Stem cells for neuroprotection in glaucoma

Introduction

The possible neuroprotective effects of stem cell transplantation are a focus of active investigation in neuroscience at the present time. There are a number of studies where such treatments have shown promise in animal models of neurodegenerative disease and applying such techniques to glaucoma has a number of attractions. Glaucoma is well suited for such investigations as it is possible to directly visualize cellular transplants in vivo and, in addition, techniques to assess small

Corresponding author. Tel.: +44 1223 331160; Fax: +44 1223 331174; E-mail: krgm2@cam.ac.uk

aNDB and TVJ contributed equally to this chapter

changes in the structure and function of the eye are well developed. Such findings may also apply to other neurodegenerative diseases with similar pathologies. In addition, current treatments for glaucoma are limited mainly to reduction of intraocular pressure, which fails to prevent further deterioration in some glaucomatous eyes and necessitates the development of new treatment approaches. Methods that have the potential for prolonged therapeutic effects after a single treatment are particularly attractive.

In this chapter, we explore the potential use of stem cells for neuroprotection in the central nervous system (CNS) and, in particular, the challenges of using such techniques for glaucoma therapy. We also consider the implications of results obtained in glaucoma models for other neurodegenerative diseases.

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Glaucoma as a model of neurodegenerative disease

Glaucoma is the most common neurodegenerative disease in the world, affecting approximately 80 million people or 1.2% of the global population (Quigley and Broman, 2006). Like many neurodegenerative diseases, glaucoma is characterized by the loss of a discrete neuronal cell population from the CNS. Similarities in the pathophysiological mechanisms of cell death appear to exist between glaucoma and other neurodegenerative diseases. For example, signs of oxidative stress, glutamate toxicity, reactive glial changes, and impaired axonal transport have been identified in glaucoma (Gupta and Yucel, 2007; Kumar and Agarwal, 2007; Nickells, 2007) as well as in other neurodegenerative diseases. As such, progress made in understanding the mechanisms of glaucomatous onset and progression may provide insight into the pathogenesis of other neuropathologies. Furthermore, successful approaches to protecting retinal ganglion cells (RGCs) could potentially translate into therapies for other cell types.

Unlike other neurodegenerative diseases, structural changes associated with glaucomatous progression can be directly visualized in live subjects. In human patients, the optic nerve head and retinal nerve fiber layer can be examined directly and longitudinal changes are routinely documented. In animal models of ocular hypertension and glaucoma, the ability to observe RGC loss directly is even more powerful. RGCs can be visualized at single-cell resolution by scanning laser ophthalmoscopy after fluorescent labeling via retrograde tracing or immunohistochemical identification of apoptotic cells (Cordeiro et al., 2004; Maass et al., 2007). If in vivo imaging of apoptosing RGCs proves to be safe, and to correlate well with RGC death, then such techniques may provide a unique method to visualize cell death longitudinally in a human neurodegenerative disease.

Why use stem cells for neuroprotective therapy?

It is reasonable to ask what advantages there may be in using stem cells, or their various derivatives, as a neuroprotective therapy in glaucoma rather

than simply using a pharmacological approach. One distinct advantage cellular therapy could provide is chronic neuroprotection after a single treatment. Once integrated into the host tissue, the ideal cellular therapy would provide lifelong support for RGCs and attenuate visual field loss. A further benefit of a cell-based therapy may be the noted ability of stem cells to migrate or ‘‘home’’ to sites of injury within the CNS and thus deliver support locally, where it is most needed. Such stem cell behavior has been observed in various neuropathological models, and has been particularly well-characterized following stroke (Felling and Levison, 2003; Tai and Svendsen, 2004). Furthermore, some stem cells have the ability to migrate extensively within the CNS, offering the potential for widespread therapeutic activity following focal delivery. Such integration with the host tissue may also allow stem cells to provide support through contact-mediated mechanisms, thereby offering a supporting niche for surviving neurons.

Cell-based therapies may function by a variety of different mechanisms. It is acknowledged that neuroprotection by stem cells involves trophic support of neurons (as discussed below). It seems very likely that such protection is not due to the secretion of single growth factors but is multifactorial. For example, transplantation of neural stem cells into the spinal cord has been shown to delay disease onset and progression in a mouse model of amyotrophic lateral sclerosis (Corti et al., 2007). The protective mechanism in this model was found to involve vascular endothelial growth factor (VEGF)- and insulin-like growth factor 1 (IGF1)-dependent signaling pathways and resulted in improved performance in behavioral tests. In addition, transplantation of immortalized human neural stem cells into the lesioned striatum has been observed to improve function significantly in a rat Parkinson’s disease model (Yasuhara et al., 2006). Again, this effect appeared to be due to trophic factor secretion by engrafted cells, although there also was evidence of neuronal differentiation by some of the transplanted cells. Interestingly, the effect of cellular transplantation was greater than that of single injections or continuous infusions of trophic factors. Such a

neuroprotective effect might be bolstered by modification or manipulation of stem cells prior to transplantation in order to control the identity and levels of factors supplied.

In addition to supplying trophic factors, transplanted cells may also be able to modify the pathological environment to promote neuronal survival. Neural stem cells appear to possess this ability inherently. As an example, stem cells derived from the subventricular zone have been found to modify the local environment directly through immunomodulatory mechanisms (Pluchino et al., 2005) or by influencing gene expression in surrounding neurons (Madhavan et al., 2008). In addition, it has been demonstrated that the integration of glial precursor cells, which possess active glutamate transporters, into organotypic spinal cord cultures enhanced glutamate uptake and reduced motor neuron cell death (Maragakis et al., 2005). This neuroprotective approach could potentially be extended to other pathologies that may involve glutamate excitotoxicity, such as amyotrophic lateral sclerosis and glaucoma.

Stem cell sources

Stem cells possess the ability to self-renew indefinitely and to differentiate into a variety of different cell types. In contrast, progenitor cells are generally considered a more restricted cell type, often multipotent but usually of limited proliferative capacity. For simplicity, in this chapter, we will use the term ‘‘stem cell’’ in its broadest sense to cover all precursor cell types. Stem cells exist within a wide array of tissues and at all developmental stages from embryogenesis to adulthood. Selection of the optimal stem cell type to be used for therapeutic applications depends upon the ultimate therapeutic goal: cellular replacement and/or neuroprotection of surviving cells. In the case of cell replacement for neurodegenerative disease, the relative levels of lineage potency and commitment are of primary concern. The ideal cell must be capable of differentiating into the specific cell type that has been depleted, but should also be restricted from differentiating into other cell types.

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If transplantation is to be used for neuroprotection, on the other hand, grafted cells need only support the surviving tissue and attenuate disease progression, rather than having to acquire neurological function themselves.

Perhaps the most well-known type of stem cell is the embryonic stem (ES) cell, which is derived from the inner cell mass of the developing blastocyst. ES cells are capable of differentiating into any somatic cell type in the body and are of great clinical interest, especially with regard to cell replacement. However, the potential for malignant transformation of transplanted ES cells, concerns regarding the ethics and logistics of obtaining ES cells, and the inherent necessity of allogeneic transplantation leading to the risk of graft rejection may ultimately limit their clinical use.

Neural stem cells are of particular interest in neurodegenerative disease. These cells can selfreplicate but are lineage-restricted and capable only of differentiating into neurons and glia. Neural stem cells have been isolated from multiple regions of the CNS, including the brain, spinal cord, and retina. In adults, the most wellcharacterized neural stem cells are from the lateral wall of the subventricular zone and the subgranular zone of the hippocampal dentate gyrus, two regions where neurogenesis occurs throughout life. Retinal stem cells residing in the ciliary marginal zone have been identified in adult birds, fish, and amphibians, and contribute to retinal repair following injury in these species (Otteson and Hitchcock, 2003; Hitchcock et al., 2004). In mammals, this process does not occur naturally; however, cells isolated from this region can proliferate and differentiate in vitro, and growthfactor induced reactivation of these cells has been reported in vivo (Abdouh and Bernier, 2006). Furthermore, mammalian Mu¨ller glia and cells isolated from the iris-pigmented epithelium, as well as the pars plana of the ciliary body, display neural stem cell-like characteristics when isolated in vitro (Asami et al., 2007; Lawrence et al., 2007; MacNeil et al., 2007; Xu et al., 2007).

Somatic cells isolated from nonneural tissue may play an important role in the future of stem cell therapy for neurodegenerative disease. First, many somatic stem cells that are seemingly

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unrelated to neural tissue reportedly hold neural competence given the proper in vitro differentiation conditions. For instance, some evidence suggests that mesenchymal stem cells (MSCs) isolated from bone marrow, blood, and umbilical cord can transdifferentiate to give rise to neuronal or glial-like cells, though this phenomenon is controversial (Krabbe et al., 2005). Second, many somatic cells have demonstrated benefit in neurodegenerative disease models in the absence of overt differentiation toward a neural phenotype. Potential mechanisms of this neuroprotective effect will be discussed in the following section. Last, very recent evidence suggests that adult human somatic cells, including terminally differentiated non-stem cells such as fibroblasts, can be reprogrammed to achieve a pluripotent, self-renewing stem cell-like state by ectopically inducing the expression of certain genes associated with ES cells (Takahashi et al., 2007; Yu et al., 2007). Importantly, somatic cells that can be easily isolated from adult patients provide the opportunity for autologous cell transplantation therapy, thereby eliminating the risk of graft rejection or lifetime administration of immune suppressant drugs.

Neuroprotection by transplanted stem cells

Numerous published examples exist where stem cell transplantation for neurodegenerative disease has led to structural and functional benefit in the absence of differentiation or functional integration on the part of the engrafted cells. In these cases, the effect is often attributed to neuroprotection of the endogenous surviving tissue. There are a number of mechanisms by which transplantation of stem cells can be neuroprotective, and each may hold potential therapeutic applications for glaucoma. The most widely cited, and perhaps most important mechanism, involves supply of neurotrophic factors and support for surviving neurons. In addition, regulation of immune activity and the promotion of endogenous CNS repair also may play a role.

It has been demonstrated, both in vitro and in vivo, that various types of stem cells synthesize and release neurotrophic factors without forced

differentiation or experimental manipulation. Transplantation of these cells into the nervous system is associated with a preservation of surviving neural structure and function in a variety of neurodegenerative conditions including Parkinson’s disease, amyotrophic lateral sclerosis, spinal cord injury, and traumatic brain injury. Specifically in the eye, stem cell transplantation has been shown to rescue retinal degeneration in a range of models. For instance, engrafted bone marrowderived MSCs have preserved photoreceptors in the rhodopsin knockout mouse (Arnhold et al., 2006). Furthermore, transplantation of ES cells (Schraermeyer et al., 2001), Schwann cells (Lawrence et al., 2000), or pigmented iris epithelial cells (Schraermeyer et al., 2000) have all reduced degeneration in the Royal College of Surgeons rat, which displays marked photoreceptor loss. In addition, neuralized ES cells have been found to attenuate retinal degeneration in mnd mice (Meyer et al., 2006). The authors of each of these studies suggest that the primary neuroprotective benefit is derived from trophic factors secreted by the grafted cells to support surviving retinal neurons.

Failure of neurotrophic support might be especially important in glaucoma where it has been shown that retrograde axonal transport of brain-derived neurotrophic factor (BDNF) is disrupted (Pease et al., 2000). In fact, an accumulation of motor proteins at the optic nerve head is suggestive of a general failure of axonal transport (Martin et al., 2006), and intraocular supplementation of BDNF has been shown to reduce RGC loss in experimental glaucoma (Martin et al., 2003). Transplantation of MSCs into rats with induced ocular hypertension has also been shown to increase RGC survival (Yu et al., 2006). In this case, the authors demonstrated a concomitant increase in basic fibroblast growth factor, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), and BDNF within the retina of treated eyes.

Some stem cells have also revealed an ability to modulate immune cell activity. Immune mechanisms have been implicated in a wide range of neurodegenerative diseases. In multiple sclerosis, autoimmune activity appears critical to the pathogenesis of the disease (McFarland and Martin,

2007). In acute neurodegenerative conditions such as stroke, spinal cord injury, and traumatic brain injury, inflammation may contribute to secondary neuronal degeneration. In more chronic conditions such as Alzheimer’s disease and Parkinson’s disease, inflammatory ‘‘bystander’’ damage is thought to contribute to disease progression (Lucas et al., 2006). Transplantation of cells able to modulate immune activity has demonstrated benefit in many of these conditions. Bone marrowderived MSCs are known for being particularly immunomodulatory and are currently the subject of clinical trials for treating multiple sclerosis (Passweg and Tyndall, 2007). Neural stem cells are reportedly capable of downregulating inflammatory processes within the CNS, leading to functional recovery in a range of neurodegenerative diseases (Martino and Pluchino, 2006). Whether these neuroprotective mechanisms are applicable to glaucoma requires further investigation.

Finally, there is some evidence that transplanted stem cells are able to influence local CNS tissue to promote endogenous repair mechanisms. Unlike the peripheral nervous system, the plasticity of the CNS is notoriously restricted and neuritic regrowth following injury is very limited. This lack of regeneration after injury appears to be due to the combination of a lack of trophic cues in the adult CNS, which signal for regenerative activity, and the inhibitory nature of the CNS environment. The production and release of certain neurotrophic factors by neural stem cells has been shown to trigger axonal regrowth in the adult injured spinal cord (Lu et al., 2003) and, therefore, release of neurotrophic factors by engrafted cells might have beneficial consequences beyond neuroprotection alone. The presence of various inhibitory molecules, such as chondroitin sulfate proteoglycans and members of the Nogo family, within the local environment of the CNS also contributes to a general lack of plasticity. Transplantation of neural stem cells has been found to reduce the expression of these inhibitory molecules by increasing the levels of various matrix metalloproteinases within the retina (Zhang et al., 2007). In turn, this has enhanced neurite outgrowth. If neurite sprouting and the formation of local synaptic connections could be triggered in the

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glaucomatous retina, it is conceivable that the receptive field size of surviving RGCs could be increased in order to improve visual function.

Enhanced neuroprotection by transplantation of modified stem cells

In addition to neurotrophic support by naive transplanted stem cells, the possibility of augmenting the neuroprotective effect by using genetically modified stem cells to deliver specific factors has been explored. This concept has attracted particular attention in stroke research. Many studies have demonstrated that transplantation of various stem cell types into the infarcted brain can ameliorate ischemic damage (Haas et al., 2005). In addition to these earlier studies, a number of groups have now demonstrated substantial protection by engineered stem cells in various models of ischemic disease. For example, a significant improvement in neurological deficiency was observed in a rat transient focal cerebral ischemia model following the engraftment of neural stem cells, modified in vitro to express VEGF, into the perifocal zone (Zhu et al., 2005). The observed improvement was greater than that of control transplantation of naive neural stem cells. Furthermore, the transplantation of human neural stem cells overexpressing VEGF into the cortex overlying an intracerebral hemorrhage lesion has been shown to improve survival of engrafted cells, to stimulate host angiogenesis and to enhance functional recovery in mice (Lee et al., 2007). Similarly, the introduction of MSCs transfected to express BDNF into the brain 6 h after permanent middle cerebral artery occlusion was found to reduce lesion size and improve functional outcome (Nomura et al., 2005). Furthermore, in this model, stem cells engineered to produce BDNF provided greater neuroprotection than that observed following the delivery of naive cells. The same group also reported similar results using MSCs modified to express GDNF (Horita et al., 2006) and placental growth factor (PIGF) (Liu et al., 2006), with PIGF also stimulating angiogenesis. The secretion of GDNF by implanted neural stem cells has also provided neuroprotection in other neurodegenerative disease models, such as Huntington’s disease