Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Cell Death

There is little evidence of glial death in remodeling, but the proportions and numbers of survivor neurons change significantly. BCs form the largest cohort of neurons (other than photoreceptors) in normal retina; however, ACs always predominate in phase 3, suggesting that BC death is far more common than AC or GC death. This may be due to the fact that BCs are the only retinal cells that lose all of their glutamatergic input. Neurons require a basal level of Ca influx to maintain homeostatic gene expression and, through self-signaling, ACs and GCs clearly possess the critical glutamatergic input required to provide both transmitter-gated Ca flux and voltage-gated Ca-channel activation; BCs clearly lack that input. As subjects age, ACs and GCs also decrease in number. In any event, the loss of neurons has strong implications for all therapeutic interventions, including epiretinal implants.

MC Remodeling

MCs make up nearly 50% of the mass of the peripheral primate retina and are one of the major drivers of remodeling. While there has been little analysis of phase 1 MC function in inherited retinal degenerations, there is abundant evidence that they respond to rapid, coherent photoreceptor stress initiated by LIRD within hours by increasing intermediate filament expression, displaying distal process hypertrophy in the ONL, and increasing arginine expression (a marker of increased protein synthesis). In phase 2, MCs play a lead role in forming a seal between the remnant RPE and/or choroid. The transport characteristics of that seal are unknown, but its formation is paralleled by a massive increase in MC glutamine levels. MCs normally export glutamine from MCs to surrounding neurons. This transport is voltage sensitive and, similar to many Na-coupled transporters, allows increasing export with depolarization. Depolarization of MCs is closely coupled to light-activated events, and the loss of photoreceptors may play some role in progressive hyperpolarization of MCs and retention of gluamine. However, as the increase in MC glutamine is temporally linked to the formation of the distal seal, the mechanism of glutamine retention appears more complex than just constraining the MC voltage.

RPE Remodeling

In classical RP, invading RPE cells are one of the hallmarks of advanced disease. After formation of the MC seal, certain RPE cells and, sometimes, choriocapillaris endothelia are able to penetrate into the neural retina, forming large complexes of hypertrophic MCs, RPE with altered melanosomes encapsulating invading

and remnant retinal capillaries, clusters of new neurites, and columns of migrating neurons. RPE cells seem to be the foci of large-scale morphologic derangements in the survivor retina, but whether they are initiators or responders is not clear. The RPE remains partially intact for long periods in many retinal degenerations, including the Royal College of Surgeons (RCS) rat defect; however, the RPE layer can become broken by patches of invading RPE cells and apical processes. In the RCS rat, apical RPE processes can extend to the GC layer.

Vascular Remodeling

New capillaries invade the neural retina in phase 3, emanating from both vitreal and choroidal sources. Little is known of the fundamental transport properties of these new vessels (e.g., whether they are fenestrated or not), but both molecular and genetic profiling show that neural retina in RP is metabolically deprived. New imaging data as well as electron microscopy suggest that the new vessels are too attenuated and too heavily invested by hypertrophic MCs and RPE to allow proper perfusion of the retina. These anomalous foci may trigger neuronal migration. The stimuli for vascular remodeling remain unknown, but VEGF secretion by invading RPE cells is one plausible source.

Impact of Remodeling on Therapeutics

The potential reversibility of remodeling events varies. For example, phase 1 and 2 changes in reprogramming of gene expression and neurite switching are reminiscent of normal plastic behavior and might respond to the appropriate therapeutic signals. However, phase 3 changes such as rewiring, neuritogenesis, and microneuroma formation, migration, and neuronal cell death are not reversible by any known means. Intermediate phenomena such as MC, RPE, and vascular remodeling are similarly challenging. Human RP patients show the full spectrum of remodeling defects; therefore, most therapies are impacted by a narrowing therapeutic window.

Primary Gene Therapy

All prospective primary gene therapies (those targeting known gene defects) depend on photoreceptor survival. However, it is clear that photoreceptor deconstruction starts long before photoreceptor death. The recent successes with gene therapy for replacing the deficient isomerase (RPE65) in that variant of Leber’s congenital amaurosis (LCA) is not likely relevant to primary rod or cone gene defects, nor to defects associated with the accumulation of genotoxic and cytotoxic debris in the

subretinal space such as those due to defects in RPE mer tyrosine kinase (MERTK). In human rod dystrophies, it is clear that mutations impacting rhodopsin trafficking also lead to changes in the inner segment function and photoreceptor architecture. Rod photoreceptor rewiring in human RP is unlikely to be reversible regardless of the success in replacing defective phototransduction genes. So far, gene therapy successes in animal models are largely restricted to prenatal or early postnatal genetic interventions. With the exception of soft diseases such as LCA and stationary night blindness that do not trigger photoreceptor deconstruction, most retinal degenerations clearly have only a tiny window for gene therapy. Primary gene therapies are currently restricted to phase 1.

Survival Factor Therapy

One strategy to retard retinal degeneration involves the use of survival factors such as neurotrophins (e.g., ciliary neurotrophic factor, CNTF) that slow photoreceptor apoptosis. These strategies were validated for slower models of adRP (the rat P23H and S344ter rhodopsin transgenic models). Some argue that the structural preservation afforded by CNTF in these models does not reflect a parallel functional rescue. There is evidence that early postnatal CNTF infusion in other rodent models of RP negatively alters photoreceptor gene expression profiles, activates MC stress signaling, and alters inner retinal organization. Simple survival factor therapy without a known cellular and molecular target is not likely to generalize well across human RP types. At present, survival factors offer little prospect of reversing or retarding remodeling. Survival factor therapies are restricted to phase 1 and early phase 2.

Stem/Neuroprogenitor Cell Therapy

Several groups have shown that isolated stem/ progenitor cells, particularly murine postnatal day 5 rod neuroprogenitor cells, have the potential to intercalate in the ONL and, possibly, repopulate the retina. The efficiency of such intercalation is low and it is not likely that these cells will survive in phase 3 retinas. Penetration of the MC seal is unlikely and several groups have failed to obtain significant numbers of exogenous photoreceptors to extend synapses through it. Direct injection into the retina is likely to activate microglial killing and, in any event, isolates any surviving photoreceptors from the remnant RPE. For photoreceptor progenitor cells to be successful in rescuing vision, they must also lead to cone survival and reconnect with existing neurons before morphologic remodeling begins. This excludes most current RP patients. Stem/neuroprogenitor cell therapies are currently restricted to phase 1 and early phase 2.

Retinal Transplantation

The team of Seiler and Aramant pioneered the effort to insert sheets of fetal retina into the subretinal space of degenerating retina. They have successfully demonstrated long-term photoreceptor survival and some visual driving in rats. Remodeling remains a major barrier in several ways. The extent of transplant-to-host neurite intermingling is small, and the glial seal predominates. Further, the transplanted retina begins to remodel and appears even more susceptible to alteration than the host. This suggests that remodeling signals emanate from the survivor retina. Certainly, after phase 3 BC dendrite truncation in the host retina, a semi-intact fetal retina cannot recapitulate normal circuitry by tandem connections. It is likely that most transplant-to-host connectivity is AC ! AC driven and probably functionally random. Even so, such networks can generate light-driven behavior. Though lacking the spatiotemporal precision normal GCs, transplants have the potential for much higher sensitivity, range, and resolution than bionic mechanisms. Retinal transplantation therapies are largely restricted to phases 1–2, but may function if the connection can traverse the glial seal in phase 3 retinas.

Secondary Gene Therapies: Photosensitive

Proteins

A recent development of import is the ability to induce expression of photosensitive proteins in survivor neurons by viral or molecular transfection, generating light responses directly in neurons. Several groups have shown that it is possible to express channelrhodopsin 2 (ChR2) in retinal BCs, and elicit ChR2-BC-driven photoresponses in GCs and light–dark preferences in behavioral search. This is an important advance and represents the potential to convert blind retinas into navigational systems without surgical intervention and ancillary equipment. The challenge is to demonstrate that this is possible in phase 3 profoundly blind patients, as they are the most obvious candidates for therapy. This technique has, once again, only been established in phase 2 or earlier models. Further, as with transplantation, there is no evidence that such secondary therapies will be resistant to remodeling, prevent cell death, or overcome signal corruption. A significant amount of basic research remains to be done but, despite promise, secondary gene therapies still seem limited to phases 1–2, although they may function in phase 3, even if randomly targeted.

Bionic Implants

The most successful schemes to restore vision to the profoundly blind from aggressive phase 3 retinal degenerations are epiretinal bionic implants. Surgically placed near the

GC layer, epiretinal implants provide direct current stimulation of retinal GCs or nearby circuitry to activate patches of visual sensation. The argument that such stimulation conflates ON and OFFresponses seems irrelevant as several implanted patients can now successfully navigate with such devices. It is clear that the severe remodeling, especially neuronal death, limits candidacy. All implants, whether they are epiretinal, intraretinal, or subretinal, seem to induce further glial and neuronal remodeling. Severe remodeling, BC death, and microneuroma formation will more severely impact subretinal models.

The Importance of Cone Rescue

In every model, the survival of cones seems to delay the onset of phase 3, holding the retina in a phase 2þ state indefinitely. The effect is local and small patches of cones preserve connected BC dendrite structure even when surrounding BCs have lost all dendrites and glutamate receptors. The preservation persists even when cones are severely deconstructed, lacking outer segments and visual pigment expression, and significantly reduced in size. This suggests that cone contact alone, perhaps through synaptic integrins, may be sufficient to preserve BC function. Expression array studies suggest that cone deconstruction may be accelerated by metabolic deprivation. While preserving cones will not rescue vision, it will permit holding the survivor retina in suspended animation, making an array of interventions such as stem/progenitor cell transplantation, fetal retinal sheet transplantation, and secondary gene therapy viable for adults suffering from advanced retinal degenerations. Revisiting survival factor research with a focus on cones may be the critical advance needed for all intervention methods.

See also: Anatomically Separate Rod and Cone Signaling Pathways; Injury and Repair: Light Damage; Injury and Repair: Prostheses; Injury and Repair: Stem Cells and Transplantation; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration; Secondary Photoreceptor Degenerations.

Further Reading

Gargini, C., Terzibasi, E., Mazzoni, F., and Strettoi, E. (2007). Retinal organization in the retinal degeneration 10 (rd10) mutant mouse:

A morphological and ERG study. Journal of Comparative Neurology

500: 222–238.

Gupta, N., Brown, K. E., and Milam, A. H. (2003). Activated microglia in human retinitis pigmentosa, late-onset retinal degeneration, and age-related macular degeneration. Experimental Eye Research 76: 463–471.

Jones, B. W., Watt, C. B., Frederick, J. M., et al. (2003). Retinal remodeling triggered by photoreceptor degenerations. Journal of Comparative Neurology 464: 1–16.

Li, Z. Y., Kljavin, I. J., and Milam, A. H. (1995). Rod photoreceptor neurite sprouting in retinitis pigmentosa. Journal of Neuroscience 15: 5429–5438.

MacLaren, R. E., Pearson, R. A., MacNeil, A., et al. (2006). Retinal repair by transplantation of photoreceptor precursors. Nature 444: 203–207.

Marc, R. E., Jones, B. W., Anderson, J. R., et al. (2007). Neural reprogramming in retinal degenerations. Investigative Ophthalmology and Visual Science 48: 3364–3371.

Marc, R. E., Jones, B. W., Watt, C. B., et al. (2008). Extreme retinal remodeling triggered by light damage: Implications for AMD.

Molecular Vision 14: 782–806.

Marc, R. E., Jones, B. W., Watt, C. B., and Strettoi, E. (2003). Neural remodeling in retinal regeneration. Progress in Retinal and Eye Research 22: 607–655.

Margolis, D. J., Newkirk, G., Euler, T., and Detwiler, P. B. (2008). Functional stability of retinal ganglion cells after degenerationinduced changes in synaptic input. Journal of Neuroscience 28: 6526–6536.

Peng, Y-W., Hao, Y., Petters, R. M., and Wong, F. (2000). Ectopic synaptogenesis in the mammalian retina caused by rod photoreceptor-specific mutations. Nature Neuroscience 3: 1121–1127.

Seiler, M. J., Thomas, B. B., Chen, Z., et al. (2008). Retinal transplants restore visual responses: Trans-synaptic tracing from visually responsive sites labels transplant neurons. European Journal of Neuroscience 28: 208–220.

Stasheff, S. F. (2008). Emergence of sustained spontaneous hyperactivity and temporary preservation of OFF responses in ganglion cells of the retinal degeneration (rd1) mouse. Journal of Neurophysiology 99: 1408–1421.

Strettoi, E., Pignatelli, V., Rossi, C., Porciatti, V., and Falsini, B. (2003). Remodeling of second-order neurons in the retina of rd/rd mutant mice. Vision Research 43: 867–877.

Sullivan, R., Penfold, P., and Pow, D. V. (2003). Neuronal migration and glial remodeling in degenerating retinas of aged rats and in nonneovascular AMD. Investigative Ophthalmology and Visual Science 44: 856–865.

Varela, C., Igartua, I., De la Rosa, E. J., and De la Villa, P. (2003). Functional modifications in rod bipolar cells in a mouse model of retinitis pigmentosa. Vision Research 43: 879–885.

Glossary

Choriocapillaris – Vascular layer of the eye, located between the sclera and Bruch’s membrane. Ganglioside – A component of the cell membrane involved in signal transduction.

Neuroprotection – Halting apoptotic cell death of neurons through delivery of diffusible growth factors. Proteoglycans – A unique class of heavily glycosylated glycoproteins.

Tetraspanins – A family of membrane proteins with four transmembrane domains.

Introduction

The use of surgical procedures for the treatment of blinding conditions has a long history, most notably for cataract. In the modern era, refinements in ophthalmologic techniques and pharmacology have greatly expanded the range of available interventions for a host of ocular conditions. Nevertheless, conditions involving the loss of nonregenerating cell types remain a persistent therapeutic challenge. In the case of diseases involving the loss of corneal endothelial cells, tissue transplantation has proven successful, even though the limited availability of donor corneas restricts the use of this approach.

More problematic are diseases involving the loss of retinal neurons and retinal pigment epithelial (RPE) cells where few, if any, options are currently available to the treating clinician. As a group, diseases of this type are common and examples include age-related macular degeneration, retinal detachment, retinitis pigmentosa, as well as various forms of optic neuropathy, including glaucoma. In addition, common retinal vascular diseases such as diabetic retinopathy and vascular occlusive disease frequently involve retinal cell loss. Given the prevalence of visual disability resulting from these conditions, novel methods of both preserving and replacing retinal neurons are sorely needed.

Early Transplant Work

Over the last several decades, experimental work in rodents has made considerable progress in neuroprotection as well as cell and tissue transplantation in the setting of retinal cell

loss. Seminal work by LaVail and Steinberg demonstrated the power of growth factor-mediated neuroprotection for retinal degeneration, both hereditary and acquired. Work by Lund’s group showed that transplanted retinal tissue can, to a certain extent, engraft and integrate with the recipient visual system. Work by the Turner and Gouras laboratories showed that RPE transplantation can confer survival benefits to host photoreceptors following placement in the subretinal space. This last observation was made in dystrophic Royal College of Surgeons (RCS) rats and was subsequently extended to a variety of manipulations in this model. Taken together, these early studies provided compelling evidence of efficacy in mammals.

Despite the above advances, however, significant limitations remained, thereby encumbering further development of these otherwise promising strategies. In the case of neuroprotection, a major challenge has been to find a method of sustained intraocular delivery that is not neutralized due to the rapid degradation of peptide growth factors by endogenous proteases. For neural transplantation, a perplexing challenge has been to find a method of promoting regeneration across injury-induced gliotic barriers. Furthermore, RPE cell transplantation was challenged by the apparent reluctance of grafted cells to reconstitute an orthotopic epithelial monolayer on Bruch’s membrane. In all these cases, a compounding impediment to translation of these strategies was posed by the limited ability of the rodent eye to model an approach with tangible clinical relevance.

Within the past 10 years, new breakthroughs pertaining to the above challenges have emerged from the nascent field of stem cell transplantation, as will be discussed presently.

Neural Progenitor Cells

The first report of successful transplantation of stem-like cells to the retina was from Masayo Takahashi, then working in the laboratory of Fred Gage. The cells used were adult hippocampal progenitor cells (AHPCs), a clonally selected line of highly mitotic cells cultured from the brain of mature rats and genetically altered to express green fluorescent protein (GFP) as a reporter gene. This study showed a remarkable degree of apparently seamless integration of donor AHPCs into the immature retina of normal recipients. Although the cells fell short in terms of marker expression, their morphology, placement, and orientation were strikingly similar to resident retinal cell types.

In addition to working with Gage, we showed that AHPCs can also integrate into the diseased or injured retina of mature rats and that at least a degree of phenotypic marker expression was achievable, thereby underscoring the potential of using stem cells for cell replacement in the setting of common retinal disorders.

Together, these two studies with AHPCs stimulated considerable interest in stem cell research within the scientific community, particularly with respect to retinal applications. Initial work on cultured cells continued to focus on AHPCs as the first cell type with a demonstrated ability to integrate into the cytoarchitecture of the neural retina. These cells were shown to exhibit a number of nonimmunogenic properties and to express a number of important cytokines, as did similar brain-derived cells of human origin. Neural progenitor cells from the brains of human and GFP-mouse brain were compared in terms of surface marker expression. It was found that cultured neural progenitor populations consistently express 3-fucosyl-N-acetyl-lactosamine (CD15), GD(2) ganglioside, and the tetraspanin proteins CD9 and CD81 across species, whereas major histocompatibility complex (MHC) expression is more variable, specifically for class I antigens. Transplantation of the mouse cells showed that they exhibited characteristics of an immune-privileged cell type. Similar cells were later derived from the brain of the pig and cat.

Retinal Progenitor Cells

A significant limitation that emerged from the work with brain-derived progenitors was the difficulty in obtaining photoreceptor marker expression from these cells following engraftment in the retina. While it has been shown that recoverin expression can be induced under exceptional circumstances, it remained to be seen whether a stem-like cell could generate cells with outer segments that co-express multiple appropriate markers such as recoverin and rhodopsin. It was this challenge that led us to explore the possibility of deriving transplantable progenitor cell cultures from the mammalian neural retina.

Starting with newborn GFP-transgenic mice, we were able to grow cells from dissociated retinal tissue in the presence of mitogenic stimulation from epidermal growth factor (EGF). These cells were capable of expressing rhodopsin, either in culture under differentiation conditions or following engraftment in the host retina. We have obtained similar findings in work with porcine and human donor tissue of fetal origin. Following differentiation, either in vitro or in vivo, these cells have been shown to be capable of co-expressing rhodopsin and recoverin, consistent with rod photoreceptor phenotype. Retinal progenitor cells (RPCs) therefore represent a stem-like cell type that provides a potential source of new photoreceptors for

retinal repair in the setting of various forms of diseases and injuries.

As mentioned above, transplantation of mature RPE cells for cell replacement has been hampered by the apparent inability of these cells to recreate an epithelial sheet and adhere to Bruch’s membrane. In contrast, retinal and various types of neural progenitor cells have also shown an ability to integrate into the RPE monolayer, although it is not yet known if such cells can replace native RPE cells at the functional level. Recent work with pluripotent stem cells suggests that these represent a source for new retinal cells with functional properties.

Induced Pluripotent Stem Cells

A new development in stem cell biology that has changed the way we think about commitment and differentiation is a process known as direct reprogramming (Figure 1). Yamanaka and colleagues were the first to demonstrate this technique, in which somatic cells are reprogrammed to become pluripotent stem cells through the addition of several stemness transcription factors. They began with a type of skin cell known as a fibroblast, and inserted four transcription factors (Oct3/4, Klf4, Sox2, and c-Myc) using retroviruses. A subpopulation of the fibroblasts began expressing other markers of embryonic stem (ES) cells, such as nanog, and formed teratomas when transplanted into immunocompromised mice. Careful evaluation of the resulting cells revealed that they indeed had all the properties of ES cells, and have been termed induced pluripotent stem (iPS) cells.

There are several important ramifications of this work. First, this technology offers the promise of creating patient-specific stem cells. One could harvest skin cells from a donor, turn these cells into iPS cells, and subsequently differentiate this population into the cells of interest, which would then be transplanted back into the donor. Importantly, this would obviate the need for chronic immunosuppression and eliminate the risk of immunorejection of the grafted cells. Many of the ethical issues associated with ES cells would also be circumvented by using this technique, although as with most such developments, new and unexpected ethical questions arise. Thus far, no one has created a viable embryo through this technology, and indeed there are many biological hurdles standing in the way of this. However, as the theoretical possibility of using iPS cells for reproductive cloning exists, this work raises novel moral issues.

A number of technical barriers must be overcome before iPS cells can be useful for the treatment of disease. Foremost among these is the use of oncogenes (cancerassociated transcription factors) to reprogram fibroblasts. These genes often lead to tumorogenesis, and indeed scientists have seen cancerous growths from the

Transplantation

Photoreceptor production

Photoreceptor production conditions

Isolated fibroblast

+Sox2 +OCT4 +KLF4

Retinal progenitor cell production

RPC induction conditions

transplanted progeny of iPS cells. In addition, the use of retroviruses to deliver the four factors to the nuclei of the fibroblasts must be replaced by other means before using these cells in the clinic. It is worth noting, however, that in the months that followed the first publication of this technique, a flurry of scientific papers were published in which only two or three of the reprogramming factors were needed, and vectors other than retroviruses were used for gene insertion. This field is developing extremely rapidly, and it is likely only a matter of time until these methodological obstacles are overcome.

Stem Cells for Neuroprotection

In addition to cell replacement, stem-like cells have potential use in retinal neuroprotective strategies. We reported evidence that murine RPCs confer survival benefits to dysfunctional rods following transplantation to the retina of rhodopsin double knock-out mice. This result indicated that RPCs, or their more differentiated progeny, possess an inherent neuroprotective influence that is revealed in this setting. Interestingly, a similar finding was obtained in retinal dystrophic mice in work with a very different type of stem cell derived from the bone marrow.

Elucidating the mechanisms underlying such effects remains a subject of active interest; however, an alternative

method for inducing neuroprotection via stem cells is to modify them genetically so as to over-express the therapeutic gene of interest. Gamm and colleagues have shown that brain-derived progenitor cells modified to ever express a GDNF transgene were capable of rescuing photoreceptor cells in the RCS rat following transplantation.

Retinal Transplantation

As suggested above, to achieve functional repair in patients afflicted with retinal degenerative disorders, there is an urgent need to reconstruct, via cellular replacement, the damaged or lost layers of the retina. During the past 30 years attempts have been made in order to achieve retinal regeneration via transplantation of developing donor material. Although significant progress and invaluable information have been acquired, visual restoration using this technique has not been achieved. For instance, although embryonic rat retina was shown to survive without immune rejection, develop normally, and continue to respond to light for up to 3 months post-transplantation, extensive host–donor integration was not identified. Similarly, comparable experiments performed in pig models of retinal dystrophy showed that although healthy retinal tissue from fetal donors survived and maintained proper laminar structure and cellular

organization for up 6 months post-transplantation, this tissue did not integrate with the host retina, and as such, did not aid in visual restoration. These findings collectively suggest that the eye is an amenable site for transplantation; however, the proper donor material or extracellular conditions have yet to be met.

Transplantation Strategies

Stem/progenitor cell transplantation as a means of inducing tissue reconstruction and functional regeneration has garnered extensive interest in the field of regenerative medicine. Unlike the solid tissue transplants mentioned above, stem cells have the ability to integrate within host tissue and develop into retinal specific cell types. For instance, in 2004, we were able to show that a subset of grafted RPCs gave rise to mature retinal ganglion and photoreceptor cells following transplantation. Since then, numerous studies reporting varying degrees of success have utilized an assortment of different cell types, including the fate-restricted photoreceptor precursor and the pluripotent ES cell. Regardless of the cell type used, efficient delivery methods are required. Traditionally, the two transplantation techniques most extensively utilized for cell delivery were vitreal cavity and subretinal space injections. Regardless of species, vitreal cavity injections generally involve insertion of a needle through the pars plana into the vitreal space, where in the case of mice, approximately 1 ml of cell suspension as a bolus is injected (Figure 2(a)). The subretinal space, unlike the vitreous, is actually a pseudo-space that is created upon detachment of the retina from the underlying RPE layer. Cellular injections into this space are performed by one of the two techniques. The first, which is very similar to that carried out during vitreal injections, involves entering the eye through the pars plana and inserting a needle beneath the retina via a precut hole, or retinotomy. Through this hole, fluid, most often saline, is injected, creating a retinal detachment and bleb into which the cell of choice can be delivered (Figure 2(b)). This approach generally requires a larger vitreal space in which to work and is often performed in larger models of retinal disease, such as the pig and human. The second technique, which is more often employed in smaller organisms such as rodents, is performed by making an incision through the sclera (sclerotomy), choriocapillaris, Bruch’s membrane, and overlying RPE, through which a needle is inserted and saline is injected. This again creates a retinal detachment/bleb into which cells can be delivered (Figure 2(c)). The reasons for choosing one approach over the other are vast and may range from personal preference to surgical competence. However, more practical reasons for choosing vitreal or subretinal approaches for stem cell transplantation exist, including the type of injury and cell/retinal layer being targeted.

For instance, subretinal transplantation would be more beneficial for targeting the retinal photoreceptor layer in retinal degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), while vitreal transplantation would be more beneficial for targeting the inner nerve fiber layer in diseases such as glaucoma and various other optic neuropathies. Tailoring the transplantation approach to the specific disease at hand allows for larger numbers of cells to be provided to the area required. For instance, performing vitreous cavity injections of retinal stem cells as a treatment regime for photoreceptor cell replacement requires that the cells migrate through the entire thickness of the remaining retina before arriving at the desired location. With an increased distance for cellular migration, there is a greater chance that cells will take up residence in unwanted retinal layers, thus decreasing the chance that they will actually reach the appropriate target.

Polymer Substrates

Despite choosing the correct transplantation technique, the use of bolus cell injection as a delivery method is actually quite inefficient and typically results in massive cellular efflux, low cellular survival, and poor integration. For example, it has previously been suggested that as little as 0.1% of transplanted retinal stem cells actually survive for an extended period of time following traditional bolus injection. Issues such as these, which hinder functional regeneration, are largely accounted for by the lack of cellular support and trauma associated with the injection processes. For instance, in a recent publication we were able to show that the shearing forces placed on retinal stem cells by passing them through the typical glass injection needle resulted in nearly 60% cell death at 3 days post-passage. When the glass injection needle was replaced with a large bore pipette, cell death was reduced to less than 2%. However, even if a large bore pipette could be used during the transplantation process, the fact remains that a large portion of the cells injected would still be lost to efflux via the implantation port. This is especially true when attempting to place cells into the subretinal space through an opening in either the retina or sclera. One approach that we have developed in an attempt to circumvent these issues is to apply tissueengineering techniques focused on the use of biodegradable polymer scaffolds as cell delivery vehicles. In one study we were able to show that the transplantation of retinal stem cells on a biocompatible poly(lactic-co-glycolic acid) (PLGA) polymer resulted in greatly reduced cell death and prevented leakage and migration of retinal stem cells away from the transplantation site. This resulted in significantly improved cellular integration when compared to bolus cell injections.

Inhibitory Barriers

Although we have developed an efficient means of cell delivery, in order to achieve functional regeneration via

retinal reconstruction/repopulation, a further increase in cellular integration and axonal extension/synapse formation is required. As suggested above, a major contributor to the lack of functional regeneration in the face of efficient

cell delivery is the presence of an inhibitory extracellular environment. Unlike the peripheral nervous system (PNS), the central nervous system (CNS) is stricken with an abundance of myelin-associated extracellular matrix (ECM) proteins, namely MAG, Nogo, and Omgp, which are well known for their ability to inhibit process extension and functional regeneration. The optic nerve, like other CNS locations, is laden with these inhibitory proteins, and as such, their chemical and/or enzymatic neutralization has been shown to significantly enhance retinal ganglion cell axon extension and optic nerve regeneration. Thus, in order for stem cell therapies to be used as an effective treatment for diseases such as glaucoma, which affect the retinal ganglion cell layer and optic nerve, inhibition of these proteins will ultimately be required.

Unlike the optic nerve, the retina is void of myelin, oligodendrocytes, and their associated inhibitory ECM molecules. Thus, these factors are not a concern when attempting to stimulate regeneration in retinal degenerative diseases such as RP and AMD, both of which predominantly affect the retinal outer nuclear layer. Why then are attempts at stimulating retinal regeneration via stem cell transplantation still largely unsuccessful? The lack of functional integration following transplantation is in large part due to the presence of an inhibitory injury induced glial scar that forms during reactive gliosis. In the retina, reactive gliosis is predominantly characterized by Mu¨ller glial activation, as indicated by upregulation of intermediate filament proteins such as glial fibrillary acidic protein (GFAP) (Figure 3(a), red) and the projection of processes from their original location (forming the outer limiting membrane) into the subretinal space. Here, these processes proceed to form a dense fibrotic barrier that contains a variety of growth inhibitory ECM/adhesion molecules,

which include the chondritin sulfate proteoglycan (CSPG) Neurocan (Figure 3(a), blue) and the hyaluronan-binding glycoprotein CD44 (Figure 3(b), red). Both Neurocan and CD44 have previously been shown to function as chemical inhibitors to axon growth and cellular migration, thus preventing regeneration and functional synapse formation. A variety of approaches have been taken in an attempt to remove these inhibitory ECM molecules, including enzymatic degradation of the proteins themselves. One such enzyme that we have utilized for this purpose is the matrix metalloproteinase 2 (MMP2). MMPs are well known for their ability to degrade a variety of ECM and cell adhesion molecules, including CD44, the CSPGs, and the aforementioned myelin-associated inhibitors. MMP2, in particular, has been shown to cleave both CD44 and Neurocan, thus releasing their negative hold on axonal extension and cellular migration. In a recent study, we discovered that endogenous MMP2 induction resulted in glial barrier-associated CD44 and Neurocan degradation at the outer limits of heavily scarred degenerating retinas, subsequently stimulating integration and synapse formation between the host and healthy transplanted tissue grafts. Conversely, chemical and/or genetic inhibition/removal of MMP2 was shown to completely abolish stem cell migration and retinal repopulation. In light of these findings, we have developed a biodegradable PLGA polymer scaffold that, as in previous permutations, is capable of efficient cell delivery and also possesses the ability to provide a sustained controlled release of active MMP2 following degradation (MMP2PLGA). Subretinal transplantation of this polymer in conjunction with retinal stem cells as a composite graft has resulted in efficient glial barrier degradation and enhanced stem cell integration. This new polymer scaffold will undoubtedly act as an invaluable transplantation tool

for targeted repopulation of the retinal and restoration of vision.

Conclusions

Regardless of the source of the transplanted material, retinal transplants aim to rescue or replace injured photoreceptors. It is likely that each of these objectives will require different cell types as donor tissue. For example, rescue of diseased rods may be accomplished by delivering nonengrafting mesenchymal stem cells, or perhaps with engrafting neural stem cells. In these cases, rescue would be achieved by the delivery of growth factors such as glial cell-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) in a nonspecific fashion, possibly to cells other than photoreceptors (e.g., Mu¨ller glia). Engraftment and long-termed survival is a requirement if rescue of the diseased photoreceptors is to be prolonged. In contrast, repair through reconstruction involves a more complicated and indeed more difficult set of concerns. Cell replacement not only requires engraftment and long-termed survival, but also necessitates differentiation.

A cell type that can differentiate into fully mature, functional photoreceptors narrows the field of players somewhat. While a hematopoetic or stromal stem cell may by somewhat ill-suited to such a task, retinal stem cells or ES cells may be ideal candidates.

See also: Coordinating Division and Differentiation in Retinal Development; Injury and Repair: Retinal Remodeling; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Retinal Ganglion Cell Apoptosis and Neuroprotection; Retinal Histogenesis; Zebra Fish–Retinal Development and Regeneration.

Further Reading

Caroni, P. and Schwab, M. E. (1988). Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading. Journal of Cell Biology 106: 1281–1288.

Kajita, M., Itoh, Y., Chiba, T., et al. (2001). Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration.

Journal of Cell Biology 153: 893–904.

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Relevant Websites

http://www.blindness.org – Foundation Fighting Blindness. http://www.hsci.harvard.edu – Harvard Stem Cell Institute. http://webvision.med.utah.edu – John Moran Eye Center, University of

Utah, Webvision.

http://www.schepens.harvard.edu – Schepens Eye Research Institute, Michael Young.