Our studies of photoreceptor precursor cell integration and survival after transplantation into a degenerating retina have resulted in several key findings. First, we established that rod precursor cells from the Nrlp-GFP mouse transplanted into the subretinal space of the Rd9 mutant mouse can integrate into the ONL and generate rod photoreceptors. Second, we showed that the extent of cellular integration increases as a direct function of Rd9 host age, with more precursor cells entering the ONL in older and more degenerated host retinas. Third, the transplanted and integrated cells have a limited survival in the Rd9 host eye, and the rate of transplanted cell death appears to be faster with younger host ages. Fourth, we showed that transfected donor cells overexpressing the antiapoptotic gene XIAP exhibit significantly increased transplanted photoreceptor survival in the Rd9 host retina. Finally, we did not detect any donor-cell specific induction of intraretinal inflammation, suggesting that the mechanism of cell death prevented by XIAP therapy is not a specific immune response against the donor cells.
The functional benefit of photoreceptor precursor cell and embryonic stem cell transplantation has been demonstrated in other models,1,6,12,13
yet the mechanism of such effects is poorly understood. One possible explanation is that the transplanted cells themselves are providing increased signal transduction of light stimuli. Our morphologic analysis shows that the transplanted cells appear to make connections with bipolar cells. The small number of integrated cells is consistent with the numbers reported by other groups.6,12,13
This number represents <1% of the normal complement of rods found in a mouse retina and may be below the threshold for measuring improvement in visual acuity. An early report of transplantation of dissociated whole retinas by Kwan et al.2
showed that the transplanted cells formed small patches of integration with the host retinal circuitry, which could mediate simple light responses. These investigators 2
showed that their retinal grafts survived for approximately 6 weeks without the induction of a significant inflammatory response. Further work by the same group has recently shown that injection of human cortical neural progenitor cells into the subretinal space of the Royal College of Surgeons rat, a rodent model of retinal degeneration, prevented host photoreceptor degeneration for prolonged periods of time up to several months.12
There was minimal integration of the donor cells into the host retina. Instead the donor cells appeared to differentiate into sheets of cells located between the host RPE and retina. The exact nature of these cells was unclear, but these data suggest an alternative explanation of how transplanted cells exert a protective effect on the host retina. Perhaps the exogenous cells exert a neurotrophic effect that prevents degeneration of the endogenous cells. This would be expected to provide a more robust benefit to vision than the direct transduction of visual stimuli by these small numbers of cells. If this mechanism indeed proves to be true, it would suggest that in future clinical applications, transplantation earlier in the course of the disease might be needed to provide a greater protective therapeutic effect.
The increased integration of rod precursor cells into older host retinas presents interesting implications with respect to optimal timing of cell-based therapies for future applications in a clinical setting. Previous reports have suggested that the extracellular matrix of the retina may present a barrier to integration. For example, disruption of chondroitin sulfate proteoglycans14,15
or of the outer limiting membrane may allow for improved integration of transplanted cells.16,17
These findings suggest that there may be a component of mechanical obstruction to the successful incorporation of the implanted cells into the host retina. In our experiments, we observed greater number of cells integrated when placed into older, thus more degenerated, eyes. We suggest that the transplanted cells fill the void created by endogenous cell loss or damage in the host retina. It is known, however, that severely degenerated retinas have secondary changes in the inner retina, such as neuronal rewiring, glial cell hypertrophy, and optic nerve degeneration.18–20
Thus, the advantage of age-dependent increased integration of cells may have to be weighed against the possible loss of functional success that may result from the secondary changes to the retina. Strategies for successful cell-based therapies may, therefore, include the use of adjunctive treatments to increase cellular integration at earlier ages, preserve inner retinal function, or both.
In the Rd9 mouse model of XLRP, we demonstrate that regardless of the age or degenerative stage of the host retina, donor cells did not survive for long periods of time after transplantation. The mechanism of transplanted cell death is unknown but is presumed to be apoptotic because of the lack of inflammatory infiltrate or necrotic debris accumulating in the treatment zone. Our screen of intraretinal changes in inflammatory gene transcript levels did not show any significant changes between the Rd9 retina receiving subretinal transplantation of cells and the Rd9 retinas undergoing sham surgery. We interpret this to mean that there was not a “donor-cell” induced inflammatory response but rather that inflammatory changes detected were caused by the surgery itself. Our analysis has several key limitations. First, we were analyzing the intrinsic gene expression profile within the retina for inflammation-associated genes. We were not measuring actual cytokine or chemokine levels present in the retina. Second, we analyzed the transcript levels at only one time point, 1 month after transplantation. This time point was chosen because the highest level of integration was detected then, and we hypothesized that that would be a large stimulus for any inflammatory response. Curiously, even at 1 month after transplantation, there were significant differences in the inflammatory gene transcript levels found even in the eyes receiving sham surgery. This suggests that surgery itself may be a relatively toxic procedure.
The proapoptotic stimulus that ultimately results in the death of the transplanted cells is unknown. One possibility is that the cells are degenerating because of a toxic signal emanating from the degenerating host retina. A second possibility is that the transplantation process itself initiates proapoptotic pathways. This seems less likely because the cells do survive for some period after the transplantation. Regardless of the signal initiating the apoptotic pathways, we find that overexpression of the XIAP gene in the donor cells significantly increases their survival in the degenerating host environment. These surviving cells have all the morphologic features of the non–XIAP-treated integrated cells, suggesting that they are functional photoreceptors. Given that XIAP targets the most downstream components of the apoptotic cascade (caspases 3, 7, and 9), we find it surprising that surviving cells are able to maintain relatively normal photoreceptor morphology. However, previous work with XIAP in photoreceptor degenerations has shown similar results.8–10
This suggests that the ability to prevent apoptosis, regardless of how far downstream in the pathway, can yield therapeutically significant results. It should be noted that the effect of XIAP is not absolute, and some cells do still die. This may suggest the need for additional, complementary methods for increasing cell survival.
In summary, we show successful transplantation of photoreceptor precursors in the degenerating retinas of Rd9 mice. Nonetheless, significant obstacles (such as the identification of a suitable cell line to inject in humans) remain before photoreceptor precursor cell transplantation can become a viable therapeutic option for patients with retinal degenerative diseases. Further investigations are necessary in this exciting field to facilitate cell-based therapies.