Safety and efficacy of human Wharton's Jelly-derived mesenchymal stem cells therapy for retinal degeneration

Purpose To investigate the safety and efficacy of subretinal injection of human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats. Methods RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies. Results No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells. Conclusions Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies

Micro-Computed Tomography Detection of Gold Nanoparticle-Labelled Mesenchymal Stem Cells in the Rat Subretinal Layer

Mesenchymal stem cells are widely used in many pre-clinical and clinical settings. Despite advances in molecular technology; the migration and homing activities of these cells in in vivo systems are not well understood. Labelling mesenchymal stem cells with gold nanoparticles has no cytotoxic effect and may offer suitable indications for stem cell tracking. Here, we report a simple protocol to label mesenchymal stem cells using 80 nm gold nanoparticles. Once the cells and particles were incubated together for 24 h, the labelled products were injected into the rat subretinal layer. Micro-computed tomography was then conducted on the 15th and 30th day post-injection to track the movement of these cells, as visualized by an area of hyperdensity from the coronal section images of the rat head. In addition, we confirmed the cellular uptake of the gold nanoparticles by the mesenchymal stem cells using transmission electron microscopy. As opposed to other methods, the current protocol provides a simple, less labour-intensive and more efficient labelling mechanism for real-time cell tracking. Finally, we discuss the potential manipulations of gold nanoparticles in stem cells for cell replacement and cancer therapy in ocular disorders or diseases

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

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

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

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

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