injection (C) at day 70. The site of injection was evident by a small remnant subretinal bump (red arrow). This showed that the preservation of outer nuclear layer is not limited to the injection site. Scale bar represents 50 μm.</p

Viability and cytotoxicity of hWJ-MSCs after gold nanoparticles loading.

<p>A) Representative LIVE/DEAD images of nanogold-loaded hWJ-MSCs taken at day 1, day 5 and day 10 at different concentration showed good viability where most cells were live cells which were labelled green compared to the very scant dead cells which were labelled red (indicated by white arrow). Scale bar 100μm. B) At optical density with the wavelength of 545 nm, different concentrations of 80 nm gold nanoparticles dissolved in water showed a regression (R²) of 0.99. C) The proliferation profile of hWJ-MSCs with various concentration of gold nanoparticles were evaluated over a 10-day period. Median values were represented by horizontal lines. Cell numbers on day 1, day 5 and day 10 are marked with blue circle, green square and pink triangle respectively. At day 1, there was no significant difference in cell count between control sample and samples from any of the gold nanoparticle concentrations. At day 5, the cell count was significantly reduced in the samples incubated with gold nanoparticle concentration of 1x10¹⁰ particles/ml or higher compared to the control sample (marked with asterisks). The cell count of the samples incubated with gold nanoparticles was also significantly reduced at day 10 compared to the control, but only at the concentration of 1x10¹¹ particles/ml (marked with asterisks). (p = 0.014; Mann-Whitney U test).</p

Tracking with micro-computed tomography.

<p>MicroCT images showing localisation of gold-loaded hWJ-MSCs in the right eye (A) at day 1 and it remained in the eye with no further migration systemically at day 30 (B) and day 70 (C) post injection. PKH 26 (labelled red) showed the subretinal location of hWJ-MSCs after the injection at week 2.</p

Confocal microscopy of the whole eye injected with hWJ-MSCs.

<p>Confocal microscopy of the whole eye (A) and magnified images of the injected site (B-D). Red box represents the magnified area and the white arrow indicates the injection site. Co-localisation of DAPI (blue) and stem 121 (red) with rhodopsin (green), GFAP (green) and PKC-α (green) was detected at day 70 post injection, indicating that hWJ-MSCs has the potential to differentiate to retinal neuronal cells. Scale bar represents 10 μm.</p

Histology of experimental groups.

<p>A representative histology at day 0 (A) and day 70 (B-E) of different experimental groups. At day 70, the ONL were clearly detectable in eyes treated with hWJ-MSCs (B) compared to a thin layer or almost absent ONL in contralateral non-injected eye (C). The ONL was undetectable in the BBS injected eye (D) or the follow control eye (E). RGC: retinal ganglion cell, IPL: inner plexiform layer, INL: inner nuclear layer (indicated by double-headed arrows), ONL: outer nuclear layer (indicated by asterisks), RPE: retinal pigment epithelium. Scale bar represents 20μm.</p

Outer Nuclear Layer Thickness.

<p>The average ONL thickness of the hWJ-MSCs and BBS injected group at day 70 post injection. The ONL thickness of the hWJ-MSCs injected eyes was significantly greater than that of the uninjected fellow eyes (p<0.001) and eyes injected with BSS (p<0.001). Error bars indicate standard error of the mean.</p