Repair of the central nervous system by cell transplantation has been a long-standing goal for a number of degenerative disorders. This strategy requires the transplanted cell population to migrate and integrate efficiently within the target tissue. The extrinsic environment is well known to play an important role in determining the success or otherwise of this integration (43
). Here, we have examined the potential for manipulating the recipient retinal environment by gene transfer in order to improve transplanted photoreceptor precursor cell integration and survival. Ectopic overexpression of IGF1, but not FGF2, in the adult wild-type recipient retina led to significantly improved numbers of Nrl.gfp+ve
precursor cells integrating following transplantation into the subretinal space. Conversely, the upregulation of CNTF significantly impaired integration, potentially due to the induction of increased reactive gliosis. Therefore, modifying extrinsic signaling within the recipient retina can both positively and negatively modulate photoreceptor precursor cell integration into the adult retina, and the extrinsic recipient retinal microenvironment represents an important consideration for photoreceptor transplantation strategies.
We have previously shown that postmitotic photoreceptor precursor cells derived from the early postnatal retina are optimal donors for transplantation (45
is expressed at higher levels in the developing postnatal retina compared with the adult (5
) and the IGF1/insulin receptor pathway is important for both retinal cell survival and photoreceptor maturation (14
). Although IGF1 levels are reduced in the adult, retinal neurons appear to retain the ability to respond to IGF1/insulin receptor signaling. Studies in the rds
mouse have shown increased photoreceptor survival in the presence of increased proinsulin production (17
), while retinal ganglion cell survival is improved in models of optic nerve transection when combined with intravitreal administration of IGF1 (17
). The increased number of integrated rod photoreceptors observed in the AAV2/2 CMV.IGF1-treated eyes described here could be due either to IGF1 acting to increase the survival of donor cells (either before or after integration) or by augmenting the frequency of precursor cell integration itself. We consider the former more likely because barriers such as the OLM still remain in the host retina, which have previously been shown to limit the number of photoreceptor precursor cells that are able to integrate into the host ONL (53
). IGF1 acts via the activation of the antiapoptotic phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and inhibition of caspase 3 in the maintenance of retinal ganglion cells following optic nerve transection (32
). Sustained PI3-K activation in transgenic mice has been reported to increase the survival of differentiated photoreceptors in the postnatal retina (55
). It is possible that the enhanced survival of migrating and integrating transplanted photoreceptor cells occurs by a similar mechanism. Consistent with this, we found a reduction in active caspase 3+ve
, apoptotic cells in the ONL of AAV2/2 IGF1-treated retinae compared with untreated retinae. In keeping with this idea, recent work in the retina has shown that XIAP (X-linked inhibitor of apoptosis protein), which inhibits apoptosis via the inhibition of caspases, including caspase 3, can reduce transplanted photoreceptor cell death in the rd9 model of retinal degeneration (77
Another potential consequence of IGF1 upregulation might be the improved or strengthened synaptic connectivity of the transplanted cells. Newly born neurons, including photoreceptors, are vulnerable to pruning and apoptosis if appropriate synaptic connections with downstream targets are not formed or maintained (65
). We observed IGF1 expression in the outer plexiform layer around the time of photoreceptor synaptogenesis in the postnatal retina. Overexpression of IGF1 has previously been shown to promote synaptogenesis in the hippocampus during postnatal development (51
). IGF1 has also be associated with the upregulation of brain-derived neurotrophic factor (BDNF), an important modulator of synaptic plasticity in the adult brain, after mechanical injury and with exercise-induced cognitive function (15
). A recent study investigating the molecular pathways involved in the increased development of visual acuity responses induced by environmental enrichment demonstrated that intraocular injection of IGF1 increased BDNF expression in the retina (36
). Moreover, both IGF1 and BDNF were required to mediate the increased maturation of visual acuity observed in rats reared in enriched environments. Although beyond the scope of this study, it is possible that the increased expression of IGF1 in the current study could mediate the subsequent upregulation of BDNF in the adult retina, and that a combination of these factors might improve transplanted photoreceptor synaptic connectivity and, subsequently, survival.
FGF2 has been implicated in retinal neurogenesis (23
), migration (7
), and photoreceptor survival (29
). Mice with a targeted disruption of FGF receptors 1 and 2 (FGFR1 & 2) undergo a progressive loss of photoreceptors (9
), suggesting that FGF2 may act as a survival factor for photoreceptors. Therefore, we considered FGF2 to be of interest in terms of the migration, integration, and survival of transplanted photoreceptor precursor cells. However, we observed no improvement in cell integration following the over expression of FGF2 in the recipient retina, compared with control transduced or untransduced retinae. Our assessment of FGF2 expression indicates that the cell transplantation procedure itself, which involves a substantial if temporary retinal detachment, leads to significant upregulation of FGF2, probably greater than that achieved by AAV transduction. In the adult mouse retina, mechanical injury results in the upregulation of FGF2 expression, which is maximal at 2–4 days posttrauma. Both injury- and light stress-related upregulation of FGF2 have been shown to protect photoreceptor cells from further light-induced degeneration in the adult mouse (49
). Similarly, AAV-mediated upregulation of FGF2 has been shown to reduce photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa (37
). Further work blocking FGF signaling will be required to determine the role FGF2 plays in the integration and survival of photoreceptors transplanted into the normal, untransduced retina.
CNTF has previously been shown to inhibit rod photoreceptor differentiation in the developing retina (20
) and here we found photoreceptor precursor cell integration to be markedly reduced in AAV2/2 CMV. CNTF-treated eyes compared with controls. CNTF/LIF signaling has been shown to block the expression of the rod photoreceptor transcription factors Nrl
), but the reduction in integrated cells did not appear to be due to a downregulation of Nrl
, as the percentage of Nrl.gfp+ve
cells in the transplanted cell mass was not significantly reduced compared with that of controls. We cannot rule out the possibility that the GFP protein remains in the absence of sustained Nrl
expression, although this is unlikely.
Expression of the α-subunit of the CNTF receptor, CNTFRα, by photoreceptors is thought to be minimal, although the literature is complicated, possibly due to the fact that CNTFRα can act as a soluble mediator for CNTF signaling (18
). Interestingly, a recent study has shown that CNTFR
α is expressed in immature cells of the ONL, but is downregulated with the onset of rhodopsin expression (26
). This may explain how CNTF signaling might affect immature photoreceptor precursor cells and not directly affect adult photoreceptors of the mammalian retina.
CNTF has been shown to be act as a chemoattractant for blood-derived macrophages (12
). We have previously found that high macrophage presence is associated with poor integration (73
). However, the extent of macrophage infiltration observed in CNTF-treated retinae was very similar to that seen in control eyes, suggesting that it was not a major factor in terms of precursor cell integration.
The most likely cause of reduced cell integration in the CNTF-treated retinae is glial scarring. In the adult retina, CNTF has been shown to exert its stress-related neuroprotective effects indirectly via the activation of Müller gial cells (54
). Others have demonstrated the upregulation of GFAP, a marker of glial cell activation, in Müller glial cells following injection of recombinant CNTF, an effect mediated by the JAK/STAT signaling pathway (54
). The ability of cytokines to induce GFAP expression varies, and CNTF has been shown to be the most potent compared with FGF2 and LIF (71
). We observed very marked increases in GFAP expression; little GFAP was observed in control AAV2/2 CBA.RFP-treated retinae, demonstrating that neither the mechanical trauma of the intravitreal viral injection nor that of the subretinal cell transplantation were sufficient to elicit significant sustained glial cell activation in wild-type eyes. Chen and colleagues demonstrated that transplanted cell integration was significantly higher in mice in which GFAP and vimentin was knocked out (33
). In keeping with this, we have observed an inverse correlation between the degree of glial scarring and the number of integrated photoreceptors (A. Barber and R. Pearson, unpublished observations), indicating that the glial scar presents a restrictive barrier that inhibits photoreceptor precursor cells from migrating into the ONL.
Here, we show that it is possible to manipulate the recipient retinal environment via rAAV-mediated gene transfer with respect to developmentally regulated neurotrophic factors. When combined with cell transplantation, IGF1 led to significantly increased levels of cell integration, while CNTF resulted in unwanted effects in both host and donor cells. It may be that different levels, both higher and lower, of these factors may have different effects and it will be of interest to establish a dose response, particularly for IGF1. Taken together, these findings demonstrate the importance of the extrinsic environment of the host retina for successful photoreceptor cell transplantation.
We have previously shown that a number of manipulations can improve the numbers of transplanted photoreceptor precursors that go on to integrate following transplantation, including transient disruption of the outer limiting membrane (OLM) (53
). Here, we tested the impact of manipulating the levels of IGF1, CNTF, and FGF2 in isolation in normal wild-type mice to permit assessment of a single variable. It is likely that a combination of factors will be required to adequately modify the retinal environment to promote optimal cell integration. These may include manipulation of the OLM, endogenous growth factor levels, and potentially even changes to the donor cell population itself. It is also important to consider that the degenerating retinal environment will present a very different and potentially hostile environment to donor cells compared with that of the wild-type retina. Thus, it will be important to determine the impact of degeneration upon cell integration but also whether manipulations of this environment such as those described here could be used to enhance transplanted photoreceptor precursor cell integration.