progenitor-like cells in the adult mammalian eye

Progress in Retinal and Eye Research 31 (2012) 213e242

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Neurogenic potential of stem/progenitor-like cells in the adult mammalian eye Stefanie G. Wohl a, b, *,1, Christian W. Schmeer a,1, Stefan Isenmann b, c,1 a

Hans Berger Department of Neurology, Jena University Hospital, Jena, Germany University of Witten/Herdecke, Witten, Germany c Department of Neurology, HELIOS Klinikum Wuppertal, Wuppertal, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 13 February 2012

The neural retina as part of the brain has received a great deal of attention since quiescent neural stem cells/progenitor cells (NSC/PCs) were discovered in this non-neurogenic region. Herein, we particularly feature the adult rodent eye and provide an overview of all putative neuronal progenitor-like cells attributed to the various ocular areas that have been identified during the last decade. These neuronal progenitor-like cells include the pigmented cells of the ciliary body (CB), as well as the pigmented cells of the iris and the retinal pigment epithelium (RPE). Within the retina, the Müller cells, the specialized macroglia of the vertebrate eye, display neurogenic potential, i.e. de-differentiation into retinal neurons following exogenous stimulation. In addition, retinal astrocytes, which are immigrants from the brain and do not arise from a common retinal progenitor show signs of de-differentiation after injury. Interestingly, microglial cells, the immune competent cells of the central nervous system (CNS), feature neurogenic potential in vitro. Moreover, it appears that this potential can also be initially induced by injury in vivo, both in the brain and the retina. This review summarizes characteristics of various endogenous progenitor-like cells reported in in vitro and in vivo studies. A focus is placed on in vivo studies with a special regard to cellular responses after exogenous stimulation, such as growth factor treatment or injury. Finally, we discuss therapeutic potential of these cells with respect to cell replacement strategies and putative clinical application. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Ciliary body Growth factors Injury Microglia Müller glia NG2-glia

Contents 1. 2.

3. 4.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .214 Retinal stem cells and retinogenesis in the vertebrate eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .215 2.1. Ciliary marginal zone (CMZ) in fish and amphibians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 2.2. CMZ in birds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 2.3. CMZ in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Progenitor-like cells in the rodent eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 Activation of dormant stem/progenitor cells through exogenous stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 4.1. Growth factor treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 4.2. Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 4.2.1. Neurotoxic injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 4.2.2. Retinal ischemia/glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 4.2.3. Optic nerve lesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 Neurogenic potential of progenitor-like epithelial cells in the adult rodent eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 5.1. Pigmented epithelial cells of the ciliary body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 5.1.1. Ciliary progenitor cells after injection of defined factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 5.1.2. Ciliary progenitor cells after neurotoxic lesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 5.1.3. Ciliary progenitor cells after optic nerve lesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

* Corresponding author. Hans Berger Department of Neurology, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany. Tel.: þ49 3641 9323425; fax: þ49 3641 9325902. E-mail address: [email protected] (S.G. Wohl). 1 Percentage of work contributed by each author in the production of the manuscript: Stefanie G. Wohl: 80%; Christian W. Schmeer: 10%; Stefan Isenmann: 10%. 1350-9462/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.preteyeres.2012.02.001

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5.1.4. Ciliary progenitor cells after retinal ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 5.1.5. Ciliary “stem”/progenitor cells: true identity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 5.1.6. Nestin expression in other ciliary cell types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 5.2. Epithelial cells of the retinal pigmented epithelium (RPE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 5.3. Epithelial cells of the iris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Neurogenic potential of progenitor-like glial cells in the adult rodent retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .227 6.1. Progenitor-like Müller glia in the retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 6.1.1. Müller glia after neurotoxic injury and growth factor treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 6.1.2. Müller glia after optic nerve injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 6.1.3. Müller glia after ischemia/glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 6.1.4. Müller glia after laser injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 6.1.5. Müller glia as true retinal progenitors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 6.2. Progenitor-like astrocytes in the retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Progenitor-like cells in the optic nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Microglia as putative progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 8.1. Neurogenic microglia in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 8.2. Neurogenic microglia in the retina? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 Cell transplantation approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

1. Introduction Regeneration of the mammalian CNS, especially the neural retina, is currently an issue of intensive research. Considerable progress has been achieved during the past 20 years, on developing strategies to protect neurons of the visual system following neuronal injury, for example, by enhancing neuronal cell survival through exogenous delivery of trophic factors (Di Polo et al., 1998; Garcia Valenzuela and Sharma, 1998; Johnson et al., 1986; Kermer et al., 1998; Klöcker et al., 1997; Mey and Thanos, 1993). Our work illustrates that application of specific neurotrophic factors saves a proportion of affected neurons from degenerating, at least for a limited period of time following injury (Isenmann et al., 1998, 1999, 2004; Kretz et al., 2005, 2006a, 2006b; Schmeer et al., 2002, 2008; Straten et al., 2002; for review see Isenmann et al., 2003). In addition, inducing endogenous stem/progenitor cells to proliferate and differentiate into new retinal neurons, thereby replacing injured cells, constitutes another potential tool for retinal regeneration. This field in particular has become an important focus for research over the last decade. Retinal repair by cell replacement is attempted either by transplanting stem/progenitor cells (for review see Bhatia et al., 2010; Bi et al., 2009; Dahlmann-Noor et al., 2010; Harvey et al., 2006; Klassen et al., 2004a; MacLaren and Pearson, 2007; Yau et al., 2007; Young, 2005) or by recruiting endogenous progenitor-like populations residing within the adult mammalian tissue. Identification and characterization of endogenous retinal progenitor-like cells is an area of active research, not only in rodents, but also in primates including humans. Rodents, in particular mice and rats, are commonly used as animal models for studies in the CNS, because of ease of maintenance, low costs, and short breeding times. Another advantage is that anatomy and physiology of the CNS share extensive similarities with their human counterparts. These species represent ideal animal models for analysing cellular and molecular aspects as well as underlying mechanisms in developmental, physiological or pathological processes. In addition, generation of transgenic mice is an important tool allowing evaluation of specific cell types or for defining functions of particular proteins in specific processes, for instance, in adult neurogenesis. In mammals, almost all neurons of the CNS are formed early during development and arise from neural stem cells (NSCs). NSCs are more committed than toti- or pluripotent stem cells found in

embryonic development (Fig. 1). By definition, NSCs are multipotent giving rise to cells of the neural lineage, i.e., neurons and glia. In this review, we do not present detailed information regarding stem cells and NSCs, their neurogenic potential, and intrinsic as well as extrinsic limitations. Several excellent reviews are available in the literature (Alvarez-Buylla and Temple, 1998; Denham et al., 2005; Galli et al., 2003; Okano, 2002; Potten and Loeffler, 1990; Temple and Alvarez-Buylla, 1999; Temple, 2001). Neural stem cells/ progenitor cell (NSC/PCs) populations in avians and mammals (often referred to as “higher vertebrates”) decrease with progressive differentiation into various neuron types, so that in adulthood only a small fraction of NSC/PCs are found in particular regions of the CNS. NSC/PCs in adult mammals have been reported in two neurogenic areas of the brain, the subventricular zone (SVZ) (Alvarez-Buylla and Garcia-Verdugo, 2002; Doetsch et al., 1999; Lois and Alvarez-Buylla, 1993) and the dentate gyrus of the hippocampus (Balu and Lucki, 2009; Eriksson et al., 1998; Gross, 2000; Kempermann et al., 2004) as well as in non-neurogenic regions such as the cerebral cortex (Gould et al., 1999; Magavi et al., 2000), spinal cord (Matsumura et al., 2010) and in various structures of the eye (Ahmad et al., 2000; Ffrench-Constant and Raff, 1986; Haruta et al., 2001; Tropepe et al., 2000). NSC/PCs in non-neurogenic areas are mitotically dormant and quiescent, but can be activated by exogenous factors, for example, following injury (Kernie et al., 2001; Magavi et al., 2000; Ming and Song, 2005; Mori et al., 2005). The regeneration potential of the vertebrate retina, in particular, in fish and birds has been extensively described in comprehensive reviews during the last ten years (Amato et al., 2004; Fischer, 2005; Fischer and Bongini, 2010; Haynes and Del Rio-Tsonis, 2004; Hitchcock et al., 2004; Klassen et al., 2004a; Moshiri et al., 2004; Ohta et al., 2008; Perron and Harris, 2000; Reh and Levine,1998; Reh and Fischer, 2006). Herein, we give an overview of the key findings regarding the neurogenic potential of retinal progenitor cells (RPCs) identified in different regions in the eye of the adult mouse or rat, in particular, in epithelial cells of the CB, iris and retina, retinal glial cells, progenitors in the optic nerve (ON) as well as progenitor-like cells at the retinal margin. Although, fundamental evidence from in vitro studies is presented here, the main focus of this review are the various methods for exogenous stimulation that are used to activate “dormant” progenitor-like cells in vivo, such as growth factor treatment and/or different injury models including ocular neurotoxic injury, ischemia/glaucoma, and ON injury. In addition,

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Fig. 1. Differentiation paths of neural, retinal, and hematopoietic stem cells. During development, stem cells (SCs) become increasingly committed. Multipotent SCs, e.g., neural stem cells (NSCs) of the central nervous system (CNS) or hematopoietic stem cells (HSCs) in bone marrow are usually restricted to the neural and hematopoietic cell lineage, respectively. NSCs give rise to neural progenitor cells (NPCs) such as neuro- and glioblasts which subsequently differentiate into neurons and glia. Multipotent NSCs of the retina are retinal stem cells (RSCs) which give rise to early and late retinal progenitor cells (RPCs). The former generate early born, the latter late born retinal cells. Retinal astrocytes derive from NSCs of the brain and immigrate into the retina via the optic nerve. HSCs give rise to several progenitor cells (PCs) that differentiate into red and white blood cells. Perivascular macrophages arise from such a PC and migrate into the brain and retina during vascularization. Microglia directly derive from more immature myeloid progenitors and migrate into CNS tissue early in development, before vascularization begins. Some of the cerebral and retinal microglia are suggested to be immature cells which possibly differentiate into mature microglia in the tissue. O-2A-PC: oligodendrocyte-type-2 astrocyte progenitor cells; RGC: retinal ganglion cells (modified after Reh and Fischer, 2006; Ransohoff and Cardona, 2010).

we introduce for the first time, retinal microglia as a putative new progenitor-like cell type of mesodermal origin, since there is increasing evidence that cerebral microglia are capable of transdifferentiating into neurons in culture. As a final point, we discuss aspects relating to improving methodology as well as possible approaches for translation of acquired data into clinical application. 2. Retinal stem cells and retinogenesis in the vertebrate eye The neural retina, the light-sensitive tissue in the eye responsible for transforming light signals into chemical and electrical

events, represents a protuberance of the diencephalon that constitutes a part of the brain. The retina, as an inner layer, is attached to the uvea (middle layer) and sclera (outer layer), and flanked at the periphery by the CB (Fig. 2A). Further, the retina comprises several neuronal and glial cell types (Fig. 2B). Firstly, RGCs, located in the ganglion cell layer (GCL), serve as projection neurons of the retina. Secondly, interneurons such as bipolar, amacrine, and horizontal cells are found in the inner nuclear layer (INL), and, thirdly, photoreceptors (rods and cones) which reside in the outer nuclear layer (ONL). The outer segments of the photoreceptors are embedded in the RPE which passes into the ciliary

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Fig. 2. Retina and ciliary body of the adult rodent eye e ciliary and retinal cell types and retinogenesis at a glance. A: horizontal section of an adult rodent eye with cilio-retinal area (red box). B: ciliary and retinal cell types: the ciliary body (CB) consists of a pigmented and an unpigmented epithelial cell layer and ciliary stroma filled with connective tissue and including the ciliary muscle. Retinal progenitor-like cells are located within the pigmented epithelium, in particular, in the pars plana/pigmented marginal zone (PMZ). The ciliary epithelium passes into the retinal pigment epithelium (RPE) in which the outer segments of the photoreceptors (PR; rods and cones) are embedded. PRs nuclei are found in the outer nuclear layer (ONL). Somata of retinal interneurons (bipolar, amacrine, and horizontal cells) are located in the inner nuclear layer (INL). Retinal ganglion cells (RGCs), the projection neurons of the retina, are located in the GCL. Retinal astrocytes are found in the GCL/nerve fibre layer (NFL). Müller glia radially span all retinal layers with their processes, their nuclei are located in the INL. Retinal microglia are found in the inner and outer plexiform layer (IPL and OPL, respectively) and the ciliary epithelium, macrophages in the connective tissue and CB stroma. C: retinogenesis. Generation of all retinal cell types. PCM: pigmented ciliary margin, (modified after McMenamin et al., 1992; Marquardt and Gruss, 2002; Klassen et al., 2004a, and http://www.mpih-frankfurt.mpg.de).

epithelium at the pars plana (Fig. 2B). All six types of retinal neurons together with the specialized radial glia, the Müller cells, arise from retinal stem cells (RSCs) during embryogenesis and early postnatal development (Fig. 2C). Retinogenesis is a highly conserved process which occurs in a defined sequence (Fig. 2C): In a first wave, RGCs, cone photoreceptors, as well as horizontal and amacrine cells are generated by early RPCs. Thereafter, in a second wave, bipolar cells, rod photoreceptors, and Müller glia differentiate from late RPCs (Figs. 1 and 2C) (Galli-Resta, 2002; Marquardt and Gruss, 2002; Reh and Fischer, 2006; Reichenbach, 1993; Reichenbach and Bringmann, 2010; Turner and Cepko, 1987). This sequence in retinal cell generation is due to a progressive change of RPCs per se (intrinsic cues, competence) as well as due to a changing environment (extrinsic cues) during development (James et al., 2003; Reh and Kljavin, 1989; for review see Livesey and Cepko, 2001). Excellent reviews on retinal cellular development and RSCs, different types of NSCs and their regulation in the mammalian eye, and transcriptional control of neuronal diversification in the retina have been

published in the last decade (Ahmad et al., 2004; Klassen et al., 2004a; Marquardt, 2003). Resembling the process in fish and in birds, retinogenesis in mammals begins in the central retina adjacent to the ON head, spreading toward the periphery. Consequently, younger cells are located in the periphery of the eye (Amato et al., 2004; Perron and Harris, 2000). Retinal astrocytes and microglia derive from other progenitors (Fig. 1) and immigrate into the retina during early development (Chen et al., 2002a; Ling et al., 1989; Watanabe and Raff, 1988). Oligodendrocytes are restricted to the ON and ensheath the RGC axons, but are not present within the “myelinfree” retina. A recent study in chicken demonstrated that retinal astrocytes and ON oligodendrocytes derive from a common progenitor (Rompani and Cepko, 2010). However, it is worth mentioning that avian and mammalian retinae display some degree of heterogeneity, e.g., different types of glial cells (Fischer et al., 2010). Mammalian RPCs are characterized by their ability to re-enter the cell cycle with subsequent cell division (self-renewal) and

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(re-) expression of specific transcription factors and developmental proteins including Pax6, Sox2, nestin, vimentin, musashi, and Chx10 which are conserved throughout the vertebrate species (Ahmad et al., 2004; Alonso, 2001; Belecky-Adams et al., 1997; Ellis et al., 2004; Hitchcock et al., 1996; Lendahl et al., 1990; Marquardt et al., 2001; Matsushima et al., 2011; Sakakibara et al., 1996). Identification of putative stem/progenitor cells is still hampered by the lack of specific and selective markers. Most available markers are expressed by immature NSC/PCs as well as by mature neuronal and glial cell types. For instance, the transcription factor Pax6 is also expressed in mature amacrine cells (Hitchcock et al., 1996; Karl et al., 2008; Ooto et al., 2004) while the homeobox gene Chx10 is found in mature horizontal cells (Belecky-Adams et al., 1997; Liu et al., 1994). Sox2, another transcription factor, is also expressed in mature Müller glia and retinal astrocytes (Fischer et al., 2010). The intermediate filaments vimentin and nestin are expressed in glial cells and, moreover, significantly increase their immunoreactivity after injury (Wohl et al., 2009; Xue et al., 2006b, 2006a). In addition, nestin is found in a variety of other cell types including endothelial cells (Mokry et al., 2008; Nickerson et al., 2007; Suzuki et al., 2010), pericytes (Alliot et al., 1999; Dore-Duffy et al., 2006) and tumor cells (Ishiwata et al., 2011; Krupkova et al., 2010; Lobo et al., 2004). Accordingly, NSC/PCs can only be identified via colocalization of more than one of these markers. Another means of identification involves using a transgenic mouse model in which NSCs are endogenously labelled by a fluorescent dye such as the nestin-GFP mouse (Yamaguchi et al., 2000). RPCs have been described for different cell types in the vertebrate retina, for both, intrinsically generated and immigrated retinal cells.

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this particular cell type (Reh and Tully, 1986; Reh, 1987). However, the regeneration potential of CMZ-derived cells in the amphibian retina decreases with age (Moshiri et al., 2004). 2.2. CMZ in birds In birds, retinogenesis is complete at hatching and all neurons are functionally integrated. Normal postnatal expansion of the avian eye has been reported to be mainly due to tissue stretching and not a result of cellular growth or addition of new neurons (Amato et al., 2004), since at least 90% of the retinal cells are generated more than 1 week before hatching (Prada et al., 1991). Nevertheless, a CMZ-like proliferative marginal zone has been identified in post-hatched chicken (Fischer and Reh, 2000) and quail (Kubota et al., 2002). The neurogenic potential of these avian marginal cells is fairly restricted compared to cells in fish and amphibians. Noticeable cell proliferation is restricted to a period lasting 2e3 weeks post-hatching, where only a few interneurons, but not ganglion cells are generated. However, this restriction can be overcome after growth factor treatment, indicating that extrinsic rather than intrinsic factors limit CMZ-mediated neurogenesis in post-hatched chicks (Fischer and Reh, 2000). In addition, ocular growth can also be induced via visual deprivation (Fischer, 2005; Reh and Fischer, 2006). In contrast to fish and amphibians, avian CMZ-derived stem cells do not contribute to cellular regeneration processes after retinal injury (Fischer and Reh, 2000; for review see Amato et al., 2004; Fischer, 2005; Moshiri et al., 2004; Reh and Fischer, 2006). 2.3. CMZ in mammals

2.1. Ciliary marginal zone (CMZ) in fish and amphibians Pioneer studies on neurogenesis and cellular repair were performed in fish and amphibians. Retinogenesis in these vertebrates occurs throughout life so that new neurons are generated and functionally integrated in existing circuits, and the retina grows along with the enlarging body (Amato et al., 2004; Hitchcock et al., 2004; Moshiri et al., 2004; Perron and Harris, 2000 for review). Highly proliferative multipotent RSCs are located in the CMZ, a perpetually self-renewing proliferative neuroepithelium (Perron et al., 1998) and peripheral growth zone of the neural retina (Amato et al., 2004; Fischer et al., 2010; Grigoryan, 2001; Harris and Perron, 1998; Perron and Harris, 2000). In the fish eye, growth factor treatment (Boucher and Hitchcock, 1998; Otteson et al., 2002) as well as injury increase proliferation and neurogenesis at the CMZ. However, the regenerated tissue shows a somewhat altered cellular organization (aberrant from the normal structure) after neurotoxic injury (Stenkamp et al., 2001). In addition, specific endogenous stem cells, exclusively generating rod photoreceptors, reside within the central retina in the fish eye. Following injury, these endogenous stem cells can give rise to the full range of retinal neurons (Amato et al., 2004; Hitchcock et al., 2004; Otteson and Hitchcock, 2003), indicating increased stem cell plasticity (multipotency) in response to lesion, owing to environmental changes. More recently, Bernardos and colleagues demonstrated by a lineage-tracing study that these endogenous retinal stem cells in a normal, growing fish retina are Müller glia progeny. Thus, differentiated Müller glia give rise to photoreceptor progenitors. However, the authors do not rule out the possibility that a quiescent retinal stem cell population (non Müller glia) might also be present in the central retina (Bernardos et al., 2007). In amphibians, CMZ stem cells also contribute to cellular regeneration after retinal injury (Kagiwada et al., 2004; Mitashov, 2001). Moreover, the selective loss of particular retinal neurons due to neurotoxic injury stimulates production and neurogenesis of

Mammals also have a CMZ-like zone with single proliferating RSCs in the peripheral retina that differentiate into glial or late neuronal cell types, however this property can only be observed during the first postnatal weeks (Moshiri et al., 2004; Nishiguchi et al., 2008; Zhao et al., 2005). A developmental in vitro study demonstrated that P1-3 dissociated rat retinal cells proliferate, increase in number, and express neuronal and glial markers with the exception of oligodendrocyte markers (Engelhardt et al., 2004). Because the proliferation capacity of these cells is already decreased at P8-14, retinal tissue harvested after P14, or from adult retinae does not give rise to neurospheres (Ahmad et al., 2000; Engelhardt et al., 2004; Tropepe et al., 2000). In vivo studies with postnatal P14 mice receiving two hourly 5-bromo-20 -desoxyuridine (BrdU) injections to label newly generated cells, show only an occasional BrdUþ cell at the retinal margin (Moshiri et al., 2004). At P14, retinogenesis in mice is already concluded (Young, 1985). Downregulation of RSC proliferation capacity at the mammalian retinal margin appears to be mediated by the sonic hedgehog (Shh) pathway (Moshiri et al., 2004). In transgenic mice with only a single functional allele of the Shh receptor patched, persistent proliferating RPCs are observed at the retinal margin. Interestingly, after crossing these transgenic mice with mice having a retinal degeneration background (pro23his rhodopsin transgenic), significantly more proliferating RPCs were found at the retinal margin. Moreover, in these patched; pro23his mice, the generation of retinal neurons including photoreceptors was observed, indicating that injury or disease triggers RPC activation (Moshiri et al., 2004). In addition, exogenous delivery of growth factors including insulin and fibroblast growth factor 2 (FGF2) significantly increases the number of proliferating progenitor cells and induces migration as well as differentiation of these cells into retinal neurons and Müller glia in the postnatal retina (P7-P21) (Zhao et al., 2005). In adult rodents, in vivo proliferation studies using BrdU labeling showed no, or only sparse S-phase cells at the retinal margin as well as

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within the central naïve retina (Ahmad et al., 2000; Kubota et al., 2002; Moshiri et al., 2004). Hence, the naïve adult mammalian eye, including the neural retina, has been described in vivo as having a deficient regenerative capacity and lacking a proliferative zone at the retinal periphery (Kubota et al., 2002). More recently, however, it has been shown that disease pathology can promote RSC/PC activation at the retinal margin (Jian et al., 2009). In adult Royal College of Surgeons (RCS) rats, an animal model for retinitis pigmentosa, significantly more proliferating “CMZ”-progenitor cells were found in vivo than in wild type rats. These results suggest that RSCs in the retinal proliferating marginal regions in mammals have the potential to regenerate retinal neurons following degeneration (Jian et al., 2009). Interestingly, in the primate retina (macaca, up to 15 years of age) all the various types of retinal cells were found in cysts formed in the pars plana suggesting that neurogenesis can occur in adult primate ocular tissue (Fischer et al., 2001). There is increasing evidence to suggest that the peripheral retina and the pars plana of adult monkeys as well as humans contain neural and retinal progenitor cells (Bhatia et al., 2009; Coles et al., 2004; MartinezNavarrete et al., 2008; Mayer et al., 2005). However, definitive evidence suggesting ongoing neurogenesis in the primate retina is currently lacking. On analysing the retinal margin/pars plana of post mortem adult human retinae, RPCs forming neurospheres in the presence of FGF2 (Mayer et al., 2005) or epidermal growth factor (EGF) were identified (Coles et al., 2004). These RPCs isolated from the pars plicata and pars plana of the retinal-ciliary margin (w10.000 cells per eye) formed spheres and displayed 100% selfrenewal and multipotency by regenerating all retinal cell types (Coles et al., 2004). After transplantation in P1 mouse eyes, human RSCs survived, migrated, integrated, and differentiated in the neural retina, especially into photoreceptors (Coles et al., 2004). Moreover, in retinal explants of humans (14e80 years of age), a ciliary maginal-like zone was observed with cells proliferating after EGF treatment in vitro suggesting that the adult human retina possesses regenerative potential (Bhatia et al., 2009). 3. Progenitor-like cells in the rodent eye Remarkably, in the year 2000, two groups independently identified quiescent retinal “stem” cells in the adult mammalian eye (Ahmad et al., 2000; Tropepe et al., 2000). Under culture conditions, these adult RSC/PCs proliferated, i.e. displayed signs of selfrenewal, and generated new retinal neurons both in the presence and absence of endogenous growth factors. However, these RSC/ PCs were not located in the neural retina, but in the non-neural ciliary epithelium (ciliary body, Fig. 2A and B), a structure described as homologous to the CMZ in fish, amphibians, and birds (Ahmad et al., 2000; Tropepe et al., 2000). It is important to point out that these ciliary “stem cells” are fully differentiated, sparsely pigmented epithelial cells (Cicero et al., 2009) in contrast to immature, undifferentiated (non-pigmented) stem cells such as those from the CMZ, found in the eyes of lower vertebrates or in adult mammalian neurogenic brain regions. Accordingly, these cells will henceforth be referred to as progenitor-like cells. Doubts recently rose concerning cell identity and neurogenic potential of these CB-derived cells (Cicero et al., 2009) will be discussed in Section 5.1.5. In the rodent, besides the progenitor-like cells of the rodent CB, pigmented cells of the RPE and iris epithelium have also been reported as being capable of transdifferentiating into neuronal cells with a primary de-differentiation into a more immature cell state and a subsequent differentiation along the neuronal lineage (Asami et al., 2007; Engelhardt et al., 2005). Within the mouse and rat retina, Müller glia, specialized radial glial cells, as well as

retinal astrocytes (which are homologous to those in nonneurogenic regions in the brain) have also been reported to possess progenitor-like characteristics both in vitro and in vivo. Fig. 3 gives a summary of neural progenitor cell-like cells identified in different regions of the rodent eye: Pigmented epithelial cells of the iris, CB (both located in structures of the uveal tract), and pigmented epithelial cells of the retina, as well as progenitor cells of the retinal margin (referred to as “CMZ”) in postnatal eyes, Müller glia and astrocytes, neural progenitor cells (NPCs) of the ON, and, as a novelty, retinal microglia are all present. Based on the reported cellular responses of these different putative progenitor (-like) cell types, we classified three main processes, (1) expression of developmental proteins indicating an immature state of the cell also referred to as de-differentiation (if cells already differentiated), (2) re-entry into the cell cycle and mitosis, i.e., cell division, and, (3) expression of neuronal or glial markers indicating differentiation into neurons, glia or cells of the RPE. Functional recovery can be considered as a possible 4th criterion, however, since there is no conclusive evidence available in the literature with respect to functional integration of newly generated neurons in the mammalian retina, we have omitted this point in our scheme. Fig. 3 illustrates the specific “transdifferentiation” path for every putative progenitor(-like) cell type and presents an overview of published studies performed on postnatal (indicated by “P”), or adult rodent eyes, both in vitro and in vivo. Listed are, to the best of our knowledge, all reports associated with naïve (untreated) eyes of mice and rats, and also studies related to exogenous stimulation, such as growth factor treatment or injury, more precisely neurotoxic injury, ON lesion, ischemia as well as laser injury. These studies will be discussed in the following sections. 4. Activation of dormant stem/progenitor cells through exogenous stimulation NSC/PCs in the mammalian eye are mitotically quiescent. Exogenous delivery of growth factors as well as various forms of retinal injury have been shown to trigger a proliferative response and reacquisition of developmental characteristics in fish (Boucher and Hitchcock, 1998), amphibians (Ikegami et al., 2002; Sakaguchi et al., 1997), birds (Fischer et al., 2002; Fischer and Reh, 2003a), as well as mammals (Karl et al., 2008; Lamba et al., 2008; Ooto et al., 2004). 4.1. Growth factor treatment Growth factors are proteins that regulate a variety of cellular processes, including cell proliferation and differentiation, but also maturation and survival by binding to specific receptors on target cells that activate various signalling cascades (Loughlin and Fallon, 1993). Several classes of growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins (NT) -3 and -4, or glial cell line-derived neurotrophic factor (GDNF), bone morphogenetic proteins (BMPs), and erythropoietin (EPO) secreted by various cell types including glia, neurons, and NSC/PCs per se have been described (Abrous et al., 2005; Arsenijevic, 2003; Barde et al., 1978, 1980, 1982, 1983, 1987; Cameron et al., 1998; Davies et al., 1986; Johnson et al., 1986; Thoenen et al., 1979, 1987; Thoenen and Barde, 1980 for review see Loughlin and Fallon, 1993). During development, these growth factors play essential roles in proliferation, migration, and differentiation processes of NSC/PCs. Therefore, many studies use exogenous growth factors to stimulate and analyse particular cellular responses. The effects of exogenous stimulation with growth factors on progenitor-like cells have been reported in a variety of vertebrate studies (Bi et al., 2009; Fischer, 2005; Karl and Reh, 2010;

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Lamba et al., 2008; Otteson and Hitchcock, 2003, for review). For example, insulin or insulin-like growth factor (IGF) strongly increases the cell proliferation potential of RSCs in fish and birds (Fischer and Reh, 2000; Otteson et al., 2002). In the avian retina, treatment with FGF2 and insulin can induce transdifferentiation of Müller cells (Fischer et al., 2002) and RPE cells (Fischer and Reh, 2001a). Moreover, insulin and FGF2 induce increased neurogenesis of cells from the CB (Fischer and Reh, 2003a) and from the peripheral retinal margin (Fischer and Reh, 2000), and stimulate the regeneration of early born RGCs after neurotoxic injury (Fischer and Reh, 2002). FGF2 and EGF, both influence and/or promote cell division or differentiation processes of NSC/PCs in the brain as well as RSC/PCs in the retina in rodents (Angenieux et al., 2006; Arsenijevic et al., 2001; Close et al., 2006; Das et al., 2005; Giordano et al., 2007; Inoue et al., 2006; James et al., 2004; Liu et al., 2005). Moreover, FGF increased stem cell-renewal and favored differentiation of RSC into photoreceptor-like cells (Giordano et al., 2007). Interestingly, comparable to the observations made in avian eyes (Fischer and Reh, 2002, 2003a), intraocular delivery of FGF2 significantly affects the progenitor-like potential of ciliary or retinal cells, i.e. increases cell division and transdifferentiation into neurons in the naïve mammalian eye (Abdouh and Bernier, 2006; Karl et al., 2008; Karl and Reh, 2010; Lewis et al., 1992). Intraocular application of insulin and FGF2 also induces proliferation and de-differentiation of progenitor-like cells in the adult rat CB (Abdouh and Bernier, 2006), whilst FGF1, insulin, and EGF induce these response in Müller glia of adult mice (Karl et al., 2008). Significantly, following injury, various cells either re-express certain growth factors, or increase their expression levels (Cao et al., 2001; Garcia and Vecino, 2003; Glanzer et al., 2007; Kostyk et al., 1994; Lewis et al., 1992; Wen et al., 1995), in turn, leading to increased cell proliferation, migration, and differentiation. The extent of growth factor release by activated cells and, consequently, the resulting cellular responses appear to be strongly dependent on the type of lesion. The well-established and most commonly employed retinal injury models will be discussed in the next paragraph. 4.2. Injury A variety of studies dealing with the avian retina (Fischer and Reh, 2002; Fischer, 2005) and the rodent brain (Magavi et al., 2000) report that injury to a specific neuronal cell type leads to increased neurogenesis of this phenotype, in other words, celltype-specific cell replacement. For example, after colchicine(induces RGC death), or kainate-mediated injury (induces cell death of amacrine, bipolar, and RGCs) in avian eyes, generation of Brn3þ RGCs was also observed (Fischer and Reh, 2002). Thus, elimination of a specific cell type by injury could be an appropriate method for analysing cell-type-specific cell replacement. 4.2.1. Neurotoxic injury Some of the most common naturally occurring brain toxins inducing neurotoxicity, as a result of excessive dosage, include oxygen radicals, beta amyloid (Ab) or glutamate. Neurotoxic injury based on excitotoxicity is a process resulting from excessive release of excitatory neurotransmitters, such as glutamate, causing damage to neurons and glia and even leading to neuronal cell death. Neurotoxicity can also be experimentally induced by glutamate receptor agonists e.g., N-methyl-D-aspartate (NMDA) or kainic acid (kainate), which are referred to as excitotoxins. Glutamate-mediated excitotoxicity via NMDA receptor activation occurs in various pathological conditions in the brain (e.g., Alzheimer’s and Parkinson disease) (Caudle and Zhang, 2009; Hynd et al., 2004) and retina (e.g., glaucoma, ischemia) (Dreyer et al.,

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1996; Perlman et al., 1996). Since glutamate is the major excitatory neurotransmitter in the vertebrate retina, disruption in the glutamate circuitry results in neuronal loss. Retinal neurons including amacrine cells, photoreceptors, and RGCs express glutamate receptors, the latter, also kainate and NMDA receptors (Osborne et al., 2004 for review). NMDA, a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors are also found on macroglia such as astrocytes (Krebs et al., 2003; Zhou et al., 2010) and Müller cells (Lamas et al., 2005; Puro et al., 1996; Reichenbach and Bringmann, 2010), on oligodendrocytetype-2-astrocyte progenitor cells (O-2A-PCs) (Wang et al., 1996) as well as on microglia (Noda et al., 2000, for review see Gallo and Russell, 1995; Gallo and Ghiani, 2000). The eye is an excellent model organ for analysing the effects of excitotoxins, since these can be injected intraocularly, and, therefore, only specifically affect the structures in the eye. Both NMDA (Karl et al., 2008; Lam et al., 1997) and kainate-based injury models (Chang et al., 2007; Sucher et al., 1991) have been applied to analyse cellular reactions, including the NSC/PC response. Recent studies have shown that NMDA treatment in chicken (Fischer and Reh, 2000, 2001b, 2002, 2003b; Fischer and Omar, 2005) and rodents (Das et al., 2006a; Karl et al., 2008; Ooto et al., 2004), resulted in proliferation of retinal stem/progenitor-like cells as well as in a reacquisition of progenitor-like properties of already differentiated cells which then undergo transdifferentiation. The effect of neurotoxic injury and/or growth factor treatment on retinal stem/ progenitor-like cells will be further discussed in paragraphs 5.1.1. and 6.1.1. 4.2.2. Retinal ischemia/glaucoma Retinal ischemia is by definition a restriction of blood supply to the retina, resulting in a lack of oxygen and glucose (Osborne et al., 2004). Aspects of retinal ischemia pertaining to mechanisms of damage as well as potential therapeutic strategies have been extensively reviewed by Osborne et al. (2004). Ischemia results in accumulation of the excitotoxins glutamate and aspartate in the extracellular fluid (see above), causing neuronal dysfunction and cell death, a process aggravated by the lack of oxygen and glucose. Retinal ischemia leads to selective neuronal cell death, more precisely of RGCs expressing both kainate and NMDA receptors, as well as of amacrine cells and photoreceptors (el-Asrar et al., 1992; Lam et al., 1997; Louzada-Junior et al., 1992; Neal et al., 1994; Perlman et al., 1996; for review see Osborne et al., 2004). As stated above, progenitor cells (Gallo et al., 1995) including O-2A-PCs (Wang et al., 1996), and several glial cells including microglia (Noda et al., 2000) also express glutamate receptors (Gallo and Russell, 1995; Gallo and Ghiani, 2000 for review). In addition, bloodretinal barrier (BRB) breakdown is accompanied by an increased infiltration of immunologic cells from the blood stream and consequently, an increased inflammatory response. Thus, the retina is an ideal structure for studying ischemiainduced neural damage, for two reasons: firstly, due to its major excitatory neurotransmitter glutamate, and secondly, anatomically, the retina is more easily accessible than other CNS tissues. Retinal ischemia can be induced by means of several methods. One, often used and a well-established method in our laboratory, comprises ischemia-induction via elevated intraocular pressure (IOP) (Büchi et al., 1991; Kawai et al., 2001; Krempler et al., 2011; Schmeer et al., 2008). Other methods available include vessel occlusion by transvitreal diathermy (Stefansson, 1990; Stefansson et al., 1990), ligature of ON bundle (Faberowski et al., 1989; Ohira et al., 1990; Stefansson et al., 1988), ligation of the two common carotid arteries (Osborne et al., 1991, 1995), or cauterization of limbal-draining veins using a small vessel cauterizer (Laquis et al., 1998; Shareef et al., 1995; Wang et al., 2000; Xue et al., 2006a;

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Fig. 3. Putative neural progenitor-like cells in the postnatal and the adult rodent eye under the particular experimental conditions. The coloured-boxes behind the citations indicate the processes analysed, i.e., de-differentiation (green box), cell division (red box), and differentiation into neurons (yellow cell/box) or glia (blue cell/box), or both (yellow/blue box), or in RPE (brown box). If a process was analysed but not found, the particular box is crossed. The data presented in this paper are displayed in blue and by an asterisk. The postnatal ages are indicated with “P” for postnatal day, studies without further information indicated were carried out in adult tissue. The generated retinal cell type is also indicated: PC: progenitor cell; RPE: retinal pigment epithelium; PRs: photoreceptors; BCs: bipolar cells; ACs: amacrine cells, MG: Müller glia, A: astrocytes, O: oligodendrocytes; RP: reprogramming, RCS (Royal College of Surgeons), tg: transgenic mouse, GF: growth factors; red citation means that the observed responses were not interpreted as progenitor features.

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for review see Levkovitch-Verbin, 2004; Osborne et al., 2004). All of these procedures lead to hypoxic ischemia and therefore, to neuronal cell loss through apoptosis or/and necrosis (Büchi, 1992; Joo et al., 1999), in particular of RGCs and amacrine cells (Kaur et al., 2008; Lafuente et al., 2002; Osborne et al., 2004). It has also been reported that there is no significant reduction in neurons of the GCL and INL, but only of photoreceptors in the ONL within 4 weeks following ischemia. Hence, retinal ischemia is a useful model for analyzing photoreceptor degeneration (Kim et al., 1998). Increased IOP can lead to glaucoma, an eye disorder that is best described as a type of optic neuropathy. In contrast to acute retinal ischemia, experimentally induced glaucoma (e.g., by means of cauterization of episcleral veins) is a more chronic retinal injury model, with immediate clinical relevance. Untreated glaucoma leads to permanent damage of the ON with ensuing visual field loss, which can progress to blindness (Liesegang, 1996). Glaucomatous retinae are characterized by a reduction of retinal thickness (compression of the retinal layers) and significant loss of RGCs (Garcia-Valenzuela et al., 1995; Kawai et al., 2001; Laquis et al., 1998). Moreover, glaucoma also leads to Müller glia and astrocyte activation, i.e., increase in glial fibrillary acidic protein (GFAP) and nestin expression (Xue et al., 2006a). 4.2.3. Optic nerve lesion ON lesion is an appropriate paradigm for inducing a cell-typespecific cell loss of RGCs, which are amongst the first cells to degenerate and die. RGCs are the projection neurons of the retina which axons fuse at the papilla to the ON. There are two basic types of experimental ON lesions, [1] complete intradural transection known as axotomy, and, [2] a crush lesion, sometimes also termed “axotomy” (albeit incomplete) considered to be a moderate injury (Agudo et al., 2008), for review see (Levkovitch-Verbin, 2004). Crush lesions can be generated to varying degrees with regard to the crush force used (Klöcker et al., 2001) and the duration of squeezing, which may range from 5s to 20s (Berkelaar, 1992; Kretz et al., 2006b; Wohl et al., 2009). In addition, both types of ON lesions can be executed either by intracranial or intraorbital surgery and lesion sites may vary with respect to distance to the eye bulb (Garcia-Valenzuela et al., 1994; Grafstein and Ingoglia, 1982; Misantone et al., 1984; Villegas-Perez et al., 1993). Therefore, variations in these parameters lead to differences in cellular responses and time courses, a critical aspect which is of consequence when comparing results from different studies. ON lesion leads to an interruption of the functional link between RGC somata and their target in the brain, as well as to their supply of necessary neurotrophic factors. Consequently, RGCs undergo death via specific delayed apoptosis following a characteristic timecourse and a peak of cell death of cells between days 5e7 after ON lesion (Berkelaar et al., 1994; Isenmann and Bähr, 1997; Isenmann et al., 1997; Villegas-Perez et al., 1993). Moreover, this type of injury does not lead to a breakdown of BRB (Garcia-Valenzuela and Sharma, 1999; Hou et al., 2004), and therefore, to an increased inflammatory response as occurs after ischemic insults. The underlying mechanisms and the molecular determinants involved in RGC death, in particular following ON lesion, are reviewed in detail by Isenmann et al. (2003). Intriguingly, after ON lesion, there was an increased proliferative cellular response of quiescent cells in the CB (Nickerson et al., 2007; Wohl et al., 2009) and retina (Wohl et al., 2009), concomitant with an increase in the expression of the intermediate filament nestin. Nestin is the major cytoskeletal protein found in the neuroepithelial stem cells of the mammalian brain, as well as in various regions of the adult rodent eye including the CB, retina, and ON (Wohl et al., 2009; Xue et al., 2006b). Nestin, a developmental protein expressed by NSC/PCs is absent in mature neurons, and is therefore often used as a marker for immature

neural cells (Ahmad et al., 2004; Dahlstrand et al., 1995; Hockfield and McKay, 1985; Lendahl et al., 1990). Moreover, it is also considered as an indicator for de- or transdifferentiation (Kohno et al., 2006b; Tackenberg et al., 2009). However, nestin is also expressed in a variety of other cells such as those of the vasculature, testis, muscle, kidney, and skin as well as in tumors. Nevertheless, there is increasing evidence that nestin is related to cell division and migration processes, and hence, to a more immature cell state of these different cell types (for review see Gilyarov, 2008; Michalczyk and Ziman, 2005). In addition, there are also a variety of studies reporting that nestin expression is correlated with neural cells such as proliferating NPCs (Sahlgren et al., 2001; Sunabori et al., 2008; Xue and Yuan, 2010), cultured neurogenic astrocytes (Sergent-Tanguy et al., 2006), reactive astrocytes (Chang et al., 2007; Wohl et al., 2009), and ependymal cells in the spinal cord (Namiki and Tator, 1999). A recent study demonstrated that reduction in nestin expression resulted in a G1 cell cycle arrest as well as in lowering of cortical neurogenesis (Xue and Yuan, 2010). Furthermore, blocking nestin expression by using nestinmorpholino showed nestin as being essential for brain and eye development in Zebrafish since loss of nestin lead to apoptosis of NSC/PCs (Chen et al., 2010). However, nestin has also been described as a “stress” protein, because it is up-regulated in activated glia and is thought to maintain the structural integrity of the tissue (Xue et al., 2006a, 2006b) as discussed in Sections 6.1.2 and 6.1.3. Why do endogenous cells re-enter the cell cycle within the adult retina after ON lesion? Following ON lesion, neurons, in particular RGCs secrete numerous factors (Kostyk et al., 1994; Wen et al., 1995) that act on both adjacent and distal cells. FGF2 (Ooto et al., 2004; Palmer et al., 1999; Zhao et al., 2005) and EGF (Anchan et al., 1991; Close et al., 2006) induce cells to re-enter the cell cycle and, moreover, promote neurogenesis in non-neurogenic CNS regions including the ON and retina. Since similar cellular reactions were observed after ON lesion (Nickerson et al., 2007; Wohl et al., 2009), specifically FGF2 seems to function as one of the important signalling molecules for lesion-induced cell activation (Kostyk et al., 1994; Wen et al., 1995), although the underlying mechanisms of the process are still elusive. 5. Neurogenic potential of progenitor-like epithelial cells in the adult rodent eye Pigmented epithelial cells of retina and the uvea, more precisely of the iris and CB, derive from the neuroepithelium of ectodermal origin from which the CNS develops. These cells are densely packed with pigment granules leading to a dark appearance of the tissue. However, pigmentation per se has no influence on stem/progenitor cell properties of these cells as shown in albino animals (Ahmad et al., 2000; Tropepe et al., 2000). 5.1. Pigmented epithelial cells of the ciliary body The CB is part of the uveal tract and flanks the neural retina in the periphery as a ring-like structure (Fig. 2A). The CB is responsible for accommodation of the lens and generation/segregation of aqueous humour. The lumen of the CB consists mainly of connective tissue as well as the ciliary muscle and blood vessels (Fig. 2B). A bi-layered epithelium outlining the vitreous consists of an inner non-pigmented (production of aqueous humour) and an outer pigmented cell layer containing the quiescent stem cells. The folded area of the CB is named pars plicata and continues in the iris epithelium. The zone between the CB and neural retina is termed pars plana or ora serrata, the border region where the pigmented epithelium ends and the RPE begins. The ciliary and the retinal

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pigment epithelium as well as the neural retina itself share a common neuroectodermal origin (Haruta et al., 2001; Hyer, 2004) and it is therefore not surprising that they possess some common properties in vitro, e.g., increased proliferation and dedifferentiation (expression of common transcription factors such as Pax6, and Chx10) (Amato et al., 2004; Ohta et al., 2008 for review). The first in vitro studies on adult RSC/PCs in the CB were undertaken by Tropepe et al., 2000 in mice and by Ahmad et al., 2000 in rats, in two independent studies. Adult RSCs were found in the pigmented cell layer (Ahmad et al., 2000), known as the pigmented marginal zone (PMZ, Fig. 2B), a structure described to be homologous to the retinal CMZ of lower vertebrates (Tropepe et al., 2000). Under culture conditions, ciliary epithelial stem/progenitor cells (as originally defined by the authors) lose their pigmentation (i.e., they de-differentiate), begin to proliferate and form cell clusters, with or without exogenous growth factor stimulation (Ahmad et al., 2000; Das et al., 2005; Engelhardt et al., 2004, 2005; Giordano et al., 2007; Tropepe et al., 2000). Cultured ciliary epithelial cells express developmental proteins such as nestin and Chx10, which is an indication for an immature neural cell state. After several days in culture, they express proteins of mature glia and neurons, which is a sign for differentiation into a neural cell lineage (Ahmad et al., 2000; Das et al., 2004, 2005; 2006b; Engelhardt et al., 2004; Tropepe et al., 2000; Yanagi et al., 2006). Moreover, generation of photoreceptors can be induced in cultured adult ciliary cells by means of gene transfer (Akagi et al., 2005; Haruta et al., 2001). Fig. 3 provides a comprehensive overview of de-differentiation and cell division of ciliary epithelial cells and their differentiation into neural cells in vitro. In contrast to observations made by Ahmad et al., 2000, Engelhardt and co-workers did not observe expression of oligodendrocyte markers and, therefore, concluded that ciliary stem/ progenitor cells display a more restricted proliferation and differentiation potential than NSCs of the adult rat SVZ (Engelhardt et al., 2004). It is noteworthy that in the “pioneer” studies by Tropepe and Ahmad, only pigmented CB-derived epithelial cells formed neurospheres. Pigmented cells of the RPE and iris, unpigmented cells of the CB, or retina-derived cells did not display such progenitor-like features, (Ahmad et al., 2000; Tropepe et al., 2000), a finding that was contradicted by other groups as discussed in Sections 5.2 and 5.3. In addition, CB-derived “stem” cells not only give rise to neuron-like cells, they can also transdifferentiate into RPE cells (Aruta et al., 2011; Vossmerbaeumer et al., 2008). However, just w0.2% of all CB-derived “stem” cells are actually able to form neurospheres (Tropepe et al., 2000). Similar fractions (w0.1%) are described for the rabbit CB that also harbour RSCs/PCs (Inoue et al., 2005). Since these first studies, other groups have confirmed that ciliary “stem” cells have significant, but very limited, proliferation potential and express characteristic progenitor markers mimicking expression profiles during early retinal development (Engelhardt et al., 2004, 2005; Lord-Grignon et al., 2006; Xu et al., 2007). Analysis of the proliferation/differentiation potential and of the transcriptome profiles of CB-derived “stem” cells from adult rat eyes suggests that these cells may represent a residual population of true RSCs with a restricted neurogenic potential in vivo (Das et al., 2005). In addition, although the proliferation potential in response to growth factor treatment in vitro is quite similar to that of early retinal precursor cells of the embryonic retina, the CB-derived “stem” cells of adult eyes only transiently express certain markers (e.g. Chx10) (Das et al., 2005). Generally, CB-derived “stem” cells can generate both early and late born retinal neurons, however, under culture conditions the generation of early retinal cells seems to be favoured (Das et al., 2005). Cultured ciliary “stem” cells and RPCs of the embryonic retina share 80% of the expressed genes (Ahmad et al., 2004; Das

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et al., 2005) and CB-derived “stem” cells have more genes in common with early rather than with late RPCs (Ahmad et al., 2004; Das et al., 2005). Further, gene expression studies have confirmed that “stem” cells of the CB of adult mice express genes that are involved in early eye development, retinal identity, and cell proliferation (Ahmad et al., 2004; Lord-Grignon et al., 2006). Interestingly, a recent study demonstrated that rare “quiescent” progenitor-like cells, identified as presumptive cone precursor cells, persisted into adulthood and were present even at P120 (Nishiguchi et al., 2008). CB-derived “stem” cells have also been described in other mammalian species. Ciliary epithelial cells derived from porcine (Gu et al., 2007; MacNeil et al., 2007) and human eyes (Moe et al., 2009; Xu et al., 2007) also displayed a putative neurogenic potential. A study of CB-derived progenitorlike cells from adult mice and rats compared to those of humans revealed that regardless of the species, mammalian ciliary “stem” cells have significant, but restricted, proliferation potential and express markers of progenitor cells during development (Xu et al., 2007). Modulation of retinal transcription factor genes such as CHX10 in human ciliary margin-derived RSCs greatly enriches photoreceptor progeny (Inoue et al., 2010). Moreover, these cells can be successfully transplanted into transducin mutant mice and promote functional recovery as shown via electrophysiologic and behavioural tests. Therefore, gene modulation in human RSCs may provide a source of photoreceptor cells for the treatment of photoreceptor disease (Inoue et al., 2010). Stem cells display two major properties or characteristics, 1) unlimited (or, at least, considerable) capacity for self-renewal and, 2) multipotency, i.e. the ability to generate various neural phenotypes from their progeny. Progenitor cells do not fulfil these criteria as they possess limited self-renewal characteristics and generate only few cell types (Reh and Fischer, 2006; Weissman et al., 2001; Yau et al., 2007). Since the reported presumptive retinal “stem” cells are limited in these two characteristics, we believe that these cells more likely represent progenitor-like cells. In addition, adult ciliary “stem” cells are more limited with regard to their selfrenewal and differentiation potential compared to NSCs of the SVZ in rats and humans (Engelhardt et al., 2004; Moe et al., 2009), rat embryonic forebrain-derived precursor cells (Yanagi et al., 2006), or neonatal mouse RSC/PCs (Klassen et al., 2004b; MerhiSoussi et al., 2006). However, this is not really surprising since adult CB-derived “stem” cells are dormant cells and, therefore, are different from permanently cycling NSCs of the brain. Moreover, as CB-derived “stem” cells do not express GFAP, these display a phenotype different from brain NSCs (Morshead et al., 2003; Wang et al., 2010; Zhao et al., 2005). Interestingly, Das et al. (2006b) demonstrated that CB-derived “stem” cells share phenotypic properties and regulatory mechanisms with NSCs elsewhere in the adult CNS, more precisely, they also express GFAP that is typical for NSCs in the brain (Das et al., 2006b). Taken together, data from currently available studies demonstrate that CB-derived “stem” or progenitor-like cells have an intrinsic but limited neurogenic potential which can be induced in vitro, implying that the mitotically quiescent cell state in the naïve tissue is obviously due to a non-permissive microenvironment. 5.1.1. Ciliary progenitor cells after injection of defined factors In the naïve adult chick retina, neurogenesis does not occur (Fischer and Reh, 2003a). However, growth factor treatment with insulin, EGF, or FGF2, stimulates non-pigmented cells of the nonneural CB to proliferate and differentiate into neurons including RGCs (Fischer and Reh, 2003a). Moreover, these growth factors had region-specific effects along the radial axis of the CB: insulin and EGF stimulate proliferation of non-pigmented ciliary cells of the

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pars plana, whilst FGF2 stimulate the proliferation of nonpigmented ciliary cells of the pars plicata. Thus, although chick CB has the capacity to generate retinal neurons, neurogenesis is actively inhibited in the naïve eye (Fischer and Reh, 2003a). Intraocular injection of insulin and FGF2 in the postnatal (P14) rodent eye demonstrated that growth factor treatment leads to an increase in proliferating (BrdUþ) cells in vivo (Das et al., 2004; Zhao et al., 2005) that can give rise to neurospheres when isolated and cultured (Das et al., 2004) (the reported studies are summarized in Fig. 3). Insulin and FGF2 injections in naïve adult rodent eyes also resulted in the re-acquisition of developmental marker expression and re-entry into the cell cycle of a subset of cells that form cell clusters (Abdouh and Bernier, 2006), an indication for dedifferentiation similar to that observed in vitro (Ahmad et al., 2000; Das et al., 2005; Engelhardt et al., 2004; Tropepe et al., 2000). However, both cell migration from the CB to the neural retina and differentiation into neurons or glia was not observed suggesting that a non-permissive environment may restrict the progenitor potential of these ciliary cells and, further, that the environment cannot be bridged by a mere addition of growth factors (Abdouh and Bernier, 2006). Therefore, only application of such factors appears to be insufficient and an inadequate measure to induce a complete neurogenic potential of these cells in vivo. A further study using paroxetine (a selective serotonin reuptake inhibitor [SSRI] and antidepressant drug) treatment showed that this pharmacological agent increased cell proliferation of nestinþ ciliary “stem” cells which formed cell clusters (Wang et al., 2010), an indication for in vivo activation of these quiescent cells. Interestingly, paroxetine had the same effect on NSCs in neurogenic regions of the brain (Wang et al., 2010). Therefore, ciliary adult “stem” cells appear to share similar mechanisms of regulation as NSCs in neurogenic cerebral zones (Wang et al., 2010). However, in the study by Wang and colleagues, cells were not further characterized regarding neuronal markers that would indicate true “transdifferentiation” of ciliary “stem” cells into neurons (Wang et al., 2010) and, hence, the answer to the question whether SSRI may induce neurogenesis remains elusive. 5.1.2. Ciliary progenitor cells after neurotoxic lesion Neurotoxic lesion by means of NMDA or kainate injections has been successfully applied in chicks to increase neurogenesis by CBderived “stem” cells (Reh and Fischer, 2001). To date, there is only one report available relating to neurotoxic injury and its inductive effect on CB-derived “stem” cells in mice (Fig. 3). Intravitreous injection of N-methyl-N-nitrosourea (MNU) causing photoreceptor degeneration leads to an increase in the number of presumptive cone receptors in the CB even in young adult (P75) mice (Nishiguchi et al., 2008). From this data, the authors suggest that the pars plana may be a potential source of photoreceptors for photoreceptor replacement therapy. 5.1.3. Ciliary progenitor cells after optic nerve lesion A first study, published by Nickerson and colleagues in 2007, describes two populations of nestin expressing cells in the uninjured eye (Nickerson et al., 2007). The first population, located around the ciliary vessels, co-expressed the endothelial specific anti-factor 8. The second population found in the epithelium adjacent to the vitreous was perceived as having ciliary progenitorlike properties. In response to ON axotomy, an increase in dividing cells was observed in the CB. Interestingly, following ON lesion, ciliary cell proliferation began before the onset of RGC death (Nickerson et al., 2007; Wohl et al., 2009). Moreover, ON transection led to an up-regulation of the developmental markers nestin and Chx10, which were often (but not always) co-expressed, whereas the progenitor markers Musashi1 or Pax6 and particular

neuronal markers, including doublecortin (migrating neuroblasts) or NeuN (immature/mature neurons) were not found (Nickerson et al., 2007). Yet, a few non-proliferating recoverinþ (a photoreceptor marker) cells were observed in the CB of lesioned eyes. Recoverin, a protein physiologically expressed by photoreceptors and bipolar cells indicates terminal differentiation into photoreceptors (Nickerson et al., 2007) as seen after neurotoxic injury (Nishiguchi et al., 2008). In the study by Nickerson et al., the majority of ciliary cells were nestinþ and some proliferated with a peak at 28 days after lesion. However, only one third of all nestinþ ciliary cells were BrdUþ (Nickerson et al., 2007). We also observed an increase of proliferating cells in the CB of adult mice after ON transection and crush lesion, although, numbers of BrdUþ cells in our study were significantly higher compared to the study by Nickerson and colleagues, and only a few of the BrdUþ cells found in the epithelium co-expressed nestin (Wohl et al., 2009). Moreover, the proliferative response of ciliary cells was stronger after ON transection and further increased over time, whilst the number of proliferating ciliary cells following crush lesion remained low and unaltered (Wohl et al., 2009). However, after both ON transection and crush injury, we observed neither cluster formation (as described after neurotoxic injury or growth factor treatment) nor signs of migration. Furthermore, in accordance with the observations made by Nickerson and colleagues, we found no indication for neuronal (“trans”)differentiation as seen in birds. 5.1.4. Ciliary progenitor cells after retinal ischemia To characterize the nestin expressing “progenitor-like cells” of the adult rodent ciliary epithelium in vivo, we used the green fluorescent protein (GFP)-nestin transgenic mouse (Yamaguchi et al., 2000), which specifically expresses GFP under the control of the nestin promoter in NSCs. Since nestin is a developmental protein expressed by NSC/PCs and is absent in mature neurons (Dahlstrand et al., 1995; Hatten, 1990, 1999; Hockfield and McKay, 1985; Lendahl et al., 1990) “true” NSC/PCs can be visualized by the green dye (Filippov et al., 2003; Keiner et al., 2010; Walker et al., 2010; Yamaguchi et al., 2000). On analysing the naïve CB of these GFPnestin transgenic mice, we did not observe any ciliary epithelial progenitor-like cells that expressed GFP (Fig. 4A, B). In fact, similar observations were already made for naïve CBs of the same GFPnestin transgenic mice at the age of 6 weeks (Kohno et al., 2006b). However, less than 1% of naïve CB-derived cells that display a weak GFP-labeling are detectable by means of FACS analysis (Kohno et al., 2006b) indicating the presence of “true” NSCs within the adult murine CB. Obviously, ciliary epithelial cells require an appropriate stimulus to become “activated” since we found GFP-nestinþ cells in the ciliary epithelium following retinal ischemia, thereby indicating transdifferentiation. Moreover, GFP-nestinþ cells had incorporated BrdU (i.e., proliferated) and were co-stained with nestin antibody, confirming the notion that “true” NPCs are present in the adult mouse CB after retinal ischemia (Fig. 4CeF). In addition, it has been shown that GFP-nestin expression of CB-derived spheres was inducible and increased under culture conditions (Kohno et al., 2006b). GFP-nestinþ cells arise from GFP-nestin cells. Moreover, the expansion of GFP-nestinþ spheres was not due to proliferation, but to integration and reprogramming of adjacent GFP-nestin spheres. Hence, it has been suggested that CB-derived cells differ from retinal and NPCs (Kohno et al., 2006b). Nevertheless, we observed BrdUþnestinGFPþ cells after ischemia in vivo indicating that these cells re-enter the cell cycle and proliferate under certain injury conditions, i.e. an appropriate stimulus. 5.1.5. Ciliary “stem”/progenitor cells: true identity? All of the studies concerning mammalian ciliary “stem” cells describe a population that can be activated after injury. But these

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Fig. 4. Ciliary progenitor-like cells in the eye of adult GFP-nestin transgenic mice. Immunofluorescent labelling with GFP (green), BrdU (red), nestin antisera (F, blue) and DAPI nuclear staining (AeD, blue) in the naïve adult CB (A,B), and 3 days after retinal ischemia (CeF). A, B: in the naïve adult CB, no nestin-GFPþ cell is found in the epithelium. CeF: 3 days after retinal ischemia, proliferating cells (BrdUþ, arrows) were found within the CB adjacent to the iris. Some of them were nestin-GFPþ progenitor-like cells in the ciliary epithelium (asterisks). The micrographs are merged z-stacked images of 1 mm optical sections to illustrate entire cell dimension. TL: transmitted light. Scale bar 50 mm.

cells constitute only a minority of putative stem cells and would not be able to replace the significant numbers of dying neurons. In contrast to the in vitro studies on isolated cells, no neurogenesis, cell migration, or any reliable indication of interaction between adjacent cells in the adult rodent CB was found in vivo, regardless of the exogenous stimulation method used (Abdouh and Bernier, 2006; Nickerson et al., 2007; Wang et al., 2010). In the adult naïve rat eye, induced differentiation of adult CB-derived cells into photoreceptor-like cells was only achieved after gene transfer by means of retroviral injections, i.e. intrinsic changes (Akagi et al., 2004). In the naïve CB, neurogenic potential was observed after growth factor treatment in vivo only in the first postnatal week, which was then seen to continuously decrease, since after P14 a prevailing inhibitory milieu prevents neuronal differentiation (Zhao et al., 2005). Thus, exogenous stimulation only activates a small minority of presumptive CB-derived “stem” cells possibly because growth factor treatment alone does not cause cellular regeneration since there is no cell loss in the uninjured eye. A lesion-induced stimulus does not appear to be sufficient to initiate cell differentiation events and the inhibitory environment in the adult tissue appears insurmountable. Combining growth factor treatment and injury may increase the stimulating effect as already

shown in the CB of chicken, but successful re-initiation of neurogenesis was only inducible shortly after birth (Fischer and Reh, 2002; Fischer, 2005). A further explanation for the neurogenic failure of these ciliary cells may be because these are not true retinal progenitor-like cells. This point of view currently appears to be readily acceptable. Recently, Cicero et al. (2009) demonstrated that CE-derived spheres from mouse and human CBs constitute proliferating pigmented epithelial cells rather than RSC/PCs. All cells found in the spheres had molecular, cellular, and morphological features of differentiated pigmented ciliary cells. After growth factor treatment, about 17% of these cells indeed expressed nestin and also very few cells expressed pan-neuronal markers such as beta-III tubulin. However, this expression, which seems to be induced by growth factors of the medium, was ectopic and transient since the cells retained their epithelial cell morphology and failed to differentiate into retinal neurons (absence of synapses or dendrites) (Cicero et al., 2009). Gualdoni and colleagues also argue against a “stem”- or progenitorlike cell character of adult CB-derived cells (Gualdoni et al., 2010). Although cultured ciliary epithelial cells from adult rats lose their pigmentation, are highly proliferative, and express neural progenitor markers (observations concomitant and compatible with

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reports on ciliary “stem” cells) (Engelhardt et al., 2005; Tropepe et al., 2000), these cells continue to express markers of differentiated ciliary epithelium and typically lack neuronal morphology (Gualdoni et al., 2010). Nicolaissen and his group confirmed that neurospheres derived from human ciliary epithelial cells retain epithelial features such as pigmentation and morphology, whilst at the same time expressing NPC markers such as nestin and Sox2 (Moe et al., 2009). Thus, these recent reports support the notion that ciliary epithelium-derived cells do not appear to represent RSCs that can be used for restoration of retinal neurons after injury or disease. However, it is conceivable that reprogramming or transdifferentiation of these cells may result in RPCs that could be used for clinical purposes (Cicero et al., 2009; Gualdoni et al., 2010). Interestingly, a study on pigmented cells of differentiated RPE cells of adult newts, which are capable of transdifferentiation into retinal neurons, also described a persisting, rudimental expression of epithelial markers during retinal regeneration (Chiba et al., 2006). Therefore, since ciliary and retina epithelium are highly related, adult CB-derived epithelial cells might rudimentarily express epithelial markers on transdifferentiation into retinal neurons, thus, explaining the retained epithelial identity. Moreover, since rodent CB-derived “stem” cells can also give rise to RPE cells (Aruta et al., 2011; Vossmerbaeumer et al., 2008), it is not surprising that characteristics of pigmented cells still persist. Consequently, ciliary epithelial cells are not “true” RSC/PCs, however, they do not appear to be irreversibly committed cells, i.e., are intrinsically restricted. Ciliary epithelial cells are capable of de-/transdifferentiating under appropriate conditions such as ischemia. However, since the cell number of ciliary progenitor-like cells is small (less than 10 nestinGFPþ cells per section and only 2e3 BrdUþ nestin-GFPþ cells per section) and a putative cellular regeneration (including neuronal differentiation, migration and functional integration) occurs fairly slowly, it is unlikely that these local CB-derived progenitor-like cells can be employed for in vivo cell replacement therapy (w300 Brn3aþ RGCs/section, not shown), especially after acute damage. 5.1.6. Nestin expression in other ciliary cell types The nestin antibody-labelled cell population within the CB appeared to be slightly different from the “true” NSCs lineage, since more nestinþ cells were detected by antibody labeling (Kohno et al., 2006b; Nickerson et al., 2007; Wohl et al., 2009). As mentioned above, nestin is also expressed by a variety of cells including ciliary endothelial cells (Nickerson et al., 2007). However, as impressively shown in another study, nestin was only expressed in newly generated, proliferating endothelial cells and not in mature vasculature, further confirming that nestin is a marker for immature cells (Suzuki et al., 2010). Interestingly, in this study nestin expression by endothelial cells was compared to that of NSCs in neurospheres using a GFP-nestin-mouse line, which expresses GFP under the control of the nestin gene neural-specific second intronic enhancer (E/nestin:EGFP) (Johansson et al., 2002; Kawaguchi et al., 2001). While NSCs expressed GFP and were immunoreactive for nestin protein, GFP was not detected in endothelial cells indicating that vascular nestin is expressed without the neural-specific nestin gene enhancer (Suzuki et al., 2010). Here, the authors conclude that both vascular and NSC/PCs nestin filaments (detectable by nestin antibodies) appear to be structurally similar, although, the mechanisms of nestin gene expression were different in endothelial cells and NSC/PCs. In addition, the CB harbours a high density of ciliary microglia and dendritic cells in the ciliary epithelium (Kezic and McMenamin, 2008; McMenamin, 1999; Wohl et al., 2010) as well as macrophages in the ciliary stroma (McMenamin, 1999; Wohl et al., 2010) (Fig. 2B), and functions as a source of immunological cells during development (Chen et al., 2002a), but also after injury (Kezic and

McMenamin, 2008). Herein, we use the term “microglia” to refer to proliferating ciliary immunologic cells, since dendritic cells and macrophages represent mature immunologic, i.e. antigen presenting cells (APCs) that are incapable of re-entering the cell cycle. In contrast, microglia are often referred as a “precursor cell” of mature APCs and is still able to undergo cell division (Guillemin and Brew, 2004). Interestingly, ciliary microglia are highly proliferative and can already divide in situ in the naïve tissue, indicating physiological self-renewal. In response to ON injury, they significantly increased in number due to local cell proliferation (Wohl et al., 2009). Thus, ciliary microglia located at some distance from the lesion site were also clearly activated after ON lesion, however, the biological significance of this “distal” activation remains elusive since there is no increased cellular migration from the CB into the retina (Wohl et al., 2010). Hence, analysis of cell proliferation in the CB after injury should include characterization of immunological cells to exclude a possible mis-identification of proliferating ciliary cells as activated ciliary retinal progenitors since immunological cells have also been shown to ectopically express neural proteins (see Section 8). 5.2. Epithelial cells of the retinal pigmented epithelium (RPE) The RPE located between the retina and the choroid (Fig. 2B) has the function of absorbing stray light and is responsible for phagocytosis of the outer segments of rod photoreceptors as well as for some metabolic functions (Strauss, 2005). In amphibians, but surprisingly not in fish, the RPE is capable of transdifferentiating into neurons and glia in vitro (Ikegami et al., 2002; Susaki and Chiba, 2007). So in these animals the RPE represents the most important source of cellular regeneration as it can regenerate the entire retina (Stone, 1950, for review see Hitchcock et al., 2004; Klassen et al., 2004a). After retinectomy (surgical removal of the retina), RPE cells de-differentiate with a concomitant loss of pigmentation and begin to proliferate, thereby generating a retinal rudiment that gives rise to retinal neurons and glia. However, this potential is only found in urodela (salamanders) and not in anura (frogs) (Hitchcock et al., 2004; Klassen et al., 2004a; Perron and Harris, 2000; Raymond and Hitchcock, 1997, 2000; Reh and Fischer, 2001 for review). RPE of embryonic and post-hatched birds (Fischer and Reh, 2001a; Park and Hollenberg, 1989; Pittack et al., 1997) and, interestingly, also of embryonic rats possesses the ability to transdifferentiate into retinal neurons and still retain the ability to develop into neural retina, but only within a certain developmental stage (Zhao et al., 1995). Whilst the research group under Derek van der Kooy failed to generate neurospheres from murine (Tropepe et al., 2000) and human RPE cells (Coles et al., 2004), Engelhardt and co-workers demonstrated that pigmented cells of the adult rat RPE express neural progenitor markers including nestin and musashi1 in vitro (Engelhardt et al., 2005). Further, these authors showed RPE proliferation, albeit to a limited extent, and differentiation into GFAP/NG2þ glia as well as into doublecortin/beta-III tubulinþ neurons with a typical neuronal morphology (Engelhardt et al., 2005). In addition, peripheral RPE cells of adult rats retained the ability to enter the cell cycle and divide in vivo, although at a low cycling rate (Al-Hussaini et al., 2008). Interestingly, there was a ten-fold increase in the number of cycling RPE cells in albino rats compared to pigmented rats, which is suggested as being due to the lack of L-DOPA (L-3,4dihydroxyphenylalanine), an upstream element in the synthetic pathway of melanin that initiates cell cycle exit (Al-Hussaini et al., 2008). However, even though mammalian RPE retains the ability to proliferate, it appears to be deficient in the regulatory elements required to control cell division and induction of transdifferentiation (Al-Hussaini et al., 2008). All studies relating to RPE

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cells are summarized in Fig. 3. Interestingly, RPE cells from humans are also reported as being capable of differentiating into beta-III tubulinþ, MAP2þ, and neurofilamentþ neurons in vitro, while no GFAPþ or rhodopsinþ cells were observed (Amemiya et al., 2004). This potential was found in young as well as in aged tissue, although the number of mature neurons was greater in the younger compared to the older cell line (Amemiya et al., 2004). 5.3. Epithelial cells of the iris The iris, a circular muscular structure in the eye (Fig. 2A) controls the amount of light entering the eye and reaching the retina by adjusting the diameter and size of the pupil. Epithelial cells of the iris from amphibians (Eguchi, 1986), birds (Sun et al., 2006), and rodents (Asami et al., 2007; Haruta et al., 2001) have also been reported as displaying progenitor-like characteristics under culture conditions, including cell proliferation and expression of developmental proteins. Studies on rodent iris cells are summarized in Fig. 3. Isolated iris-derived cells from postnatal (at least P3) and adult mice and rats displayed a multipotent stem cell character (Asami et al., 2007). Within the bi-layered iris epithelium, nestinþ progenitors were only found in the inner layer, adjacent to the eye chambers, (Asami et al., 2007). By using the transgene GFP-nestin mouse (Yamaguchi et al., 2000), iris epithelial cells were indeed shown to be true NSCs, however nestin-GFP expression declined with age (P5: 6e10%, P30: w0.3%) (Asami et al., 2007). Iris-derived cells proliferate in response to growth factors and retain the capacity to transdifferentiate. Moreover, under culture conditions and in the presence of FGF2, iris tissue from adult rats displayed signs of neuronal transdifferentiation, in particular into photoreceptor-like cells (Haruta et al., 2001). By means of gene transfer, iris-epithelial cells can be induced to generate photoreceptors in rats (Akagi et al., 2004). A comparative study of adult iris-derived cultured progenitor cells of both rats and primates revealed that iris epithelial cells display similar properties in both species, i.e. differentiation into photoreceptor-specific phenotypes by induction of transcription factors (Akagi et al., 2005). Therefore, iris-derived progenitors have been suggested as possible candidates for photoreceptor regeneration for use in patients (Akagi, 2005). More recently, MacNeil and colleagues demonstrated that pigmented porcine iris cells also posses RPC characteristics, i.e. proliferate and form spheres, express progenitor markers, and may give rise to neurons and glia (MacNeil et al., 2007). In addition, human iris tissue proliferated and expressed markers of RPCs such as Pax6, Sox2 and nestin in vivo (Froen et al., 2011). However, many cells retained properties of differentiated epithelial cells and lacked central properties of somatic stem cells (Froen et al., 2011). Therefore, although iris epithelial cells also possess “immature” cell features, they represent differentiated cells rather than stem cells and are very limited in number. Functional studies are necessary to determine whether iris-derived cells fully differentiate into genuine photoreceptors. 6. Neurogenic potential of progenitor-like glial cells in the adult rodent retina The two main macroglial cell types within the retina constitute the specialized Müller cells which are only found in the retina and the retinal astrocytes. Stem cell/progenitor cell characteristics have been described in the adult rodent eye for both macroglial cell types. 6.1. Progenitor-like Müller glia in the retina Müller cells are a type of specialized radial macroglia in the vertebrate retina that form the majority of retinal glia comprising

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about 5% of all retinal cells (Jadhav et al., 2009). Müller glia somata located in the INL have processes that span the retina from the vitreal surface to the RPE, exhibiting a radial, bipolar shape (Fig. 2B). Müller glial cells are responsible for maintaining homeostasis of the retinal extracellular milieu, supplying neurons with nutrients, contributing to the establishment of the BRB, providing structural support, and regulating the blood flow. An extensive characterisation of Müller glia cells in the healthy and in diseased retina is given in (Bringmann and Reichenbach, 2001; Bringmann et al., 2006, 2009; Newman and Reichenbach, 1996; Reichenbach et al., 2007) and is also described at length in an excellent book by Reichenbach and Bringmann (2010). Similar to astrocytes in the brain, Müller glia express GFAP and glutamine synthetase. Following injury, Müller glia become activated and build a protective scar that structurally separates degenerating from healthy tissue. Reactive gliosis is correlated with proliferation and changes in the protein expression profile, mainly by up-regulation of cytokines, neurotrophic factors or enzymes. Reactive glia also participate in wound healing, stabilizing damaged tissue, attracting inflammatory cells and enhancing neuronal survival. Finally, reactive glia also promote cell death (Bringmann et al., 2006; Eddleston and Mucke, 1993). In the vertebrate retina, Müller cells also exhibit neurogenic properties. In the naïve fish retina, occasional Müller glia dedifferentiate and give rise to rod photoreceptor progenitors that generate rods throughout life (Bernardos et al., 2007). After injury, Müller cells re-enter the cell cycle (Braisted et al., 1994; Raymond et al., 2006; Yurco and Cameron, 2005), de-differentiate, and can function as multipotent RSCs that generate retinal neurons in vivo (Bernardos et al., 2007; Fausett and Goldman, 2006; Fimbel et al., 2007; Hitchcock et al., 2004; Raymond et al., 2006; Wu et al., 2001; Yurco and Cameron, 2005). In this aspect, naïve fish Müller glia are GFAPþ unlike those of birds and mammals which are GFAP. Since GFAP is reported to be expressed by NSCs of the neurogenic niches in mammals, this could be an indication for a more immature cell state of fish Müller glia per se. However, fish Müller cells also function as “mature” glia and it is also conceivable that only a subpopulation is able to “transdifferentiate” into neurons. Further research is certainly required to illuminate this aspect. In the avian retina, following NMDA-mediated lesion (which induces amacrine cell death), more than half of all Müller cells became activated, proliferated, and de-differentiated. Although the majority of cells remained undifferentiated, a small number transdifferentiated into retinal neurons, in particular into amacrine and bipolar cells (Fischer and Reh, 2001b). Interestingly, there is cell-type-specific cell replacement of neurons that have been selectively injured (Fischer and Reh, 2002). Generation of early born Brn3þ RGCs was observed after colchicine- or kainatemediated injuries which both cause death of RGCs (Fischer and Reh, 2002). However, additional treatment with FGF2 and insulin was required for this cellular “regeneration” (Fischer and Reh, 2002). Noteworthy, in particular Müller cells located at the periphery, and to a lesser extent those of the central retina respond to neurotoxic injury and/or growth factor treatment by transdifferentiation, indicating that peripheral Müller cells are more plastic and immature than the cells in the center (Fischer and Reh, 2002, 2003b). Müller glia transdifferentiation, however, also occurred without injury simply by stimulation with growth factors (FGF2 and insulin) whereby the majority of transdifferentiated Müller glia cells remained undifferentiated, w24% differentiated into Müller glia and only 4% express neuronal markers (Fischer et al., 2002), for review see (Fischer and Reh, 2003b; Fischer, 2005). It is notable that Müller glia of the mature mammalian retina express genes for NPCs as shown by specific gene expression assays (Livesey et al., 2004; Roesch et al., 2008). In addition, they also

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possess stem cell potential, i.e., they have the ability to generate all three basic glial cell types of the CNS as well as retinal neurons including photoreceptors, a feature that has recently been demonstrated in vitro (Das et al., 2006a; Monnin et al., 2007; Nickerson et al., 2008; Takeda et al., 2008; Wan et al., 2008). In the naïve adult rodent retina, Müller glia maintain a dormant state (Das et al., 2006a), however, in response to injury they become activated, proliferate (Dyer and Cepko, 2000; Karl et al., 2008; Ooto et al., 2004; Takeda et al., 2008), and change their gene expression pattern, e.g. they up-regulate GFAP (Kim et al., 1998; Lewis and Fisher, 2003; Ooto et al., 2004; Panagis et al., 2005), and nestin (Kohno et al., 2006a; Wohl et al., 2009; Xue et al., 2006a, 2006b; for review see Bringmann et al., 2006, 2009; Reichenbach and Bringmann, 2010). Further, rodent Müller glia can also generate retinal neurons after neurotoxic injury (Karl et al., 2008; Ooto et al., 2004; Takeda et al., 2008). Additional growth factor delivery increased Müller glia transdifferentiation into retinal neurons (Karl et al., 2008), while growth factor treatment alone rarely activates Müller cells (Close et al., 2006; Karl et al., 2008). This issue will be addressed in detail in the next section. Interestingly, in human retinae, a population of Müller glia possessing stem cell characteristics have been identified, suggesting that human Müller glia could also act as endogenous stem cells after injury or disease (Lawrence et al., 2007; Limb et al., 2004, 2005). 6.1.1. Müller glia after neurotoxic injury and growth factor treatment Essential information from all published studies relating to Müller cells are summarized in Fig. 3. The first study on neurotoxic injury reported by Dyer and Cepko (2000) involved injecting the neurotoxins ouabain (an inhibitor of the Na/K exchange pump) and domoic acid (a kainic acid analogue) which resulted in increased Müller glia proliferation that was associated with changes in gene expression (downregulation of the tumor suppressor protein p27Kip1) indicating reactive gliosis (Dyer and Cepko, 2000). A few years later, Ooto and colleagues (2004) demonstrated that Müller cells proliferate, de-differentiate and are capable of producing bipolar cells and photoreceptors, though in limited numbers, after neurotoxic injury via injecting NMDA (Ooto et al., 2004). Moreover, NMDA- and kainate-induced injury in P21 rats (referred to as adult tissue) revealed Müller glia as possessing NSC properties, including cell cycle re-entry and expression of NSC/PC markers such as Pax6, nestin, and musashi1, which are influenced by the Notch and Wnt pathways (Das et al., 2006a). In adult rats, proliferating nestinþ Müller cells were found only a few days after kainate treatment and expressed the brain lipid-binding protein (BLBP) known as a marker for radial glial in the immature brain (Hartfuss et al., 2001), indicating Müller cell de-(trans)differentiation (Chang et al., 2007). More recently, Thomas Reh’s group, involved in extensive studies of neurogenesis in the chicken retina (for review see Fischer and Reh, 2003b; Fischer, 2005; Reh and Fischer, 2006), demonstrated that Müller glia of adult mice proliferate and dedifferentiate in response to NMDA treatment (Karl et al., 2008). However, an additional stimulation by means of growth factor treatment including EGF, FGF1, or the combination of FGF1 and insulin was required to enhance stimulation of cell division and possibly further support amacrine differentiation, a cell fate which has not previously been detected (Ooto et al., 2004). After intraocular injection of the toxin MNU which induces photoreceptor degeneration, de-differentiation and proliferation of Müller glia was observed leading mainly to transdifferentiation into photoreceptor-like cells in both P7 (Wan et al., 2007) and adult mice (Wan et al., 2008). Intraocular delivery of Shh-protein, a potent Müller glia mitogen in vitro, also stimulates proliferation of Müller glia-derived progenitors after retinal injury (Wan et al.,

2007). Interestingly, in adult mice, intraocular injection of glutamate and its analogue a-aminoadipate directly stimulate Müller glia to re-enter the cell cycle and transdifferentiate into neurons both in vivo and in vitro (Takeda et al., 2008). Moreover, a-aminoadipate induces expression of neuronal markers including b-III tubulin, PKCa and recoverin in cultured Müller cells, and, further, stimulates Müller cell migration towards the ONL which subsequently differentiate into photoreceptors in vivo (Takeda et al., 2008). Therefore, Müller cells appear to be restricted to giving rise to interneurons or photoreceptors following neurotoxic injury in adult mice in vivo (Karl et al., 2008; Ooto et al., 2004; Takeda et al., 2008; Wan et al., 2008). However, it has to be pointed out that only very small numbers of neurons are generated. Accordingly, Karl and his colleagues reported that only 0.04% of all Müller cells transdifferentiated into neuronal (amacrine) cells (Karl et al., 2008). 6.1.2. Müller glia after optic nerve injury After ON lesion, Müller cell proliferation was also observed in adult rats (Panagis et al., 2005) and mice (Wohl et al., 2009) although they made up only a minority of all proliferating cells (Wohl et al., 2009). Interestingly, BrdUþ Müller cells were observed in both, ipsi- and contralateral eyes (Panagis et al., 2005). Moreover, in response to ON lesion, Müller glia in adult rats (Xue et al., 2006b) and mice (Wohl et al., 2009) display an increase in nestin expression throughout the retina (summarized in Fig. 3). However, the significance of nestin expression in damaged retinas remains controversial, and it has been argued that increased expression of this intermediate filament may be responsible for maintaining structural integrity of these support cells (Xue et al., 2006b) rather than indicating a more immature cell state, as has been suggested by others (Kohno et al., 2006a; Walcott and Provis, 2003). In our own ongoing study, we found vimentin, also known as neural progenitor and “stress” marker, co-expressed with nestin in Müller glia (Wohl et al., 2009). However, in long-term studies, we observed no “ectopic” neuronal marker expression after ON injury and we also found no signs of transdifferentiation of Müller glia (unpublished results). These observations are in contrast to those made after neurotoxic injury in other studies (Karl et al., 2008; Ooto et al., 2004) and suggest that the injury type and, consequently, the underlying cellular mechanisms are relevant with regard to stimulation of the neurogenic potential of Müller glia. In other words, “neuro(neo)genesis” can not be induced to a similar extent by every type of exogenous stimulation. 6.1.3. Müller glia after ischemia/glaucoma Müller cell reaction in adult rats after ischemia induced by a transient increase of intraocular pressure for 60 min showed that at already 1 h after ischemia, Müller glia significantly up-regulate GFAP and become hypertrophic, a transient reaction that persists for a few days (Kim et al., 1998). Interestingly, 1 day after reperfusion, a few Müller glia somata appear to migrate into the IPL as they do during retinogenesis (Baye and Link, 2008). This interkinetic nuclear migration could point to a progenitor-like property of Müller glia (Kim et al., 1998) (see Fig. 3 for summary). After induction of transient retinal ischemia (duration 75 min) in adult albino rats, we observed single proliferating (BrdUþ) Müller cells. These dividing Müller cells were hypertrophic and were mainly located in the center of the retina (unpublished results). Further, our examination of retinae of naïve adult GFP-nestin transgenic mice for visualization of true NSCs (Yamaguchi et al., 2000) demonstrated that some, but not all, Müller glia express nestin and remain in the adult tissue, further suggesting that these Müller glia might be “true” NSC/PCs (Fig. 5A). However, GFP-nestinþ Müller glia are not located in the retinal periphery where the more

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Fig. 5. Müller cells in the retinal of adult GFP-nestin transgenic mice. Immunofluorescent labelling for GFP (green), BrdU (red), and DAPI nuclear staining (blue) in the naïve adult mouse retina. AeC: in the unlesioned retina, nestin-GFP labelling, indicating an immature cell state, was found in some Müller glia cells which are arranged in cell clusters. In the periphery, nestin-GFPþ Müller glia were hardly found, while in the central retina, the density of nestin-GFPþ Müller glia was increased (A). In the naïve retina, Müller glia were long and filigree (B, C). Three days after retinal ischemia, they became hypertrophic (D, E), however did not proliferate. The micrographs are merged z-stacked images of 1 mm optical sections to illustrate entire cell dimension. Scale bar 50 mm.

immature cells are located as confirmed in previous reports (Bhatia et al., 2009; Karl et al., 2008). Instead, they unexpectedly reside in the central retina, a notion that casts doubt on their alleged immature state. Notably, these GFP-nestinþ Müller glia are arranged in cell clusters of about 5 to 20 cells in both naïve (Fig. 5B, C) and injured retinae (Fig. 5D, E). After retinal ischemia, the long and filigree Müller glia cells became hypertrophic, but proliferation was not observed in the acute phase (Fig. 5D, E). The question as to whether these cells are capable of transdifferentiating into latestage retinal neurons such as amacrine cells after retinal ischemia is a major issue of ongoing investigation in our laboratory. Cauterisation of episcleral veins results in a chronic increase of intraocular pressure leading to enhanced expression of nestin and GFAP in Müller glia. Upregulation of these intermediate filaments was interpreted as an indication for the reactive state/gliosis in response to injury and was not discussed in the context of proliferation and neurogenic potential (Xue et al., 2006a). In a transgenic glaucoma mouse model (DBA/2J), it was shown that progressive glaucoma leads to GFAP up-regulation in Müller glia and astrocytes, and the proliferating cells were identified as microglia and pericytes (Inman and Horner, 2007). Thus, in contrast to reactive gliosis in response to trauma in other CNS regions, the chronic model of glaucoma is not accompanied by macroglial proliferation (Inman and Horner, 2007).

Finally, a recent study has reported that transplantation of cells from the human Müller stem cell line (MIO-M1) into glaucomatous rat eyes causes these cells to differentiate into neurons and glia. However, functional integration of these cells into the retina requires a local modulation of the retinal environment using chondroitinase ABC or EPO (Bull et al., 2008). Thus, cell replacement therapy using Müller glia in ischemic/glaucomatous retinae in rodents/mammals is still complex and challenging, requiring further research. 6.1.4. Müller glia after laser injury After laser injury to adult rat retinae, an increased expression of nestin and vimentin in retinal Müller glia was observed that was concomitant with an increased local cell proliferation, indicating Müller glia de-differentiation (Kohno et al., 2006a). Laser photocoagulation performed in the region of the posterior pole lead to spots ranging from 150 to 200 mm. By means of anti- Ki67 and cyclin D1 labeling, in situ Müller cell division was observed 3 days after injury, but not after 7 days (Kohno et al., 2006a). However, more recent data from retinal laser photocoagulation confirmed local proliferation of nestinþ Müller cells at the lesion site and nuclear migration towards the injury site, although no evidence for transdifferentiation was seen within one week after laser injury (Tackenberg et al., 2009) (see Fig. 3 for summary). Retinal

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progenitor markers including Sox2, Chx10 were not expressed after laser injury. Therefore, the authors concluded that this injury model is not appropriate for stimulation of transdifferentiation or neurogenesis in the retina (Tackenberg et al., 2009). However, longterm observations of up to at least 4 weeks after injury should be considered to confirm this statement. 6.1.5. Müller glia as true retinal progenitors? All studies discussed herein show that rodent retinal Müller cells possess progenitor-like characteristics. However, these cells represent only a very small population that can be induced to “transdifferentiate” and function as a source for new neurons in vivo. A few investigators have recently started to shed some light on the cell signalling pathways that control the ability of mature Müller glia to become progenitor-like cells (Fischer and Bongini, 2010 for review). Interestingly, Müller glia express a large repertoire of genes for late RPCs (Livesey et al., 2004; Roesch et al., 2008). A possible explanation for this fact has been supplied by Jadhav et al. (2009), who suggest that late RPCs may develop into Müller glia without undergoing an irreversible cell fate determination event (Jadhav et al., 2009). Retinal neurons irreversibly exit the cell cycle, turn off most progenitor genes, enter a differentiation program, and appear to be irreversibly locked in their fate. In contrast, RPCs that transform into Müller glia retain much of their progenitor gene expression program, particularly genes of the late retinal progenitor cells (Jadhav et al., 2009). The authors further suggest that Müller glia do not irreversibly differentiate because some genes expressed at high levels, which appear to be necessary for Müller glial function, can be turned off under certain (e.g. after injury) conditions. Therefore, late progenitor cells that give rise to Müller glial cells do not irreversibly leave the progenitor cell state, but add the expression of genes that are required for glial function to the repertoire expressed by late retinal progenitor cells (Jadhav et al., 2009). This appears to be plausible and could explain why more immature Müller cells were found in the periphery (Bhatia et al., 2009; Karl et al., 2008), the region where late progenitors persist (Nishiguchi et al., 2008). Moreover, after retinal injury there is no hyperproliferation of Müller glia in contrast to glial proliferation in other regions of the CNS. This is obviously due to a highly regulated mechanism preventing cell cycle re-entry as protection against uncontrolled cell division, and hence of tumor formation (Dyer and Cepko, 2000 for review see Jadhav et al., 2009). The group around Andreas Reichenbach convincingly demonstrated that Müller cells possess not only typical glial functions such as maintenance of homeostasis (Bringmann et al., 2006), but also operate as “living optical fibers” and are able to conduct light (Franze et al., 2007, for detail see Reichenbach and Bringmann, 2010). Hence, since these specialized retinal macroglia appear to be essential in the visual process, maintenance of their functional phenotype is necessary, and therefore, they might be impeded to act as stem cells for cell replacement after injury in the highly complex rodent retina.

and Mucke, 1993; Panagis et al., 2005; Wohl et al., 2009; Xue et al., 2006a), but often to a lesser extent than Müller cells (Vazquez-Chona et al., 2004; Wen et al., 1995). Interestingly, the majority of proliferating macroglia after an ON lesion in rats were astrocytes, not Müller glia (Panagis et al., 2005; Wohl et al., 2009). All relevant studies pertaining to this aspect are summarized in Fig. 3. After ON lesion, there was also an up-regulation in nestin immunoreactivity which may indicate the reactive state, but could also be interpreted as a characteristic for a more undifferentiated/ immature state (Wohl et al., 2009). Following kainate-induced lesion, retinal astrocytes express nestin and the early neuronal marker doublecortin several days after injury (Chang et al., 2007). This could point to a transdifferentiation potential of retinal astrocytes into neurons after injury similar to that of cerebral astrocytes of non-neurogenic regions (Costa et al., 2010). Recent reports from Magdalena Götz’s group state that parenchymal type1 astrocytes of non-neurogenic regions of the brain have stem cell potential (Costa et al., 2010). In the naïve brain, these parenchymal cerebral astrocytes are also mitotically quiescent, but proliferate in response to injury and participate in glial scar formation. Nevertheless, they retain the ability to generate neurons in vitro (Buffo et al., 2008; Costa et al., 2010) and therefore, display stem cell properties like the astrocytes in neurogenic brain regions, i.e. SVZ (Doetsch et al., 1999) and dentate gyrus of the hippocampus (Seri et al., 2001; for review see Costa et al., 2010). Since retinal astrocytes are immigrants from the brain, they are expected to possess intrinsic stem cell potential comparable to that of cerebral astrocytes. However, after NMDA-induced neurotoxic injury, none (Ooto et al., 2004) or hardly any (Karl et al., 2008) dividing and/or nestinexpressing retinal astrocytes were observed and, to date, there are no further reports available with regard to retinal astrocytes and their possible neurogenic potential after neurotoxic lesion. On analysing transgenic nestin expression via GFP fluorescence in GFPnestin transgenic mice (Yamaguchi et al., 2000), we observed that reactive astrocytes express nestin protein after retinal ischemia (Fig. 6AeB0 ). However, we found no GFP-nestin expression in retinal S100betaþGFAPþ astrocytes both in naïve and lesioned retinae (Fig. 6B), indicating that retinal astrocytes are not “true” NSCs. GFP-nestin was restricted to the Müller glia end-feet that were also arranged in clusters. Therefore, we conclude that antibody-labeling of nestin, which was observed in retinal astrocytes in a variety of studies including our own (Wohl et al., 2009; Xue et al., 2006a, 2006b), indicates a reactive cell state with gliosis as already discussed (Xue et al., 2010), although glial scar formation occurs at the lesion site behind the eye ball and within the ON (Sun et al., 2010; Wohl et al., 2009). Hence, it may be conceivable that retinal astrocytes can also be genetically manipulated and reprogrammed to function as progenitors after injury as shown in the brain (Buffo et al., 2008; Costa et al., 2010). However, the low cell numbers of retinal astrocytes, make it quite unlikely that they can be used for clinical approaches or cellular therapies. 7. Progenitor-like cells in the optic nerve

6.2. Progenitor-like astrocytes in the retina In mammals, retinal astrocytes, more precisely type-1 astrocytes, are located in the NFL/GCL (Fig. 2B). During early development, they migrate from the brain to the eye and do not arise from RPCs (Ling et al., 1989; Watanabe and Raff, 1988, see Fig. 1). Type-1 astrocytes are also present in the ON where they build a framework as well as participate in BRB and can re-enter the cell cycle after injury (Ling et al., 1989; Miller et al., 1989; Watanabe and Raff, 1988). After injury, retinal astrocytes become activated, i.e. they increase in number and cell size and up-regulate GFAP (Eddleston

The adult ON harbours glial progenitor cells, namely O-2A PCs, which derive from neural stem cells (radial glia) and migrate from the brain into the ON during CNS development (Ffrench-Constant and Raff, 1986; Wolswijk and Noble, 1989, Fig. 1; for review see Miller et al., 1989; Richardson et al., 2011). In vitro perinatal (P6/7) (Kondo and Raff, 2000; Raff et al., 1983) as well as adult O-2A progenitors (Ffrench-Constant and Raff, 1986; Wolswijk and Noble, 1989) have the ability to proliferate, migrate, and differentiate into oligodendrocytes or type-2 astrocytes depending on culture conditions. Compared to perinatal O-2A-PCs, those of adult nerves are distinct in morphology and display a more restricted

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Fig. 6. Astrocytes in the retina and the optic nerve (ON) in ischemic adult GFP-nestin transgenic mice. Immunofluorescent labelling with GFP-nestin (green), S100b (A, B: red), nestin (AeC: blue; D, red), and DAPI nuclear staining (C, D blue) of the adult mouse retina (AeB0 , flat mount) or ON (C, D, horizontal cross section) after injury. After retinal ischemia, nestin-GFP expression was not found in retinal astrocytes or in cells within the ON. However, 7 days after ischemia, nestin protein immunoreactivity was found in reactive astrocytes (B, B0 arrow). The micrographs are merged z-stacked images of 1 mm optical sections to illustrate entire cell dimension. ON: optic nerve. Scale bar in A, C, D 50 mm, in B, B0 20 mm.

proliferation, differentiation, and migration potential (Wolswijk and Noble, 1989; Wren et al., 1992). However, adult O-2A-PCs express at least some properties of stem cells (Wren et al., 1992). Interestingly, following growth factor treatment in vitro, newborn (Omlin and Waldmeyer, 1989), perinatal (P6) (Kondo and Raff, 2000) and also adult O-2A PCs from the ON (Palmer et al., 1999) display a neurogenic potential, i.e. express neuronal markers like beta-III tubulin or neurofilaments (Kondo and Raff, 2000; Omlin and Waldmeyer, 1989; Palmer et al., 1999) (studies are summarized in Fig. 3). O-2A PCs from the ON belong to the NG2-glia (Butt et al., 1999, 2002; 2004, 2005; Nishiyama et al., 1999, 2001; 2009; Trotter et al., 2010; Zhu et al., 2011) which express the chondroitin sulphate proteoglycan 4 (CSPG4), in rats also termed nerve/glial antigen-2 (NG2) (Stallcup and Beasley, 1987). Whether NG2-glia can act as NSCs is presently a matter of debate. A few studies report that proliferating NG2-glia from the piriform and neocortex express the neuronal marker doublecortin (Guo et al., 2010; Tamura et al., 2007). Moreover, transdifferentiation of postmitotic NG2-glia

into neurons of the piriform cortex has been suggested (Rivers et al., 2008), however validation of this finding requires further research (for review see Richardson et al., 2011). Regarding the ON, one issue that needs to be addressed is whether progenitor cells remaining in the adult ON have the potential to give rise to retinal neurons, in particular after injury. Following injury, reactive proliferating astrocytes release growth factors such as FGF2 (Kostyk et al., 1994) which promote “transdifferentiation/reprogramming” into neurons in vitro (Kondo and Raff, 2000; Palmer et al., 1999). Therefore, it is conceivable that ON oligodendrocyte progenitor cells (NG2-glia) reprogramming might also be feasible in vivo and could function as a potential source for retinal neurogenesis as well. In a previous study, we observed nestinþBrdUþGFAP cells in the ON (labelled with a specific antibody) and assumed that some of these cells are O-2A-PCs (Wohl et al., 2009). Nestin antibody labels nestinþ cells independent from the origin of tissue. Thus, not only neural cells including glia were stained, but also endothelial

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cells and pericytes. Therefore, we used the GFP-nestin mouse in which specifically “true” NSC/PCs, but not endothelial cells are labelled (Yamaguchi et al., 2000). However, we did not observe any GFP-nestinþ cell within the nerve which would indicate presence and response of true NPCs either in naïve or in lesioned ON (Fig. 6C, D). Interestingly, NPCs of the dentate gyrus of the hippocampus or the SVZ only rise to new neurons and are accordingly neuroblasts. In contrast, O2-A-PCs whose progeny are exclusively glial cells (type 2 astrocytes and oligodendrocytes) constitute glioblasts. Thus, it is likely that GFP-nestin is only expressed by neuroblasts, but not by glioblasts. Moreover, after ON lesion, there was no indication of cell migration from the ON into the retina of any of the cell types (Wohl et al., 2009). A possible immigration of cells, progenitors or other types from the ON into the retina as occurs during development is prevented by specialized astrocytes in the lamina cribrosa that create an environment hostile to immigrating cells or processes in the adult state (Miller et al., 1989; Perry and Lund, 1990; Wolburg and Buerle, 1993). These astrocytes with their numerous processes form a dense meshwork and, therefore, represent a mechanical barrier adjacent to the nerve head (Miller et al., 1989; Sun et al., 2010; Wolburg and Buerle, 1993). In addition, at the lesion site, inhibitory molecules such as glycoproteins or cytokines prevent functional neurogenesis or regenerative events (Alonso, 2005; Fawcett and Asher, 1999; Monje et al., 2003; Streit et al., 1999). Following brain injury, oligodendrocyte precursor cells are stimulated to re-enter cell cycle, divide, accumulate at the lesion site, and participate in glial scar formation together with microglia and astrocytes (Chen et al., 2002b). There is no evidence that OPCs/NG2-glia differentiate into neurons in the brain in vivo. Nevertheless, the possibility that single progenitor cells migrate from the ON into the retina can not be excluded and future studies have to clarify the possible neuronal potential of adult OPCs/NG2glia in vivo. 8. Microglia as putative progenitors Microglia, the immunocompetent cells of the CNS, are of mesodermal origin and invade the retina through the ON and the CB via blood vessels during development (Barron, 1995; Chen et al., 2002a). Ramified parenchymal microglia enter the retina prior to vascularization (embryonic stages), while perivascular microglia immigrate thereafter (Chen et al., 2002a for review, Fig. 1). Vascularization begins in the first week of life in mice (Gariano and Gardner, 2005). In the naive brain, microglia are not as inactive as previously assumed, but are highly motile cells that continuously survey the environment (Nimmerjahn et al., 2005). After injury, microglia become activated within few hours, i.e. they proliferate, change their morphology from highly ramified to ameboid, upregulate specific receptors on their surface, secrete a variety of factors including cytokines and chemokines and attract other cells (Hanisch and Kettenmann, 2007; Kim and de Vellis, 2005; Kreutzberg, 1995; Streit et al., 1999). Levels of microglial activation i.e. the resulting microglial response also depend on the mode of injury and the injury model. Interestingly, an undifferentiated/ immature character of microglia cells was described more than 10 years ago (Carson et al., 1998; Santambrogio et al., 2001). It was demonstrated that adult microglia derive from primitive myeloid progenitors that arise before embryonic day 8 (Ginhoux et al., 2010). Therefore, it is thought that microglia represent a separate population that is different from specialized macrophages of the CNS (Ransohoff and Cardona, 2010 for review), as illustrated in Fig. 1, and indeed possess a more immature state (Butovsky et al., 2006). Reasons for this hypothesis include the fact that adult microglia are similar to immature neonatal microglia because in the presence of growth factors microglia begin to differentiate into

mature, professional APCs, and moreover, display a self-renewal potential that is normally lost during differentiation (Carson et al., 1998; Guillemin and Brew, 2004; Santambrogio et al., 2001). Immature microglia differ from mature APCs with regard to their gene and protein expression patterns, especially of certain receptors which are responsible for immunologic function (Santambrogio et al., 2001). More recently, several studies show that cerebral microglia express neural markers under certain conditions and possess a neurogenic potential at least in vitro which will be discussed in the next paragraph. 8.1. Neurogenic microglia in the brain Evidence indicates that microglia of the neonate rat brain (i.e. cells derived from the hematopoietic system) may also represent a source for neurons, astrocytes, and oligodendrocytes (Yokoyama et al., 2004). These microglia express immature neural markers such as nestin and NG2 (Almazan et al., 2001; Sahin Kaya et al., 1999; Yokoyama et al., 2004, 2006) and can give rise to neurons and glia in vitro (Butovsky et al., 2006; Matsuda et al., 2008; Yokoyama et al., 2004, 2006). It has also been demonstrated that long-term administration of the pro-inflammatory cytokine interferon g in low dosages results in expression of neuronal proteins including doublecortin and beta-III tubulin, indicating transdifferentiation of microglia (Butovsky et al., 2006). Interestingly, Matsuda et al. (2008) further confirmed that these transdifferentiated microglia-derived neurons are indeed able to generate action potentials and display properties of mature neurons in vitro (Matsuda et al., 2008). In vivo experiments showed that after neuronal lesion, microglia express the hematopoietic stem cell marker CD34 (Ladeby et al., 2005), the stem cell receptor c-kit (Zhang and Fedoroff, 1999), and also show a notable capacity for self-renewal (Hailer et al., 1999; Ladeby et al., 2005). After a stab wound to the adult rat brain, activated microglia isolated from the lesion site can also give rise to neurons and glia in vitro, and possess properties similar to neonate microglia, and hence, show neural progenitor potential (Yokoyama et al., 2006). Interestingly, it has been demonstrated that nestinþ microglia already exist in the naïve brain with varying cell numbers according to the particular region analysed (Takamori et al., 2009). These microglia co-express vimentin, another intermediate filament of progenitor cells, which has been suggested as being necessary for maintaining structural integrity and cell shape (Takamori et al., 2009). This ectopic expression, however, could also indicate a more immature state of these microglia, albeit under naïve conditions, and not necessarily a neural progenitor-like phenotype. Taken together, various in vitro studies demonstrated that cerebral microglia have an intrinsic potential to give rise to neurons and glia. However, to date, there is no study available reporting on microglia-derived neurogenesis in vivo. 8.2. Neurogenic microglia in the retina? In the naïve retina, microglia are present in the plexiform layers (Fig. 2B). In a recent study, we demonstrated that a nestinþNG2þvimentinþ subpopulation of microglia also exists in the naïve adult rat retina, suggesting an immature cell state (Wohl et al., 2011). We described for the first time, that the majority of parenchymal microglia (about 60%) in the naïve adult rat retina express nestin, thereby displaying a similar subcellular distribution of nestin filaments as has recently been reported for the brain (Takamori et al., 2009). However, the fraction of nestinþ retinal microglia was much higher than previously reported for different brain regions (Takamori et al., 2009), probably because the retina differs from other brain regions in its function as a sensory organ

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(Wohl et al., 2011). After injury, there was a further increase of about 14% in the nestinþ microglial fraction, suggesting that the nestinþ population is the responding fraction after injury. Moreover, a particularly interesting finding consisted of the fact that only nestinþ microglia divide locally in response to ON axotomy. Fig. 7 illustrates in situ dividing (Ki67þ) microglia cells in the GCL and IPL which were exclusively nestinþ. We, therefore, concluded that microglial nestin expression plays a role in microglial proliferation pointing to physiological self-renewal of this endogenous cell population (Wohl et al., 2011). The involvement of nestin expression in cell cycle re-entry has previously been demonstrated for other cell types from different tissues (Daniel et al., 2008; Namiki and Tator, 1999; Sahlgren et al., 2001; Sergent-Tanguy et al., 2006; Sunabori et al., 2008; Suzuki et al., 2010; Wiese et al., 2006; Xue and Yuan, 2010). Nestin expression persisted over several weeks after injury and it appears that the nestinþ and nestin fraction of retinal microglia maintain a physiological equilibrium which may be intrinsically regulated (Wohl et al., 2011). Noteworthy is that retinal nestinþ microglia co-express vimentin and NG2 under both naïve and injury conditions, which makes them unique since cerebral microglia have consistently been reported as being NG2 in naïve adult tissue (Bu et al., 2001; Zhu et al., 2010). However, like microglia of the brain (Fiedorowicz et al., 2008;

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Yokoyama et al., 2006) and spinal cord (Matsumoto et al., 2008; Pouly et al., 1999; Zhu et al., 2010) retinal microglia increase their NG2 immunoreactivity and increase in number (Wohl et al., 2011). Thus, for the rat retina, we provide novel evidence that these NG2þnestinþ microglia already reside in naïve tissue and increase through in situ proliferation after lesion. What is the function of these microglia? We demonstrated that nestinþNG2þ microglia give rise to immature (still nestinþ) but also possibly more mature microglia (which became nestin). Furthermore, nestinþNG2þ microglia are involved in phagocytosis, and as such they have a cleaning up function (Wohl et al., 2011). Involvement of the microglial population in important physiological functions raises the question as to whether these microglia might also represent putative immature progenitors that can also give rise to neural cells in vivo. Since there is increasing evidence that CNS injury can induce neural progenitor characteristics in activated microglia of non-neurogenic regions in vivo (Ladeby et al., 2005; Wu et al., 2005; Yokoyama et al., 2006), we hypothesized that this newly identified retinal microglial subpopulation may represent an “intermediate” cell type, which could act as an endogenous neural progenitor-like cell after injury. Interestingly, Harris et al. (2006) demonstrated that physical (breaching Bruch’s membrane and inducing vascular endothelial growth factor (VEGF) expression) or chemical injury

Fig. 7. In situ dividing nestinþ microglia 3 days after ON transection. Immunofluorescent labelling for Ki67 (red), nestin (green), tomato lectin (A, C, D, G blue) and DAPI nuclear staining (B, E, F, blue) in the injured retina. AeD: 3 days after ON lesion, Ki67þ (cycling) microglia were found in the GCL, IPL and superficial INL (arrows), all of them were nestinþ. EeG: In situ dividing nestinþ microglia in the GCL. The micrographs are merged z-stacked images of 1 mm optical sections to illustrate entire cell dimensions. Scale bar 20 mm.

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(sodium iodate injection) to murine RPE results in recruitment of cells arising from hematopoietic stem cell/progenitor cells (HSC/ PCs), which differentiate into pigmented epithelial cells and incorporate into the RPE layer, i.e. regenerate a proportion of the damaged RPE (Harris et al., 2006). This study further supports the notion that bone marrow derived progenitors possess an intrinsic potential to generate cells of the neural lineage. Therefore, it is also conceivable that microglia, as not fully committed cells, may possess a similar lineage plasticity. Thus, proliferating nestinþ microglia may be progenitor-like cells with a bivalent cell fate. More precisely, they could give rise to immunological but also to neural cells as reported for cerebral microglia. Nevertheless, one question remains unsolved: Do nestinþNG2þ retinal microglia represent an endogenous progenitor-like cell that can generate new neurons after injury? In our study, we observed no ectopic expression of neuronal markers in retinal microglia that might have indicated a possible transdifferentiation within a time period of 8 weeks after ON lesion, as has been reported under culture conditions and could, thus, not corroborate our hypothesis. Therefore, ON lesion alone does not appear to be sufficient to induce the putative multipotent progenitor features of retinal microglia in vivo (Wohl et al., 2011). However, a variety of reports show that injury alone has no or only a minor effect on the transdifferentiation potential of progenitor-like retinal cells such as Müller glia (Karl et al., 2008; Ooto et al., 2004). As has recently been shown, Müller glia cells also have to be reprogrammed to display “true” progenitor characteristics and to give rise to retinal neurons (Pollak et al., 2011). Thus, reprogramming of microglia could also be a possibility to increase the number of resident retinal progenitors which can give rise to neurons, in particular after injury. Further research in our laboratory is merited to elucidate this important aspect. 9. Cell transplantation approaches Anatomical integration and differentiation of progenitor cells into the eye after heterologous or autologous transplantation has already been reported (for reviews see Amato et al., 2004; Bi et al., 2009; Dahlmann-Noor et al., 2010; Harvey et al., 2006; Klassen et al., 2004a; Locker et al., 2010; Yau et al., 2007; Young, 2005). Takahashi et al. (1998) showed that adult rat hippocampus-derived NSCs can be successfully transplanted into neonatal retinas, where they differentiate into neurons and glia (Takahashi et al., 1998). Retinal injury promotes a successful incorporation of grafted hippocampus-derived NSCs including subsequent differentiation into neurons (albeit not in retinal neurons) which actually form synapse-like structures (Nishida et al., 2000). Moreover, transplantation of hippocampus-derived NPCs in diseased retinae of RCS rats resulted in widespread migration and functional integration of the grafted cells in the degenerating retina (Whiteley et al., 2001). Thus, injury-induced or disease-related changes appear to be responsible for neuronal differentiation of grafted immature cells because NSCs do not integrate into naïve adult retinae (Takahashi et al., 1998; Tropepe et al., 2000). This could probably be due to a developmentally changed environment (age-dependent restricted plasticity) but also to the fact that an intact retina does not need additional neurons. On the one hand, injury triggers integration of grafted cells, but on the other hand, neural implantation also triggers reactive gliosis and in particular the formation of astroglial scars. Reactive astrocytes in turn prevent successful integration of grafted cells as well as the formation of neuronal projections (Kinouchi et al., 2003). Interestingly, this inhibitory astrocytic environment can be overcome by depletion of the intermediate filaments GFAP and vimentin, i.e. modifying retinal astrocytes (Kinouchi et al., 2003). Moreover, engraftment of neural

precursors can be also improved by selective deletion of cell types. Here, Mellough and colleagues showed that selective depletion of RGCs in neonate mice increases the incorporation of grafted NSClike cells into the GCL. However, there was no sign of differentiation into RCS and moreover, this integration was not found in adult animals indicating that the host age is indeed a limiting factor for successful cell integration (Mellough et al., 2004). Thus, in particular the age of the host retina seems to play a key role in the efficiency of stem cell integration as reported by Van Hoffelen et al. (2003). Furthermore, also the age of the grafted cells (recipient) is a crucial point. Interestingly, it has been shown that more committed cells can be better integrated than immature cells (MacLaren et al., 2006). However, it has become apparent that brain-derived cells do not differentiate into authentic and functional retinal neurons in the micro-environment of the mature retina (Dahlmann-Noor et al., 2010; Yau et al., 2007 for review). HSC/PCs represent another potential stem cell source (Bi et al., 2009; Yau et al., 2007). HSC/PCs have already been reported as displaying overlapping genetic programs with neural stem cells (Terskikh et al., 2001) and additionally to give rise to neurons and glia in vitro and after transplantation into the brain (Brazelton et al., 2000; Goolsby et al., 2003; Mezey and Chandross, 2000; Mezey et al., 2000, 2003). Interestingly, it has recently been demonstrated that adult bone marrow stem cells grafted into the adult degenerative RCS rats (Kicic et al., 2003) or injured rat eye (Tomita et al., 2002) differentiate into photoreceptors. However, exogenous cell transplantation is accompanied by various disadvantages including a very limited performance time window, as well as a required immunosuppression to prevent reactivation of other cell types including macro- and microglia (Lund et al., 2001). Therefore, several studies are aimed at identifying an endogenous pool of retinal “stem”/progenitor cells which can be used for autologous cell transplantation. In fact, successful integration and subsequent differentiation of adult CB-derived cells into photoreceptors in injured retinae has already been demonstrated in P10 rats (Chacko et al., 2003). However, ciliary “stem” cells, which have the ability to give rise to neurons, are very few in number, even if cultured under appropriate conditions (Tropepe et al., 2000). Moreover, after isolation (which requires complicated microsurgical procedures, in particular for isolation of cells from the CB), cell yield has to be carefully increased in culture in order to obtain adequate cell numbers for administration to the injured retina. Moreover, transplantation per se is a delicate procedure. Such procedures are time and labour intensive and improving culture yields as well as transplantation techniques are factors that will determine whether this approach represents a suitable option for autologous cell replacement therapy. Therefore, identification and utilization of an endogenous cell pool that can be intrinsically activated and properly expanded would represent a promising therapeutical option. 10. Conclusions and future directions The rodent retina displays a retinal constitution of cell types similar to the human retina. More importantly, several studies have shown that human stem/progenitor-like cells display similar characteristics to those of rodents. Therefore, rodents, and in particular mice are appropriate candidates for analysing the endogenous retinal regeneration potential of the mammalian eye. There are a variety of dormant putative progenitor-like cell populations present within the adult rodent eye that possess some degree of neurogenic potential in vitro and can be activated by exogenous stimulation in vivo. However, in contrast to fish and avians, the potential of mammalian progenitor-like cells is fairly restricted and transdifferentiation in vivo is hardly inducible. Expression of neuronal proteins after injury indicating

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neurogenesis in the adult rodent eye has only been reported for Müller glia after NMDA-induced neurotoxic injury combined with appropriate growth factor delivery. Injury alone appears not to be a sufficient stimulus to induce transdifferentiation of already functional mature cells into new neuronal cells. Moreover, it was stated by Fischer and Bongini (2010) that it does not make sense to stimulate neural regeneration from mature retinal cells, in particular, from Müller glia, in slowly degenerating retinae by inducing severe, acute damage. In addition, transdifferentiation of Müller cells, i.e. the processes of de-differentiation, re-entry in the cell cycle, and differentiation into retinal neurons is accompanied by a loss of essential glial functions (Fischer and Bongini, 2010; Reichenbach and Bringmann, 2010). Furthermore, endogenous cell populations, i.e., pigmented epithelial cells, regardless of the type of epithelium, and macroglial cells represent only a minor responding population after exogenous stimulation. Thus, isolation and in vitro expansion is required to obtain sufficient cell numbers to be transplanted into the injured tissue. However, this approach is associated with several obstacles that includes a very short time window for performance (MacLaren and Pearson, 2007; Young, 2005). A newly developed approach involves the reprogramming of somatic cells such as fibroblasts, astrocytes or RPE cells (Berninger, 2010; Dennis, 2003). However, these cells need to be transplanted after in vitro modulation and this procedure may be accompanied by tumor generation (Berninger, 2010). Therefore, a large cell population residing in the tissue which rapidly responds to neuronal loss with increased proliferation and which has the ability to (trans)differentiate and to migrate would be a perfect candidate for in vivo cell replacement research, in particular after acute damage. Most of these features are displayed by mesodermderived retinal microglia. These constitute a majority of responding cells, in particular following injury, and display a more immature cell state. Cell culture studies and analysis of transgenic mice by means of gene transfer using viral vectors (Harvey et al., 2006), or the use of small silencer RNA (siRNA) could shed light on the issue of whether microglia possess true intrinsic or inducible stem cell properties and whether they can be employed for cell replacement strategies. To achieve a breakthrough in regenerative medicine would require the recruitment of intrinsic cells for specific neuronal replacement in the near future. However, the mammalian CNS, including the neural retina, is a highly developed, complexly organized structure whose putative neurogenic potential is even to date not wholly understood. Thus, even if methods for successful cell replacement with new neurons could be established, there would be no guarantee that the developing axons would grow specifically towards the correct targets (Bähr and Bonhoeffer, 1994; Drescher et al., 1995; Müller et al., 1996; Walter et al., 1990) and correctly and functionally integrate new neurons into existing neural networks. Moreover, in the adult CNS, an inhibitory environment caused by myelin-associated glycoproteins and extracellular matrix (ECM) molecules such as chondroitin sulphate proteoglycans strongly prevent axonal, and therefore also, cellular regeneration. In addition, after injury, these inhibitory molecules are expressed by activated microglia and astrocytes, further promoting this detrimental environment (Bähr and Bonhoeffer, 1994; Dahlmann-Noor et al., 2010; Fawcett and Asher, 1999). Therefore, successful migration, integration and synapse formation of transplanted or endogenous cells need also to incorporate pharmacological approaches to overcome these inhibitory factors (Bull et al., 2008; Geisert et al., 1992; Singhal et al., 2008, 2010; Suzuki et al., 2007). Taken together, the most feasible approach for endogenous cell replacement and retinal regeneration must rely on a combination of methods involving exogenous stimulation of endogenously modified progenitor-like cells (reprogramming/gene transfer),

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