Human retinal progenitor cell transplantation preserves vision

Abstract

Cell transplantation is a potential therapeutic strategy for retinal degenerative diseases involving the loss of photoreceptors. However, it faces challenges to clinical translation due to safety concerns and a limited supply of cells. Human retinal progenitor cells (hRPCs) from fetal neural retina are expandable in vitro and maintain an undifferentiated state. This study aimed to investigate the therapeutic potential of hRPCs transplanted into a Royal College of Surgeons (RCS) rat model of retinal degeneration. At 12 weeks, optokinetic response showed that hRPC-grafted eyes had significantly superior visual acuity compared with vehicle-treated eyes. Histological evaluation of outer nuclear layer (ONL) characteristics such as ONL thickness, spread distance, and cell count demonstrated a significantly greater preservation of the ONL in hRPC-treated eyes compared with both vehicle-treated and control eyes. The transplanted hRPCs arrested visual decline over time in the RCS rat and rescued retinal morphology, demonstrating their potential as a therapy for retinal diseases. We suggest that the preservation of visual acuity was likely achieved through host photoreceptor rescue. We found that hRPC transplantation into the subretinal space of RCS rats was well tolerated, with no adverse effects such as tumor formation noted at 12 weeks after treatment.

Keywords: Eye; Neuroprogenitor Cell; Retinal Degeneration; Retinal Progenitor Cells; Stem Cells; Transplantation.

FIGURE 1.

Characterization of hRPCs. A, representative images of immunocytochemical analysis for hRPC cell line GS086p9. Scale bars, 100 μm. The cells expressed immunological markers consistent with neural progenitor and glial cells. B, dot plots showing analysis by flow cytometry. Cells were stained with anti-SOX2 APC (x axis) and phycoerythrin (PE)-conjugated antibodies (y axis). C, gene expression of pluripotent stem cell and retinal progenitor markers assayed by RT-qPCR. The graph indicates increased gene expression (x-fold) in hRPCs compared with hESCs. D, gene expression of retinal progenitor and mature retinal markers assayed by RT-qPCR. The graph indicates increased gene expression (x-fold) in hRPCs compared with adult retina. E, gene expression of growth factors BDNF and FGF2 assayed by RT-qPCRs. The graph indicates increased expression (x-fold) of growth factor genes in hRPCs compared with adult retina. Error bars, S.E.

FIGURE 2.

Visual acuity of RCS rats at 5 weeks (P56) after transplantation. OKR studies showed that hRPC-grafted eyes had preservation of visual acuity 5 weeks following transplantation compared with the untreated contralateral eyes (OD versus OS: 0.46 ± 0.04 versus 0.38 ± 0.05 c/d, p = 0.029). Vehicle-treated eyes also had preserved visual acuity at 5 weeks compared with the untreated contralateral eyes (OD versus OS: 0.44 ± 0.03 versus 0.33 ± 0.03 c/d, p = 0.017). In contrast, the mean visual acuity in untreated control rats had deteriorated to 0.34 ± 0.04 c/d in the right eyes and 0.30 ± 0.03 c/d in the left eyes. *, p < 0.05. Error bars, S.E.

FIGURE 3.

Visual acuity of RCS rats at 12 weeks (P105) after transplantation. A sustained protective effect on visual acuity was seen in hRPC-treated eyes (OD) compared with untreated contralateral eyes (OS) (0.47 ± 0.06 versus 0.22 ± 0.05 c/d, p = 0.008). In contrast, preservation of visual acuity was not observed in vehicle-treated eyes (OD versus OS: 0.29 ± 0.02 versus 0.26 ± 0.02 c/d, p > 0.05). hRPC-treated eyes had a mean visual acuity that was significantly better than that of vehicle-treated eyes (hRPC versus vehicle: 0.47 ± 0.06 versus 0.29 ± 0.02, p = 0.012). Untreated RCS rats had deterioration of visual acuity that was equal in both eyes (OD versus OS: 0.25 ± 0.026 versus 0.26 ± 0.031, p > 0.05). **, p < 0.01. Error bars, S.E.

FIGURE 4.

Representative images of photoreceptor rescue after transplantation. Retinal sections at P105 were stained with H&E. Arrows show ONL layers. Scale bars, 100 μm. A and B, extensive photoreceptor rescue was observed in hRPC-transplanted eyes. In grafted areas, there were 5–9 layers of rescued ONL. C and D, in vehicle-treated eyes, there were only 1–3 layers of ONL (C) or no ONL layer at all (D). E, the distribution of hRPC at P2 after subretinal injection shows that donor cells formed lumps of cells in the subretinal space. F, the distribution of hRPCs at P105 after subretinal injection shows that donor cells remained in only several layers in the subretinal space.

FIGURE 5.

Confocal microscopic images of retinal sections of hRPC-transplanted eyes at P105 after transplantation. A, retinal sections were double stained with human nuclear marker (red) and recoverin (green), which showed no colocalization. Donor cells were distributed throughout multiple neural retinal layers. Grafted areas had several layers of ONL. Scale bars, 100 μm. B, retinal sections stained with antibody bestrophin showed that host RPE cells were positively stained (white arrows), whereas donor cells were negative for bestrophine (brown arrows). C, retina sections were stained with human nuclear marker (red) and nestin (green). D–F are high magnification images of A–C, respectively.

FIGURE 6.

Measurement of ONL thickness. A, hRPC-transplanted eyes had the highest preserved ONL thickness (AUC) of 1789 ± 182 μm². The AUC value was significantly lower in vehicle-treated eyes (1377 ± 153 μm², p = 0.019) and in untreated RCS rats (1252 ± 156 μm², p = 0.007). There was no significant difference between vehicle-treated eyes and untreated eyes. B, the total numbers of cells in the ONL in the hRPC-treated group, vehicle-treated group, and control group were 101 ± 11, 73 ± 12, and 62 ± 12, respectively. hRPC-grafted eyes had a significantly higher cell count than vehicle-treated eyes (p = 0.0203) and the control group (p = 0.010). There was no significant difference between the vehicle group and control group. *, p < 0.05; **, p < 0.01. Error bars, S.E.

FIGURE 7.

ONL spread distance and cell count in hRPC-grafted eyes and vehicle-treated eyes. A, the average ONL spread distance in the hRPC-grafted eyes was 1676 ± 198 μm. The ONL spread distance in vehicle-treated eyes was statistically smaller at 1222 ± 218 μm (p = 0.019). The ratio of the spread distance in hRPC-grafted eyes to that in vehicle-treated eyes was 1.37. B, hRPC-grafted eyes had an ONL cell count of 794 ± 123, which was significantly higher than that of 481 ± 103 in vehicle-treated eyes (p = 0.008). The ONL cell count ratio of hRPC-grafted eyes to vehicle-treated eyes was 1.65. *, p < 0.05; **, p < 0.01. Error bars, S.E.

FIGURE 8.

The correlation coefficient of the b-wave response and ONL cell count or ONL spread distance. A, the ONL cell count correlated well with the b-wave response, Pearson r = 0.8763. B, the ONL cell spread distance correlated well with the b-wave response, Pearson r = 0.8905.