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Stem Cells: A Future Glaucoma Therapy? THOMAS V JOHNSON and KEITH R MARTIN

Summary As with many new approaches in medicine, there is the danger that advocates of stem cell research may promise more than can conceivably be delivered in a realistic timeframe, leading to disappointment, disillusionment and a loss of confidence in the potential of the approach. We must acknowledge that the technology and understanding needed to achieve functional improvement in glaucoma using stem cell therapy is in its infancy. However, in the absence of any other treatment with the potential to restore vision in glaucoma, further research on the therapeutic use of stem cells in glaucoma remains both justifiable and eagerly awaited.

Introduction Stem cells are a current focus of intense scientific and clinical interest, particularly in the central nervous system (CNS) where inherent repair is inadequate and functional damage is often permanent. The prospective therapeutic power of stem cells lies in their ability to generate new cells of many types and to effect tissue repair. By definition, a stem cell is multipotent, with the capacity to self-renew and to produce daughter cells capable of differentiating into multiple mature cell types (Fig. 63-1). However, progenitor cells, which possess the ability to generate a more limited range of adult cell types, may also contribute to tissue repair. Thus, stem and/or progenitor cells offer new hope for treating historically incurable diseases, such as glaucoma, via the selective replacement of degenerated cells to restore function.1–3 In addition, certain stem cell classes may confer neuroprotection to endogenous tissue without overt cell replacement. This chapter will discuss research relevant to the use of stem cell therapy for glaucoma treatment. Potential stem cell sources and therapeutic targets are considered, and obstacles to clinical translation are discussed.

Objectives for Stem Cell Therapy in Glaucoma RETINAL GANGLION CELL REPLACEMENT The clinical endpoint for uncontrolled glaucoma is total visual field loss as a result of progressive RGC death. Despite 642

aggressive treatment, a significant proportion of glaucoma patients experience considerable visual field reduction within their lifetimes. Theoretically, the most direct approach to treating these patients would be to stimulate endogenous repair mechanisms within the retina. In fish and amphibians, retinal regeneration is an automatic process that proceeds via differentiation of ocular stem cells located in the ciliary marginal zone. In adult mammals, however, retinal regeneration after injury or in neurodegenerative disease does not occur; retinal progenitor cells that have been identified in vitro appear to remain quiescent in vivo. Within the adult mammalian CNS, neurogenesis is limited to discrete regions (for example the hippocampus) and outside these areas the environment is notoriously resistant to the generation and integration of new neurons. One potential application for stem cells is to circumvent the human retina’s inability for self-repair by replacing neurons via transplantation. Proof of principle for this approach has been obtained in the outer retina where functional photoreceptor replacement was achieved using cells derived from developing retina,4 embryonic stem (ES) cells,5 and induced pluripotent stem (iPS) cells6,7 (Fig. 63-2). In contrast, RGC replacement has been more difficult to achieve. Retinal and neural precursor cells derived from embryonic stem cells, when transplanted into the eye, can migrate into the retina and express markers of mature retinal neurons including RGCs.8,9 Transplanted fetalderived hippocampal progenitors also demonstrate the ability to localize to the RGC layer, from whence they may extend neurites into the inner plexiform layer and towards the optic nerve head.10 Despite these initial successes, terminal differentiation and functional integration of stemcell-derived RGCs has not been convincingly demonstrated in the adult mammalian retina. Furthermore, the challenge of RGC regeneration does not end at the optic disc. In order to achieve complete functional repair in glaucoma, transplanted cells would not only need to integrate into the existing retinal circuitry but re-establish functional connections with target neurons in the brain. This would involve the extension of RGC axons through the optic nerve to the lateral geniculate nucleus (a matter of several centimeters in the human) and the precise regeneration of the retinotopic map. Furthermore, new axons need to be myelinated within the optic nerve to allow signal conduction at the appropriate velocity. Obviously, significant progress must be made before stem cell therapy can be used to repair the visual pathway so comprehensively. However, the hope is that even a small functional benefit in patients with severe visual loss could translate into a meaningful improvement

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63  •  Stem Cells: A Future Glaucoma Therapy? Brain: subventricular zone of lateral ventricle, hippocampus

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Increasing restriction or differentiation B Figure 63-1  (A) (1) Embryonic stem (ES) cells are isolated from the inner cell mass of the developing blastocyst and can be expanded indefinitely in culture. ES cells can also be manipulated in vitro to limit their fate and trigger their differentiation into potentially all cell types. (2) During development, stem cells with the potential to differentiate into neural cells can be isolated from many tissues including the brain, retina, and umbilical cord. (3) In the adult body, stem cell populations can be found within various tissues and many of these stem cells, including those from the CNS, blood, bone marrow and skin, are reportedly potential sources of neural precursor cells. (4) Stem cells can be found throughout development from the zygote to the adult; however, the potential of stem cells is inversely related to their stage of differentiation. Thus, stem cells isolated from the zygote or blastocyst display the greatest plasticity and have the potential to generate all cell types. In contrast, stem cells isolated from the adult animals are more restricted and can normally only generate cell components of the tissue type they were isolated from. In addition, stem cells may divide asymmetrically (as opposed to symmetrically to generate more stem cells) to produce progenitor cells, which are subject to further restrictions. These, in turn, differentiate into blast cells and finally mature cells, such as neurons or glia. (B) Induced pluripotent stem (iPS) cells can theoretically be generated from any cell type in the body. Donor cells, for example skin fibroblasts, are isolated and then reprogrammed by the forced expression of transcription factors associated with the control of pluripotency. Initial protocols involved retroviral transduction with the genes encoding Oct3/4, Sox2, c-Myc, and Klf4, but newer techniques to induce pluripotency with enhanced efficiency continue to be developed.

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Figure 63-2  GFP-positive P1 retinal progenitor cells integrated into the outer nuclear layer of wild-type P1 littermate recipients three weeks after sub-retinal transplantation and developed morphological features typical of mature photoreceptors. Low- (A) and high- (B,C) magnification images of P1 donor cells integrated into adult wild-type recipients. Examples of cells with rod (open arrow) and cone (filled arrow) morphologies are shown. INL, inner nuclear layer; IS, inner segments; OS, outer segments. Scale bars, 10 m. (Reprinted from MacLaren RE, Pearson RA, MacNeil A, et al. Retinal repair by transplantation of photoreceptor precursors. Nature 2006; 444(7116):203–7.)

in quality of life. Whether this hope is realistic remains to be seen.

RGC Neuroprotection While unlikely to restore vision that has been lost, neuroprotective strategies could be used adjunctively with IOP control to slow optic nerve degeneration and more effectively preserve residual vision. Research has identified multiple target pathways that might promote RGC survival including neurotrophic factor delivery, modulation of inflammation, buffering of oxidative stress, and attenuation of excitotoxicity. Several classes of cells have been shown to modulate these pathways and confer a survival benefit when transplanted into sites of CNS degeneration, including in experimental models of glaucoma.1,11,12 Stem cells of mesenchymal and glial origin have been a particular focus of this approach because of their natural neuroprotective properties.13–16 Genetic engineering techniques have broadened the potential pool of cell sources, by providing a means to generate grafts with specific neuroprotective activity [for instance, secretion of targeted neurotrophic factors],17–19 raising the possibility that

transplantation-mediated neuroprotection for glaucoma need not be limited to stem and progenitor cells.

OPTIC NERVE HEAD RESTORATION The optic nerve head (ONH) is the origin of the optic nerve within the eye. Important changes to the ONH and the surrounding extracellular matrix occur in glaucoma. Structural alterations include excavation of the optic disc, loss of elastin fibers and changes in collagen regulation.20 In addition, glaucoma has been found to stimulate activation of resident astrocytes, increase nitric oxide secretion and induce vascular changes.21 These changes place biomechanical, toxic, and ischemic stress on RGCs. Stem cell therapy directed toward repairing the structure and function of the ONH has been proposed as a possible way to slow disease progression. For example, given that fibroblasts are responsible for maintaining the ONH extracellular matrix, it is conceivable that fibroblast precursor cells could modulate the environment of the glaucomatous ONH to enhance RGC survival. Indeed, such cells have been shown to benefit some models of wound healing. It has been noted that

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Figure 63-3  Transplanted olfactory ensheathing cells (OECs, green) migrate into the optic nerve to form a dense mass occupying most of the width of the optic nerve head to a depth of up to 0.5 mm at 4 weeks after transplantation. Association with red (neurofilament-expressing) RGC axons appears yellow at higher magnification. v: vitreal surface; arrowheads in (A) highlight OECs in the subretinal space; small arrows in (E) point to solitary highly flattened cells in the outer plexiform layer; ip: inner plexiform layer; (E) shows OECs in stratum opticum moving towards the optic nerve head (arrow); (F) and (G) depict OECs in the stratum opticum (so) ensheathing RGC axons (yellow fluorescence). Scale bars: 100 µm (A, B, E), 50 µm (C, F, G), 20 µm (D). (Reprinted from Li Y, Li D, Khaw PT, Raisman G. Transplanted olfactory ensheathing cells incorporated into the optic nerve head ensheath retinal ganglion cell axons: possible relevance to glaucoma. Neurosci Lett 2008; 440(3):251–4.)

following transplantation, olfactory ensheathing cells tend to migrate to the optic nerve head where they ensheath RGC axons,22 though the functional significance of this observation remains to be determined (Fig. 63-3).

TRABECULAR MESHWORK RESTORATION Glaucoma often involves disruption to aqueous outflow through the trabecular pathway, which contributes to elevated IOP. Outflow resistance is increased by a reduction in the clearance of fibrillar material and sheath-derived plaques by trabecular meshwork cells.23 In addition, agerelated loss of trabecular meshwork cells seems to be exaggerated in glaucoma. Restoration of trabecular meshwork function is, therefore, also a potential target for stem cell transplantation. Progenitor cell populations isolated from the trabecular meshwork can be expanded in culture,24 and stem cells isolated from the trabecular meshwork have been proposed to play a maintenance role within that tissue.25 Trabecular meshwork stem cells can be expanded and differentiated into mature trabecular meshwork cells with phagocytic properties in vitro,26 raising the possibility that transplantation of these cells into the anterior chamber could improve conventional outflow in glaucoma patients.27

CONJUNCTIVAL RESTORATION AND GLAUCOMA FILTERING SURGERY A relatively common complication of glaucoma filtering surgery is the development of a thin, cystic bleb at the surgical site, which increases the risk of infection and hypotony.

Although good surgical technique and the application of antiproliferative agents to a wide treatment area can dramatically reduce the incidence of such leaking blebs, this surgical complication remains a significant problem for many ophthalmologists. One emerging technique to repair leaky blebs is through ocular surface grafting of tissue equivalents. Tissue equivalents can be generated by the isolation and in vitro expansion of conjunctival specimens isolated from the superior fornix, which contains a population of conjunctival progenitor cells. Engraftment of conjunctival progenitor cells to repair and replace damaged conjunctival tissue around the bleb have been found to reduce leakage.28 Expansion of this technique may help to reduce a major complication of glaucoma surgery.

Sources of Stem Cells for Transplantation Therapy EMBRYONIC STEM CELLS Embryonic stem (ES) cells are derived from the inner cell mass of the developing blastocyst and are capable of indefinite self-renewal in culture (Fig. 63-1A.1 and 63-1A.4). They are also pluripotent, meaning that they can generate all cell types of the body. As such, ES cells are a powerful transplantation source for stem cell therapy and, consequently, are the subject of intense research in many different medical fields, including neurodegeneration. To date, transplantation of ES cells into the mammalian retina has yielded mixed results. While transplantation of

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undifferentiated ES cells has demonstrated some potential for survival within the retina, it has not achieved robust retinal integration or neuronal differentiation, and may be tumorigenic.29 Lineage-restriction of ES cells towards a particular cell class prior to transplantation may offer a more controlled cell source. In the context of glaucoma, differentiation of ES cells in vitro has yielded both glial and neuronal cell types.30–32 Differentiation may be accomplished in vitro by mimicking the molecular events that occur during development via exposure of cells to signaling molecules. Thus, ES cells have been differentiated into retinal precursor cells that express markers of retinal development including Pax6, Lhx2, Rx/Rax and Six3/6.30,32 Furthermore, terminal differentiation of these cells can produce progeny with characteristics of various mature retinal neurons. This includes RGC-like cells as evidenced by the expression of RGC markers (HuC/D, Neurofilament-M and Tuj-1) and the generation of glutamate-induced calcium fluxes.30,32 Ectopically induced gene expression offers an alternative technique to manipulate ES cell fate in vitro. This approach has yielded retinal progenitor cells with the ability to produce RGC-like cells as assessed phenotypically and electrophysiologically.31 Recently, the ability of mouse and human ES cell aggregates to form self-organizing three-dimensional optic cups Day 1

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in culture has been characterized.33,34 Tissue morphogenesis in this system mimics the molecular events that occur during development where invagination of a spherical optic vesicle gives rise to a bi-layered cup consisting of retinal pigment epithelium underlying a neural retina (Fig. 63-4). The neural retinae of these eye cups appear able to generate multiple classes of retinal neuron, including RGCs. This technique will undoubtedly provide a useful model for studying retinal development, physiology, and disease, and might eventually provide an avenue for the generation of tissue for therapeutic transplantation. The use of ES cells has important limitations. The ethical issues surrounding the isolation of ES cells from human embryos are well publicized and a matter of intense debate. In addition, ES cell transplants are necessarily allogeneic and therefore carry the risk of graft rejection. This is particularly important when the transplant is placed in a location such as the vitreous, which is less immune privileged than the brain. Furthermore, tissue sources are limited and ES cell transplants potentially carry a risk of malignant transformation.

Induced Pluripotent Stem Cells Induced pluripotent stem (iPS) cells were first generated from mouse and human fibroblasts via cellular

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B Figure 63-4  A schematic diagram depicting self-organization of eye cups from embryonic stem cells expressing Rx-GFP is presented in (A). Optical cross-sections of Rx–GFP images (top) and laser-scanned bright-field images (bottom; dotted lines indicate the basal side) are shown in (B) and depict the four phases of the invagination process, which include spherical vesicle formation, distal flattening, narrow-angled flexures, and apically convex invagination. (Adapted and reprinted from Eiraku M, Takata N, Ishibashi H, Kawada M, Sakakura E, Okuda S, Sekiguchi K, Adachi T, Sasai Y. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 2011; 472(7341):51–6.)

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reprogramming induced by retroviral transduction of the transcription factors Oct3/4, Sox2, c-Myc, and Klf4 (Fig. 63-1B).35 Since then, numerous advances have improved the efficiency and safety of cellular reprogramming techniques.36 Like ES cells, iPS cells are pluripotent and may represent a powerful new approach to stem cell transplantation therapy as well as disease modeling. While the benefits of iPS cells include their relative ease of isolation and expansion, as well as a possibility for autologous transplantation and fewer ethical implications, there is increasing awareness that inherent differences between ES and iPS cells exist. Continued research will be necessary to determine the optimal approaches to utilizing these cell types for therapeutic purposes. Protocols have been developed to differentiate iPS cells into retinal neurons including cells with features of RGCs. Treatment of iPS cultures with soluble signaling factors and forced overexpression of RGC-specific transcription factors results in progeny that express mature RGC markers Thy1, Brn3, and Islet-1.37 RGC-like cells derived from iPS cells have also been shown to possess target selectivity for superior but not inferior colliculus explants in vitro.38 Of note however, these cells integrate poorly into the retina following intravitreal transplantation. Therefore, as for other stem cell sources, the use of iPS cells for RGC replacement will likely require not only continued research aimed at developing more robust differentiation protocols, but also methods for improving graft integration into the retinal circuitry, as discussed below.

SOMATIC STEM CELLS Adult tissues that undergo constant turnover possess discrete niches where their stem cells, termed somatic stem cells, reside and divide to generate cells required for tissue maintenance (Fig. 63-1A.3). This has been particularly well characterized in organs such as the skin, intestinal tract, and bone marrow. In vivo, somatic stem cells are restricted, perhaps by their environment, to the production of cell types native to the tissue in which they dwell. Their applicability for cell replacement therapy in other tissue types needs to be established. Interestingly, somatic stem cells derived from the blood, skin, bone marrow, and umbilical cord have been reported to be capable of neuronal differentiation. For instance, bone-marrow- and blood-derived mesenchymal stem cells have been shown to express neural cell markers such as nestin, βIII-tubulin and GFAP following neural induction.39 Intraocular transplantation of mesenchymal and hematopoietic stem cells has been shown to improve outcomes in various preclinical models of retinal neurodegenerative disease. In these cases however, the mechanisms responsible are more likely to have involved neuroprotection as opposed to functional integration, as retinal neuron replacement by somatic stem cells has yet to be demonstrated. Somatic stem cells are an attractive transplantation source for stem cell therapy for a number of reasons. Significantly, many types of somatic stem cells can be relatively easily obtained from the recipient prior to transplantation. This would facilitate autologous grafting and avoid the ethical concerns surrounding other sources, such as ES cells. In addition, an autologous source avoids graft

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rejection and negates the need for immune suppression. Such benefits sustain research into the difficult task of determining how to reprogram somatic stem cells in order to exploit them for repair of different tissue types or to maximize their ability to protect endogenous neural tissues.

NEURAL STEM CELLS Neural stem (NS) cells are somatic stem cells that give rise to neurons, astrocytes, and oligodendrocytes in the CNS. A number of phenotypic markers are used to identify NS cells including nestin (an intermediate filament), Sox2, Notch, and CD133.40 NS cells can be isolated and expanded in vitro from various neural tissues throughout development, from embryonic stages through to adulthood, although their proliferative potential decreases with age. The developing cerebral cortex, midbrain, and retina have been identified as sources of fetal NS cells (Fig. 63-1A.2). In adult animals, NS cells are commonly isolated from the subventricular zone of the lateral ventricle. In this region in vivo, NS cells continually proliferate in order to repopulate olfactory bulb neurons. The hippocampal dentate gyrus also exhibits extensive neurogenesis throughout life and NS cells may also be isolated from this CNS region. Outside these welldocumented adult neurogenic regions, the presence of NS cells is more controversial; however, it has also been reported that NS cells may be cultured from various cortical regions and the spinal cord. Despite demonstrated potential in vitro, so far there is little direct evidence that transplantation of NS cells or their progeny into the eye can achieve functional improvement. This is especially true of adult NS cells, which do not assimilate into healthy adult retinas but can show a modest level of retinal integration in damaged tissue with concomitant expression of early neuronal cell markers.41 In vitro modulation of differentiation conditions may be necessary to guide NS cell differentiation toward a retinal fate. Indeed, treatment of cultured NS cells with TGF-β3 can induce opsin expression and a photoreceptor-like phenotype. However, in vitro differentiation of NS cells into mature RGCs has yet to be achieved.

ADULT OCULAR CELLS The adult eye contains a number of potential somatic stem cell niches. Cells located in the basal limbal area undergo continuous differentiation and migration to support corneal epithelial maintenance. As mentioned above, trabecular meshwork stem cells are a focus of ongoing research and may play a future role in the control of aqueous humor dynamics for IOP reduction in glaucoma. A number of cell types have also been isolated from the posterior segment that exhibit neural potential in vitro, although these cells appear quiescent in vivo.42 Progenitor cells have been isolated from the pigment epithelium at the ciliary margin and induced to differentiate into neural retinal cell types.43 Proliferative cells capable of generating neural retinal cells have also been cultured from the pigmented ciliary body44 and the pigmented iris epithelium.45 Interestingly, the pigmented iris epithelium shares developmental origins with the pigmented ciliary body and neural retina. The role that these cells might play in the treatment

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of retinal disease either by transplantation or stimulation of endogenous regeneration is the subject of ongoing research.46 Müller glia within the neural retina may also possess the ability to produce new neuronal cells. In chicks, injury induces Müller cells to re-enter the cell cycle in order to proliferate and de-differentiate; that is they stopped expressing mature Müller cell proteins and instead expressed classical markers of developmental retinal progenitor cells such as Pax6 and Chx10. Such Müller-cell-derived progenitor cells can generate neuronal-like cells, which express Hu, calretinin or cellular retinoic acid-binding protein. Interestingly, induction of RGC-specific death promotes the synthesis of cells morphologically similar to RGCs that express RGC-type cell markers such as Brn3, Islet 1, RPF1, and neurofilament-M.47 This observation suggests that lesions which target specific neuronal classes may provoke an environment that is permissive for regeneration of that particular cell type. In adult mammals, Müller cells also display some stem-cell-like properties. For example, when cultured in vitro they can self-renew and differentiate into phenotypically neural cell types. Furthermore, when transplanted in vivo they can generate new neurons within lesioned retinas.48 Recent evidence reveals that Müller cells can also be stimulated to divide in vivo following the intraocular application of appropriate mitogenic factors, which hints at the possibility of inducing endogenous retinal repair. As such, the potential of Müller cells for retinal repair appears promising. In addition, using adult ocular stem cells as a source for stem cell therapy could facilitate autologous transplantation with the added benefit that these cells may be inherently restricted to an ocular fate.

research on this topic is required to determine the optimal transplantation technique.

MORPHOLOGICAL INTEGRATION One of the first tasks transplanted cells must accomplish is morphological integration into the host tissue (Fig. 63-5), a feat that appears to become more difficult with increasing host age. In studies on the Brazilian opossum, for example, exogenous stem cells readily integrated into the retina of early postnatal animals, but this receptivity decreased as the eye developed. By 35 days after birth, little incorporation of transplanted cells was observed.49 Similarly, integration into the intact rodent retina occurs much more readily in young animals than in adults, although some cells can survive in the posterior segment of adult eyes for weeks.50 This lack of integration may be partially overcome by injuring the adult retina.41 Many different disease models, including mechanical injury to the retina or optic nerve, chemical toxicity, ischemia and inherited retinal degeneration, have all demonstrated an ability to enhance graft integration into adult retinas. Furthermore, degeneration of a specific neuronal type tends to trigger donor cell migration to the site of neurodegeneration, an effect that has also been observed in the brain. Thus, it appears that endogenous signals from the injured retina play a key role in determining the potential for integration of engrafted cells. Other barriers to the migration of transplanted cells into the retina include reactive gliosis51,52 and components of the extracellular matrix.53,54

Strategies for Stem Cell   Therapy in Glaucoma TRANSPLANTATION The possible replacement of photoreceptors by transplantation in diseases such as macular degeneration and retinitis pigmentosa has received much recent attention. Restoration of photoreceptors by transplantation has several advantages not shared by RGC transplantation. In particular, the only afferent input to photoreceptors is light, and the target neurons to which connections must be established are in close proximity within the retina. In contrast, the complete functional replacement of RGCs requires the establishment of appropriate local afferent connections within the inner retina and also long-range efferent connections to the brain. Another important consideration for transplantation approaches is graft location. Some studies have suggested that the subretinal environment favors the selective differentiation of grafted cells into a photoreceptor phenotype.8 Subretinal transplants enjoy a more immuneprivileged milieu than those in the vitreous, and subretinal placement also ensures that the engrafted cells are held in close proximity to the retina. Alternatively, intravitreal introduction theoretically provides the transplanted cells with direct access to the inner retina. This route may, therefore, prove to be more appropriate for glaucoma-directed therapy, as opposed to outer retinal therapy. Further

Figure 63-5  Neural progenitor cells transplanted into a host retina integrate throughout all layers but mainly localize to the nuclear layers. Neurites extend into the plexiform layers and respect the ‘on’ and ‘off’ sublamina. RPE = Retinal pigmented epithelium; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. (Reprinted from Klassen H, Sakaguchi DS, Young MJ. Stem cells and retinal repair. Prog Retin Eye Res 2004; 23(2):149–81.)

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NEURONAL DIFFERENTIATION Transplantation of highly undifferentiated stem cells into the intact or lesioned mammalian retina has yielded limited success with regard to the production of appropriate retinal cell types. This may be due, in part, to molecular suppression of RGC differentiation in the mature retina. In addition, highly undifferentiated cells, such as ES cells, may not detect all of the necessary RGC differentiative cues due to an absence of appropriate receptor expression. In contrast, directing differentiation toward a particular cell lineage prior to transplantation may enhance generation of the desired cellular identity. Retinal-specific neural progenitors with RGC characteristics have been derived from ES and iPS cells in vitro.30–34,37,38 Continued work should aim to optimize protocols for the efficient generation of functional RGCs from suitable precursors in sufficient quantities for therapeutic transplantation, as has been developed for other retinal cell types. The ideal stage of differentiation for cellular transplants remains uncertain and it is likely that a compromise between plasticity and maturity will be required. It may not be necessary to create fully differentiated RGCs in vitro for transplantation as it is possible that some of the required signals may remain endogenous to the adult retina, allowing less mature cells to be used. For instance, grafted cells that migrate to different layers of the retina tend to express markers specific to local cell types. This suggests that differentiation may be modulated by endogenous factors within the retina. A key to directed in vivo differentiation may be cell-specific depletion, and subsequent induction of a microenvironment conducive to the generation of that particular cell class. Whether the glaucomatous retina can provide the necessary cues to guide the migration, differentiation and integration of transplanted cells remains to be established.

FUNCTIONAL INTEGRATION To our knowledge, the establishment of functional synapses by grafted cells in the glaucomatous eye is yet to be demonstrated. Interestingly, hippocampal-derived neural precursor cells have been observed to populate all retinal layers when transplanted into the vitreous of neonatal rats suffering from inherited photoreceptor degeneration (Fig. 63-5).10,49 In the ganglion cell layer, new neurons sent neurites into the inner plexiform layer where they associated closely with host dendrites, although functional synapse formation was not confirmed (Fig. 63-6). Grafted cells in the ganglion cell layer also extended processes through the nerve fiber layer and into the optic nerve head. Despite these promising results in neonatal rats, very little retinal integration of hippocampal-derived neural precursor cells was observed in adult animals and this was only achieved following injury. Thus, how to achieve an environment conducive to dendrite/axon extension and connection in the adult retina remains to be elucidated. This is not to say that functional connectivity is impossible. Studies of photoreceptor replacement have demonstrated an impressive level of functional integration by transplanted photoreceptor precursors in the adult retina.4,55 Stem cell therapy in this context provided measurable functional benefit in mice with inherited

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retinal degeneration. These results provide hope that the generation of functional connections for RGCs will be possible, albeit much more complicated than for photoreceptors. A further challenge to the complete functional integration of transplanted cells within the visual system will be directing growing axons through the optic nerve and into the brain. Furthermore, the development of meaningful synaptic connections within the lateral geniculate nucleus must be addressed. The challenge of establishing functional connections in the brain is increased by the fact that glaucomatous damage extends beyond RGC axons to their target neurons. It has been observed that raised IOP and subsequent RGC loss in a primate glaucoma model correlate with magno-, parvo-, and koniocellular loss in the lateral geniculate nucleus.56 In addition, pathological changes to the visual cortex were observed. Therefore, the development of neuroprotective strategies in glaucoma will need to consider the entire visual pathway as higher-level visual processing must be preserved in order to ameliorate blindness using a stem-cell-based therapy.

TRANSDIFFERENTIATION AND ENDOGENOUS REPAIR An alternative to transplantation-based approaches to stem cell therapy would be the activation of endogenous retinal repair mechanisms. Endogenous repair may proceed in two ways: transdifferentiation of mature cells or proliferation of a resident precursor cell population. Transdifferentiation is a process whereby fully committed somatic cells de-differentiate to produce cells with a phenotype resembling that of an immature cell (that is, cells stop expressing mature cell markers and begin expressing immature markers). The newly formed precursor cells may then re-differentiate, often after first proliferating, into a new cell type that is different from the original cellular identity. Transdifferentiation seems to play a role in retinal repair in some animals and may be a target for clinical modulation in order to treat neurodegenerative retinal disease.46,57 As discussed earlier, there are a number of cell types within the mammalian eye that retain the ability to proliferate. For instance, a quiescent population of proliferative cells exists in the peripheral retina which may be analogous to the regenerative cells of the ciliary marginal zone in fish and amphibians; although their ability to differentiate in response to injury in vivo appears to have been lost during evolution. Furthermore, in chicks, injury-triggered transdifferentiation of Müller cells leads to the generation of new cells which differentiate into cell types similar to those lost due to pathology.43–48 In mammals, signaling through the Notch and Wnt pathways may perpetuate the dormancy of these cells as stimulating these pathways increases the proliferation of Muller cells in vitro and in vivo.48,58 Little is known about what signaling pathways control activation of other proliferative ocular cells, such as those residing in the iris and ciliary body. Conceivably, therapy aimed at modulating the activity of these endogenous proliferative cell populations may provide an avenue for retinal repair. Alternatively, in vitro transdifferentiation of ocular precursor cells may provide donor cells that are appropriate for transplantation.

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Figure 63-6  Transplanted neural progenitor cells localized to nuclear layers in the retina and extended neurites into the plexiform layers. A grafted cell extending a large projection (A) is shown in higher power and reconstructed to demonstrate the extent of neuritic branching (B). The extended process of a grafted cell (C) is shown under higher power (D). (Reprinted from Young MJ, Ray J, Whiteley SJ, Klassen H, Gage FH. Neuronal differentiation and morphological integration of hippocampal progenitor cells transplanted to the retina of immature and mature dystrophic rats. Mol Cell Neurosci 2000;16(3):197–205.)

STEM-CELL-MEDIATED NEUROPROTECTION Recent research suggests that intraocular transplantation of stem cells can confer neuroprotection to surviving retinal neurons in the context of injury and neurodegenerative disease.1,11 The most widely accepted explanation for stemcell-mediated neuroprotection is the local secretion of neurotrophic signaling molecules, which are produced by a variety of cell types including mesenchymal stem cells and glial cells (Fig. 63-7). Experimental efforts to increase neurotrophic factor secretion through gene therapy have also led to demonstrations of enhanced retinal neuroprotection following transplantation. Neurotrophic factor delivery via a number of strategies has been proposed for the treatment of neurodegenerative conditions including glaucoma. The benefits of employing stem cell transplantation to accomplish this goal include the potential for longterm delivery of a cocktail of therapeutic factors following a single injection without the added complication of patient adherence to treatment. The safety of an intravitreal cell graft could be enhanced by encapsulation of cells in a device permeable to diffusible proteins, as is being trialed for the treatment of retinitis pigmentosa.59 Abnormal immune activity within the CNS contributes pathologically to a variety of neurodegenerative disorders

and is thought to play a role in glaucoma.60 Modulation of the inflammatory response in glaucoma may be an alternate mechanism by which stem cell transplantation could provide RGC neuroprotection, as transplanted cells have also demonstrated an ability to modulate immune cell behavior to reduce tissue damage.61 Most studies to date have involved transplantation into the injured brain, however, it is conceivable that such protection may also translate to the retina. Other potential mechanisms of stem-cell-mediated neuroprotection include buffering of reactive oxygen species and attenuation of excitotoxic damage to RGCs, both of which have been implicated in the pathogenesis of glaucoma.

Potential Hurdles REJECTION AND INFLAMMATION A major complication of allogeneic transplantation is the potential for graft rejection by the host. Immunesuppressant drugs can alleviate graft rejection, however, long-term therapy with these drugs carries its own risks. Preferentially, autologous transplantation of the recipient’s

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Elevated IOP + MSC graft

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own somatic stem cells (either from the eye or elsewhere) or patient-derived iPS cells would be a desirable future therapy. The role of inflammation in glaucoma progression and retinal repair must necessarily be examined as both surgical intervention and pre-existing disease conditions stimulate immune reactivity. The contribution of inflammation to injury, neuroprotection and regeneration in the CNS is complex. On one hand, the immune system and regeneration may be in opposition as inflammation has been shown to suppress neurogenesis that occurs naturally in the adult brain.62 Conceivably, inflammation may also hamper attempts at therapeutic induction of neurogenesis in nonregenerative CNS regions. Conversely, inflammation may be beneficial to CNS regeneration. Immune cells which infiltrate the site of a CNS injury can release chemokines that attract transplanted progenitor cells to the damaged area.63 Furthermore, the induction of a controlled autoimmune response specific for CNS-self antigen may be neuroprotective following CNS injury.64 Therefore, precise inflammatory modulation may be required to achieve maximal benefit from stem cell therapy in the retina.

Figure 63-7  Bone-marrow-derived mesenchymal stem cells (MSCs) secrete a number of neurotrophic factors and appear to confer neuroprotection in the context of CNS stress and injury. Depicted here are representative micrographs of optic nerve cross-sections from rats with normal IOP (A), 4 weeks after the onset of ocular hypertension by laser photocoagulation of the trabecular meshwork and intravitreal transplantation of dead MSCs as a negative control (B), and after 4 weeks of ocular hypertension with intravitreal transplantation of live MSCs (C). In this case, local MSC transplantation reduced RGC axonal death by more than 65%. Scale bar = 20 µm. (Reprinted from Johnson TV, Bull ND, Hunt DP, Marina N, Tomarev SI, Martin KR. Neuroprotective effects of intravitreal mesenchymal stem cell transplantation in experimental glaucoma. Invest Ophthalmol Vis Sci 2010; 51(4):2051–9.) Copyright: Association for Research in Vision and Ophthalmology (ARVO).

REACTIVE GLIOSIS Following neural injury, reactive gliosis, and subsequent glial scarring, erects physical and environmental barriers between healthy and diseased CNS tissue. These barriers obstruct endogenous neurite regrowth and can impede the migration and integration of engrafted stem cells. The potency of the inhibitory milieu created by gliosis has been demonstrated by comparing the integration of transplanted cells injected into the vitreous of normal mice compared to those with suppressed glial reactivity. Grafts into mice lacking the GFAP and/or vimentin genes (both involved in reactive gliosis) demonstrated greater neuronal integration into the ganglion cell layer when compared to control eyes. Furthermore, new neurons in the retinas of these knockout animals extended processes further into both the nerve fiber layer and inner plexiform layer than those implanted into normal eyes.52 Use of a glial toxin to selectively inhibit gliosis dramatically improved migration of grafted stem cells into the neural retina (Fig. 63-8).51 The clinical translation of cell-transplantation approaches to glaucoma will

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Control

A

GFAP

B

Nestin

C

Vimentin

AAA

Figure 63-8  Reactive gliosis has been identified as an impediment to the migration of stem cell grafts into neural retinal tissue. The use of alphaaminoadipic acid (AAA), a glial-specific toxin, has been shown to improve morphological integration of stem cell grafts into the host retina. Here, bone-marrow-derived mesenchymal stem cells (green) were co-cultured on the inner retinal surface of organotypic retinal explant cultures, mimicking intravitreal grafts. Treatment with AAA disrupts the reactive gliosis that occurs with co-culture, exemplified by upregulation of glial intermediate filiaments, glial fibrillary acid protein (GFAP), nestin, and vimentin in red. This is associated with greater penetration of transplanted cells into the host neural tissue. Tissue is oriented with the RGC layer towards the top and the outer nuclear layer towards the bottom. Nuclei are counterstained in blue with DAPI. Scale bar = 100 µm. (Reprinted from Johnson TV, Bull ND, Martin KR. Identification of barriers to retinal engraftment of transplanted stem cells. Invest Ophthalmol Vis Sci 2010; 51(2):960–70.) Copyright: Association for Research in Vision and Ophthalmology (ARVO).

63  •  Stem Cells: A Future Glaucoma Therapy?

likely necessitate the development of more targeted and reversible methods of circumventing the glial barrier to graft integration.

AXONAL GUIDANCE AND MYELINATION The adult CNS environment, outside regions of continued neurongenesis, is inherently inhibitory to the integration of new neurons. This provides a challenge for stem cell therapy, especially in diseases where the replacement of functional neurons requires considerable neurite outgrowth. For example, in glaucoma the strict replacement of RGCs will require extension of dendrites into the inner nuclear layer and axons from the inner retina through the optic nerve and into brain targets including the lateral geniculate nucleus. Because adult mammalian RGCs retain the ability to regenerate neurites in culture and through peripheral nerve grafts in vivo, much of the obstacle to RGC axonal regeneration is thought to reside in the local environment. Growth-inhibitory proteins such as myelin-associated protein (MAG), oligodendrocyte-myelin glycoprotein or Nogo are associated with CNS myelin and act directly on neurons to trigger growth cone collapse. Heparin and chondroitin sulfate proteoglycans have also been revealed as potential barriers to CNS regeneration. These proteoglycans encapsulate mature neurons and their synapses within the brain, contribute to glial scar formation and inhibit neurite growth. Such proteoglycans are present in the retina and, thus, enzymes that digest them may facilitate optic nerve regeneration. Despite the numerous obstacles that exist to axonal regeneration in the CNS, regeneration of endogenous RGC axons within the optic nerve has been induced to impressive levels by experimental manipulations carried out on RGCs themselves, presumably by putting them in a state of enhanced regenerative capacity capable of overcoming the inhibitory environment. Knock-out of the transcription factor Klf-4 in RGCs dramatically increases axonal regeneration in the optic nerve following crush injury.65 To date, the most robust effects have been obtained with a combination approach of (1) intraocular injection of zymozan to trigger an inflammatory response; (2) intraocular injection of a cAMP analog to promote RGC axonal regeneration signals; and (3) knock out of the transcription factor PTEN.66 This protocol resulted in the complete regeneration of RGC axons to brain targets including the lateral geniculate nucleus and superior colliculus, as well as the partial return of visually guided behaviors and circadian rhythm entrainment in mice with optic nerve damage. While the experimental methods employed in this work are not directly translatable to human patients at this time, these data provide proof of principle that axonal regeneration from transplant-derived RGCs is feasible. Should RGC axonal extension from the eye to the brain be achieved, myelination of the optic nerve fibers will be essential in order to maintain physiological conduction velocities. Research focused on demyelinating diseases, such as multiple sclerosis, may provide clues as to possible mechanisms for triggering remyelination. Inflammation is known to play a positive role in remyelination of damaged CNS neurons by oligodendrocyte precursor cells. It is

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possible that modulation of endogenous oligodendrocytes or transplantation of oligodendrocyte precursor cells will be required to complete the functional integration of new RGCs.

ASSESSING VISUAL IMPROVEMENT IN ANIMAL MODELS Assessing functional improvement in animal models of glaucoma presents a particular challenge. Some clinical methodologies for measuring general visual function have been adapted to many different species, including electroretinography, visual evoked potential measurement and pupillometry. In addition, behavioral tests, such as observing head tracking responses to visual targets, have also been exploited. Unfortunately, the sensitivity of these tests is relatively low and a visual benefit that may be clinically relevant to a human patient may currently be below the threshold for detection in animal models. As such, efforts to develop more sensitive and reliable visual function assessments for experimental models are required.

CONTINUED DISEASE PROGRESSION While stem cell therapy may offer the hope of restoring visual field function in glaucoma, one must remember that the pathology itself has not been ‘cured’ by such treatment. Thus, clinical management of the disease would necessarily continue to prevent renewed neurodegeneration. Presumably, the new neurons would be as vulnerable to the pathophysiological conditions of glaucoma as their predecessors. Furthermore, depletion of endogenous RGCs will also likely persist. As such, glaucoma may require life-long ocular hypotensive, and possibly neuroprotective, treatment, even after stem cell therapy. Perhaps new advents in neuroprotection and/or gene therapy will offer additional tools to the glaucoma specialist.

Summary Stem cells offer the potential to develop powerful new therapies for treating historically incurable neurodegenerative diseases, such as glaucoma, by providing a possible means of replacing dead cells to effect functional recovery. In addition, stem cell therapy may possess other less direct therapeutic benefits such as providing trophic support for remaining neurons, modulating immune responses, buffering oxidative stress, or preventing excitotoxicity. Stem cell therapy could be achieved via the transplantation of cultured stem cells or by manipulating endogenous repair mechanisms. A variety of potential stem or progenitor cell sources, each with inherent strengths and weaknesses, are available for transplantation-based therapies. In order to cure glaucoma completely, stem cell therapy would necessarily replace degenerated retinal neurons and re-establish the visual pathway. This means stem cells would ideally integrate into the retinal ganglion cell layer, differentiate into mature RGCs, establish connections with appropriate afferent neurons, extend axons through the optic nerve to the lateral geniculate nucleus and make functional connections within the brain to preserve the

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Spotlight 1  Brain Perspective Katie Gill and Alice Pébay The molecular mechanisms underlying the progressive damage and loss of retinal ganglion cells that is characteristic of glaucoma are not well understood. A major barrier to this understanding is the difficulty in obtaining retinal ganglion cells from living patients. Human induced pluripotent stem cells can be valuable cell models for this purpose, as these cells can be patient-derived and then differentiated into cells of interest, such as retinal ganglion cells, for disease modeling and understanding mechanisms underlying cell pathophysiology, thus facilitating more rapid and efficient drug screening. Another strategy is to use models of nerve damage present in other central nervous system (CNS) conditions, such as brain and spinal cord injuries. A trauma to the CNS often results in permanent damage at the site of the injury that results from the initial trauma as well as by inflammatory and other processes that prevent neuronal regeneration and recovery. Neurotrauma can affect all CNS cell types, thus understanding their biology may prove key to improving regeneration. Neurons are very sensitive to changes in their microenvironment. Following injury, neurons can degenerate and die. This can be due to both direct effects on these cells and through the modification of the properties of the cells and environment around them. Astrocytes play prominent roles in the control of the neuronal microenvironment, regulating neuronal energy demand by glucose and glutamate uptakes, neural vulnerability by secretion of neurogenic and pro-inflammatory molecules and neuronal activity via regulation of the extracellular concentration of neurotransmitters, such as glutamate, in the synaptic cleft, hence also controlling excitotoxicity.1 Following neural injury, surviving astrocytes are activated by inflammatory molecules, undergo rapid proliferation and exhibit hypertrophy, allowing formation of a glial scar. As a consequence of this environment, the insulating and protective oligodendrocytes can also degenerate, leading to axonal conduction failure. Microglia, often characterized as the immune cells of the CNS, are also activated in

retinotopic map. Such complete RGC replacement is likely to remain a formidable challenge. However, it is also possible that the survival and partial integration of transplanted cells within the retina may provide alternative benefits by enhancing the survival and function of host RGCs. In addition, glaucoma stem cell therapy could be used to treat other complications of glaucoma including reversal of optic nerve head pathology, restoration of the trabecular outflow pathway or to heal leaky blebs following surgery. There are also a number of possible obstacles to the development of a successful stem-cell-based therapy for glaucoma. Given allogeneic transplantation would necessitate long-term immune suppression in order to avoid graft rejection, it would be highly desirable to develop autologous stem cell therapies, possibly through the use of iPS cells. In addition, techniques to promote RGC axon extension, connection and myelination may need to be optimized to achieve significant visual field restoration. Finally, the issue of continued disease progression will need to be addressed in order to protect new and host neurons.

neurotrauma, further contributing to the global response to CNS injury.2 Endogenous neural stem/progenitor cells might also play a role in neurogenesis following trauma, although their success is severely hindered by the CNS inflammatory response.3 Finally, the blood–brain barrier is often disrupted following injury, hence allowing the entrance of blood cells, including platelets, from the bloodstream to the injury site, events that will contribute to the poor outcome of neurotrauma.4 Various experimental models are currently used for assessment of neurotrauma, from cell-based assays to animal models of trauma. There are many approaches underway to investigate mechanisms to improve neural regeneration and promote functional recovery, including blockage of axonal growth-inhibitory molecules, treatment with growthpromoting molecules, applying measures to reduce glial scar, and developing methods to limit diffuse inflammation and cell death.5 Thus, the key to an effective therapy after neural injury is to identify factors that contribute to both acute and secondary events that limit regeneration and functional recovery. This ‘CNS’ perspective will most likely be applicable to the optic nerve survival and regeneration. References 1. Belanger M, Allaman I, Magistretti PJ. Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metab 2011;14(6): 724–38. 2. Ransohoff RM, Cardona AE. The myeloid cells of the central nervous system parenchyma. Nature 2010;468(7321):253–62. 3. Martino G, Pluchino S. The therapeutic potential of neural stem cells. Nat Rev Neurosci 2006;7(5):395–406. 4. Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood–brain barrier. Nat Rev Neurosci 2006;7(1):41–53. 5. Fitch MT, Silver J. CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure. Exp Neurol 2008;209(2):294–301.

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