In the present study, we used in vitro and in vivo methods to investigate the barriers to the migration of intravitreal cell grafts. We used MSCs in these experiments, as these cells are currently attracting much attention as a potential therapy for CNS diseases, because of their neuroprotective properties,29–31
their ability to home to degenerating tissue,32
and their possible, though controversial, neural transdifferentiation potential.33
Since migration from the vitreous cavity into the neural retina is an early event that is likely to be important in stem cell therapy that provides both neural regeneration and neuroprotection, we focused the present study on cellular migration rather than on more specific events such as neural differentiation. We found that the ECM of the inner basal lamina is neither necessary nor sufficient to prevent retinal engraftment of stem cells but that reactive glial processes appear to play a dominant role in this process. Furthermore, we found that exogenous manipulation of the inhibitory environment can overcome inhibition of transplant migration and propose that suppression of glial reactivity will be a necessary component of intraocular stem cell transplantation therapies in the future. This is a major step forward in the development of cell therapies for retinal disease, as suboptimal graft integration has been a major stumbling block to date. As glial reactivity is a ubiquitous phenomenon throughout the CNS, our results are likely also to apply to potential cell-based therapies for a range of other CNS conditions.
Intraocular transplantation of stem cells for retinal therapy can be achieved via two approaches, either subretinally or intravitreally, with each technique possessing advantages and disadvantages for particular applications. Subretinal injections leave cells physically constrained adjacent to the outer retina and near to rich blood supply, whereas intravitreal injections are technically simpler and provide direct access to the inner retina. Most research into improving the outcome of intraocular grafts has focused on subretinal injections, in part because of an initial focus on diseases of the photoreceptors. However, we have an interest in applying stem cell therapies to glaucoma, a common neurodegenerative disease of the inner retina that is the leading cause of irreversible blindness worldwide.3
In the context of inner retinal disease, intravitreal injections are likely to be more applicable than subretinal injections. Although studies involving subretinal transplantation have identified both ECM molecules and cellular factors as inhibitory to graft migration, it is unclear whether these elements play the same role, if any, when the graft is placed intravitreally. Besides providing useful information for developing treatments for inner retinal disease, determining the commonality of barriers to cell transplantation in different regions of the retina may provide insights that will aid in developing cell-based therapies in other CNS compartments.
Components of the ECM have been identified as potential barriers to the integration of transplanted stem cells in the CNS. For example, enzymatic degradation of chondroitin sulfate proteoglycans has been shown to enhance stem cell engraftment in the spinal cord34
and also to augment the integration of neural stem cells after intraocular transplantation, although the effects have been modest.16,17
Matrix metalloproteinase-2 has a similar effect in vitro.18
In the present study, we focused on degradation of proteins concentrated at the retinal ILM, as this appears to be the site of blockade for intravitreally transplanted cells. Although our enzymatic treatments effectively digested the inner basal lamina ECM proteins laminin and collagen, they did not enhance the migration of cells into the retina. This contrasts with data from subretinal approaches where destruction of physical impairments to cell integration has proven beneficial at the outer limiting membrane.19
This effect may be due to fundamental differences in the microenvironment of the inner and outer retina. It is possible that glial obstacles are more prominent in the inner retina, rather than inhibitory ECM factors and physical barriers as in the outer retina, such that enzymatic ECM digestion has a negligible effect on intravitreal graft migration. Morphologic localization of glial intermediate filaments supports this view, given that immunoreactivity of these proteins is much higher in the inner retina, compared with the outer retina, under both normal and pathologic circumstances.15
Indeed, this concurs with the data presented by West et al.,19
who studied the effects of AAA on subretinal transplantation. They noted that AAA treatment led to an approximately threefold increase in the number of photoreceptor progenitors that integrated into the outer nuclear layer 3 weeks after injection. Of importance, the authors of this study attributed the effect to a structural disruption of the outer limiting membrane, which is composed primarily of heterotypic and homotypic adherence junctions between Müller glia and photoreceptors. Furthermore, that report indicated that GFAP immunoreactivity in healthy eyes that had not received transplants was localized exclusively to the inner retina and was not affected by AAA treatment; however, GFAP immunoreactivity in transplanted eyes was not investigated. In contrast, the present study demonstrated a dramatic increase in reactive gliosis after retinal explant culture, the onset of ocular hypertension, and intravitreal transplantation. The effects of AAA on highly reactive retinal glial cells appear to be different from normal control retinal tissue,19,36
as we demonstrated a dramatic downregulation in reactive intermediate filaments after treatment in the current models. That we also demonstrated improvement in retinal engraftment of intravitreally transplanted cells after AAA treatment indicates that glial reactivity appears to predominate over ECM-mediated effects on cell graft migration in the context of inner retinal disease. However, it is possible that combinatorial treatments would produce an even more robust effect on intravitreal transplant migration than suppressing glial reactivity alone.
In contrast to the lack of intraretinal migration of MSCs observed after enzymatic disruption of the retinal ILM, suppression of glial reactivity using a selective toxin greatly potentiated retinal integration of intravitreally transplanted cells. Similar results have been found previously using transgenic techniques to knockout glial expression of the proteins GFAP and vimentin.13
The authors reported that both subretinal and intravitreal transplantation of neural stem cells into adult mice resulted in minimal retinal engraftment, as has been well documented previously. However, they observed a more than sixfold increase in stem cell migration into the retina from subretinal transplantation after knockout of GFAP and vimentin while simultaneously preserving retinal structure and function. Suppression of Müller cell expression of GFAP and vimentin has been associated with a reduction in their reactivity.28
The data presented in our study indicate that a relatively comparable increase in the number of engrafted stem cells can be attained by a transient, rather than permanent, disruption of glial cell function. A return of GFAP expression by Müller cells after in vivo AAA treatment confirmed the transient nature of our intervention. Such acute environmental manipulation is preferable, given that permanent loss of filament protein expression by Müller cells increases retinal vulnerability to mechanical damage.37
In contrast to these results, Nishida et al.14
demonstrated a high degree of retinal integration by intravitreally transplanted neural stem cells after mechanical retinal injury. In that paper, transplanted cells integrated near the injury site and also in regions of structurally intact retina, which also exhibited reactive gliosis, up to 1200 μm away from the injury site. They suggested that glial reactivity facilitated graft integration, possibly by local production of growth factors and/or chemokines. However, other reports have suggested that reactive gliosis, or components thereof, constitutes a barrier to the retinal integration of numerous transplanted cell types in a variety of circumstances.10,13,15,17,18,38–40
An interesting hypothesis to explain this apparent discrepancy was proposed by Zhang et al.,41
when they observed that neurites from abutting retinas in culture would cross-integrate only when certain glial-associated structures were disrupted, but when this did occur, it tended to take place near areas of high GFAP expression. They admitted that neurite integration may have triggered GFAP upregulation, but suggested that, alternatively, reactive glial cells facilitated neurite integration as long as separate glial barriers at the interface of the abutting retinas were disrupted. Thus, they proposed that glial cells possess both inhibitory and facilitative components. If this is the case, then it is possible that the AAA treatment used in the present study suppressed beneficial glial mechanisms that could further enhance integration of grafted cells, such as chemokine secretion. As such, future research should identify methods of blocking the inhibitory effects of reactive gliosis while preserving any potential facilitative effects. Nonetheless, the current results demonstrate that reactive gliosis is a significant component of the inhibitory barrier to retinal integration of intravitreally transplanted stem cells. Moreover, suppression of glial reactivity had a net overall benefit for grafted cell integration.
It should also be noted that the present study was conducted with MSCs used as the transplanted cell type. Although reactive glial processes have been implicated in blocking the integration of numerous classes of transplanted stem cells in the retina,10,13,15,17,18,38–40
it is unclear whether AAA would have the same effect for other stem cell types of interest. If different classes of cells respond to different migratory cues within the host retina or have different intrinsic migratory potentials, then it is possible that protocols aimed at enhancing engraftment would be met with various levels of success, depending on the cell type transplanted.
Although we have clearly shown that disruption of glial reactivity enhances transplanted MSC engraftment in the retina, we observed remarkable differences in the effects of AAA treatment in vitro and in vivo. Most notable is that AAA was at least 10 times more effective in the culture system than in vivo, perhaps because of differences in the duration of glial reactivity suppression, as reactivity remained low in the explant cultures for the duration of the experiment yet reverted to a high level in vivo, presumably after AAA clearance. It is likely that, in contrast to the eyes of living animals, the artificial culture conditions did not permit glial cell recovery after AAA treatment, thereby providing a longer window of opportunity for the migration of cells into the retina in vitro. Thus, it is conceivable that suppression of glial reactivity for a longer period in vivo produces a stronger effect. Furthermore, transplants in vivo are exposed to an active immune system that is mostly absent in vitro. Indeed, even though the present study used pseudoautologous grafts (cells from different animals of the same strain), we observed a strong degree of macrophage/monocyte infiltration and identified some inflammatory cells that co-labeled for GFP, implying phagocytosis of engrafted cells by host cells. Immune activity has been identified as a process that limits cell transplant efficacy in the CNS,42
and immunosuppression with indomethacin in conjunction with ECM modulation has been shown to improve subretinal graft survival and integration.17
Thus, protocols to safely and efficiently suppress immune responses against intraocular grafts should be investigated.
Although the results presented herein demonstrated for the first time that inner retinal stem cell engraftment may be facilitated by acute downregulation of glial reactivity, the compound used is unlikely to be of clinical interest. In mice, 100 μg/μL AAA injected intravitreally has been shown to produce a transient disruption of Müller cells, with recovery observed 2 weeks after treatment and a peak effect at 72 hours.19
An important finding was that AAA was toxic to Müller glia exclusively, whereas the rest of the retina appeared normal. However, AAA is a gliotoxin and besides suppressing glial reactivity, it also disrupts normal physiological function of the Müller cells, which is likely to severely affect vision. Moreover, it is likely to suppress the production of a variety of chemokines (such as stromal cell-derived factor-1) that are produced by glial cells, which have been shown to be important in guiding the migration of transplanted cells in the brain after ischemic insult, and may play a role in cell therapy for other neurodegenerative conditions.43–45
Instead of such a general approach, more targeted efforts are needed. This may be approached from at least two directions. First, it may be better to block reactive glial changes without disrupting other physiological functions. The JAK/STAT3 signaling pathway has been implicated in upstream signaling of glial activation,46,47
and modulation of this pathway may overcome glial inhibition to retinal engraftment without affecting visual function. Second, specific downstream processes that occur in reactive glial cells must be identified. Glial reactivity is a blanket term that is associated with a wide variety of changes that occur in stressed glial cells, including hypertrophy; upregulation of intermediate filament expression; alterations in the production of neurotrophins, cytokines, chemokines, and reactive oxygen species; and changes in buffering properties for extracellular ions and molecules.48–50
It is unlikely that all these changes contribute equally to the poor integration of intraocular grafts. Instead, it may be that the effect noted in our study was mediated primarily by a physical blockade of cell migration by hypertrophic Müller cell processes and, therefore, targeted reduction of hypertrophy may be helpful. In addition, reactive glial cells may produce inhibitory molecules that block the integration of grafted cells and suppressing either the production or activity of these molecules may allow for a high level of stem cell engraftment in the CNS.
In summary, we have demonstrated for the first time that the predominate block to retinal integration of intravitreally transplanted stem cells is glial cell reactivity, as opposed to physical barriers contained within the ILM. An important finding was that even a transient reduction in glial reactivity could significantly enhance engraftment of stem cells into the retina in vivo. These findings have direct implications for the development of stem cell therapies for common irreversible neurodegenerative retinal diseases such as glaucoma. However, given that glial reactivity also inhibits the integration of stem cells in the brain,51–53
these findings are also applicable to the CNS as a whole. Finally, although identification of the major barrier to stem cell integration in the mature inner retina is a major step forward, it is unlikely to be sufficient for clinical therapy. Therefore, further research is necessary to characterize other inhibitory factors of interest, as it is likely that a combinatorial approach will be necessary for optimization of transplanted cell engraftment.