Cell transplantation for the treatment of neurodegenerative diseases is an area of intense current investigation. However, transplantation into the mature CNS faces a major hurdle—the limited ability of transplanted cells to survive, migrate, and establish morphological and synaptic connectivity with the recipient tissue. There has been considerable interest in trying to generate specific cell types for transplantation, but for these findings to be successfully translated into clinical therapies we also require a much better understanding of how the donor cells interact with the recipient microenvironment. A number of reports have demonstrated the potential for a variety of cell types to migrate into the immature retina (35
). However, this ability is largely lost as the recipient animal matures, whereupon stem cell populations appear able to migrate only when the recipient retina is damaged in some way (20
). Here, we provide strong evidence demonstrating that the OLM of the recipient retina is a physical barrier to donor cell migration and integration and presents a potential target for improving transplantation into both the wild-type and, importantly, the degenerating retina. Moreover, we present a novel way of inducing a transient disruption of this barrier that, when combined with cell transplantation, led to significant improvements in integration in models of retinal degeneration.
Recessive mutations in the human CRB1
gene cause retinal diseases including retinitis pigmentosa and Lebers congenital amaurosis (7
). Currently, there is no effective treatment for patients with CRB1
mutations. The Crb1rd8/rd8
mouse has a phenotype similar to human patients with missense CRB1 mutations. We found that donor cell migration was significantly improved in these animals compared with wild-type animals and that the level of integration increased with increasing OLM disruption, up to 6 weeks of age. This contrasts with the very low levels of cell integration observed following transplants into other models of retinal degeneration, such as the rho−/−
and the Prph2rd2/rd2
) mouse, where typically only a few hundred cells integrate (24
). Together these data demonstrate that the OLM presents a physical barrier to transplanted cell migration and integration [and it is important to note that the OLM is likely to be one of a number of such barriers (40
)]. Secondly, they show that the degenerative apoptotic environment itself is unlikely to be promoting transplanted cell integration. Thirdly, the Crb1rd8/rd8
mouse provides a relevant model of a specific type of retinal dystrophy, which may be more amenable to cell transplantation therapy than other inherited retinal degenerations.
While retinal dystrophies involving CRB1
may prove ideal candidates for cell transplantation therapy, retinal degeneration caused by many other gene defects, such as rd1
, is not associated with marked OLM disruption until very late stages of the disease (12
). This suggests that the OLM might present a significant barrier to transplanted photoreceptor precursor cell migration and integration in the majority of retinal degenerations, particularly at early stages. Therefore, we must seek ways to overcome the physical barrier presented by the OLM. The pharmacological induction of OLM disruption by aminoadipic acid (AAA) we have reported previously (51
) would not be suitable due to detrimental side effects within the recipient retina, including very significant apoptosis in both the Müller glia and photoreceptor populations (18
). Furthermore, the overall increase in integration, while consistently higher than the vehicle-injected eyes, was only similar to that seen for standard control injections. However, the results presented here suggest that if appropriate methods of OLM disruption are used, significant improvements in cell integration above that previously reported (3
) can be achieved. Moreover, such improvements are also possible in models of retinal degeneration.
RNAi strategies have been successfully applied to the treatment of mouse and rat models of hepatitis and cancers (25
). A feature of siRNAs that is that they are typically unstable, being removed by endogenous RNases, and so have a short duration of action. The strategy employed here utilized this characteristic and used naked siRNA to demonstrate a proof of principle, namely that the OLM could be reversibly disrupted by siRNAs in both wild-type and degenerating retinas. ZO-1 was the first tight junction protein identified and functions as a junctional adaptor that interacts with multiple transmembrane proteins, components of the junctional plaque and actin filaments (1
). Because ZO-1 is also expressed at the tight junctions of the RPE, it is possible that a subretinal injection of ZO-1 siRNA will also impact on these junctions, although we did not observe any obvious signs of leakiness or oedema. For this strategy to be taken forward, it is therefore important to identify other targets for siRNAs that may have less impact on the overlying RPE tight junctions. Recent work suggests that there are differences in the expression of tight junction proteins in the RPE compared with the adherens junctions of the OLM (8
), which may provide alternative targets. Indeed, Crb1 itself is worthy of further consideration, because its expression in the eye is restricted to the OLM (9
Although ZO-1 proved an effective target for inducing reversible OLM disruption here, high concentrations were associated with a moderate level of cell death. Recently, Yang and colleagues demonstrated that double-stranded RNAs greater than 21 nucleotides can bind with the Toll-Like receptor 3 (TLR3) and induce apoptosis (52
). It is unlikely that the apoptosis observed in this study was due to interactions with TLR3, because the levels observed were similar in both the siRNA-treated (targeting and nontargeting) and vehicle-treated eyes. Moreover, the cell loss declined with time, unlike the progressive degeneration observed by Yang and colleagues. Nonetheless, further work is required to optimize the surgical procedures involved to minimize any associated cell loss.
Of interest is the finding that integration in the 12-week-old Crb1rd8/rd8
mouse was not significantly better than wild-type controls, despite the continued disruption of the OLM. We also observed very high levels of GFAP and vimentin staining at the edge of the ONL in these older animals, with numerous Müller glial processes running along the outer limits of the ONL. This level and pattern of expression is similar to that seen in 4–6-week-old rho−/−
mice and strongly indicative of glial scarring. Such scarring is often associated with neural injury and is known to obstruct regenerative processes, such as axonal regrowth, by forming physical and diffusional barriers that separate undamaged regions from the area of injury (13
). We propose that in the Crb1rd8/rd8
mouse there is a trade-off between the progressive disruption of the OLM, permitting increased integration and an increase in glial scarring, ultimately inhibiting cell migration, despite the disruption of the OLM. There is thus a window of opportunity around 6 weeks of age, at which cell integration is increased. GFAP expression itself has been both positively and negatively correlated with cell migration. Kinouchi and colleagues (19
) demonstrated that elimination of both GFAP and vimentin in wild-type mice permitted increased integration following transplantation of retinal progenitor cells. We have also observed that integration is negatively correlated with increasing gliosis in a number of models of degeneration (A.C.B and R.A.P, unpublished observations). Conversely, Perez and colleagues (54
) suggest that cells migrate at sites of GFAP upregulation following transplantation of fetal retinal sheets. Here, we observed similar levels of glial cell activation, as indicated by GFAP staining, following application of either ZO-1 siRNA, nontargeting siRNA, or vehicle alone, but integration was only significantly improved those eyes receiving the ZO-1 siRNA. Similarly, integration was significantly lower in control rho−/−
mice, a model in which gliosis is prominent, and only enhanced in animals receiving ZO-1 siRNA. Together with the findings from the Crb1rd8/rd8
mouse and the AAA work reported previously, these results point strongly towards the improvements being as a result of OLM disruption. However, it is important to remember that it is highly unlikely that any one single barrier is responsible for preventing transplanted cell integration per se. Thus, any effective therapeutic treatment must encompass a number of different factors, including the reduction of reactive gliosis and subsequent glial scarring and transient disruption of the OLM.
In conclusion, we have provided strong evidence that the OLM represents a significant physical barrier to transplanted photoreceptor cell integration. Moreover, we present a novel use of RNAi technology to introduce a transient and reversible disruption of this barrier that when used in combination with cell transplantation leads to substantial increases in the number of successfully transplanted cells in both wild-type and degenerating retinas.