Lens and Retina Regeneration: Transdifferentiation, Stem Cells and Clinical Applications

Experimental Eye Research 78 (2004) 161–172 www.elsevier.com/locate/yexer Review Lens and retina regeneration: transdifferentiation, stem cells and clinical applications

Panagiotis A. Tsonisa,*, Katia Del Rio-Tsonisb

aUniversity of Dayton, Laboratory of Molecular Biology, Department of Biology, Dayton, OH 45469 2320, USA bDepartment of Zoology, Miami University, Oxford, OH 45056, USA

Received 17 July 2003; accepted in revised form 24 October 2003

Abstract In this review we present a synthesis on the potential of vertebrate eye tissue regeneration, such as lens and retina. Particular emphasis is given to two different strategies used for regeneration, transdifferentiation and stem cells. Similarities and differences between these two strategies are outlined and it is proposed that both strategies might follow common pathways. Furthermore, we elaborate on speciﬁc clinical applications as the outcome of regeneration-based research q 2003 Elsevier Ltd. All rights reserved.

Keywords: eye; lens; retina; regeneration; transdifferentiation; stem cells; cataracts; retinal diseases

An old Greek proverb says that when you have something clear evolutionary advantage (tail regeneration in lizards) precious you should guard it as you do your eyes. Vision, and some with no obvious evolutionary advantage (i.e. lens among all the other senses, provides the link to the outside regeneration in newts). In recent years, however, intense world which is extremely important for survival of species research, especially on stem cells, has shown that the body and is much valued by humans. So it should not come as a has more remarkable reparative capabilities than previously surprise that nature must have devised back-up strategies to thought. The same we believe is true with repair of eye loss or damage of the eye tissues. Why are then, among tissues and in this review we intend to popularize this view. vertebrates, regenerative abilities of the lens and retina so Before we examine the regeneration process and mechan- pronounced only in some amphibia? Why is regeneration of isms involved in lens and retina, let us take a note of the two the lens or retina an advantage to some salamanders and not major strategies that animals use to repair damaged tissues. to the rest of the vertebrates? Thinking along these lines we Regeneration occurs by two strategies. One strategy uses are dealing with an evolutionary paradox. differentiated cells neighbouring the damaged site. These When it comes to evolution, regeneration of body parts cells restore the damaged tissue by proliferation or by must have been an advantage, especially in asexually transdifferentiation. Transdifferentiation is the process by reproduced animals (Tsonis, 2000; Brockes et al., 2001). In which cells are able to dedifferentiate (lose the characteristics of their origin) and subsequently redifferentiate. This many cases regeneration in asexual animals is very similar strategy is used in many cases, such as liver, pancreas and is to their mode of reproduction. As species became more characteristic of epimorphic regeneration as well (Tsonis, advanced and reproduction became sexual, regenerative 2000, 2002). As we will see transdifferentiation is the capabilities diminished. Several species, however, have strategy used in lens regeneration. The other strategy is by retained remarkable regenerative capabilities, some with stem cells. See later section for a discussion on the two regeneration strategies. In retina regeneration, however, * Corresponding author. Dr Panagiotis A. Tsonis, University of Dayton, Laboratory of Molecular Biology, Department of Biology, Dayton, OH both strategies can be used. As we will see depending on 45469 2320, USA. species, transdifferentiation or progenitor cells can be E-mail address: [email protected] (P.A. Tsonis). recruited to populate damaged retina.

1. Lens regeneration

As it was mentioned above, lens regeneration was first observed in adult newts (Colluci, 1891; Wolff, 1895). These animals have been the major experimental material for the study of lens regeneration. Lens regeneration is also possible in certain frogs, but the process differs considerably from the one in newts. In these frogs, lens regeneration is possible only during the pre-metamorphic stages of development (see below). Adult frogs are not capable of regeneration. The only adult animals with that capability are some urodeles. In Section 1.1, we will examine the two animal models and compare the mechanisms in both. In mammals such regenerative properties are absent. Regen- eration, however, can be surgically manipulated. In rabbits if the lens is removed, but the capsule stays behind and rather intact, remaining lens epithelial cells differentiate and fill the capsule, thus reconstructing the lens. Lens regeneration in the adult newt begins with proliferation and dedifferentiation of dorsal iris pigment epithelial cells (PECs). By dedifferentiation we mean the loss of characteristics that define the PECs, such as pigmentation (Eguchi, 1963). Dedifferentiation initiates molecular events, such as cell cycle re-entry, which is necessary for cell proliferation and the subsequent regeneration of the lens. So far, the fastest known event that occurs after lentectomy is thrombin activation in the dorsal iris. Such activation cannot be seen in the ventral iris or in the irises of other salamanders that are incapable of lens regeneration (Imokawa and Brockes, 2003). At about 10 days post-lentectomy, a lens vesicle is formed from the depigmented dorsal PECs (Fig. 1(A)). Around 12–16 days post-lentectomy, the internal layer of the lens vesicle thickens and synthesis of crystallins begins (Fig. 1(B)). This marks the beginning of primary lens Fig. 1. Lens regeneration in newts via transdifferentiation of the PECs fiber differentiation. During days 15 –19, proliferation and from the dorsal iris (di). (A) Ten-days post-lentectomy. Note an early lens depigmentation of PECs slows down. In the internal layer, vesicle (arrow) formed by dedifferentiation of the PECs from the dorsal the lens fibre complex is formed and in the margin of the iris. (B) Fifteen-days post-lentectomy. The cells at the posterior part of the vesicle (arrow) elongate to form lens fibres. (C) Twenty-days post- external layers non-dividing secondary lens fibres appear. lentectomy. A well differentiated lens with lens fibres (lf) covered by the By 18–20 days the PECs have stopped proliferating, and the lens epithelium (le). lens fibres continue to accumulate crystallins (Fig. 1(C)). Lens regeneration is considered complete by day 25–30 Among other amphibians frogs can regenerate their lost (Eguchi, 1963, 1964; Reyer, 1977; Yamada, 1977; Tsonis, lens, but in contrast to the newt, regeneration occurs via 1999, 2000). Lens regeneration, therefore, is a clear case of transdifferentiation of the inner layer of the outer cornea. transdifferentiation. A very interesting restriction is that the Another important difference is that lens regeneration in ventral iris, which is seemingly comprised by the same frogs is possible only during premetamorphic stages and PECs is not capable of regenerating a lens. The process of ceases after metamorphosis (Freeman, 1963; Filoni et al., transdifferentiation has been proven beyond any doubt in 1997; Henry and Elkins, 2001). Also, in Xenopus laevis, the this system. These processes can also be observed when capacity seems to depend on factors that are provided by the single PEC cells are placed in culture (Eguchi et al., 1974; retina (Filoni et al., 1982). When a piece of outer cornea is Kodama and Eguchi, 1995; Tsonis et al., 2001). The implanted in the vitreous chamber, even in the presence of restrictions that we see in the in vivo newt model do not the host lens, transdifferentiation can occur. It is possible apply for the in vitro models. PECs from the whole eye that the rapid closure of the inner cornea after metamor- (including from the ventral iris) and from any species, phosis is an inhibitor to regeneration (Reeve and Wild, including aged humans are capable of transdifferentiating to 1978; Filoni et al., 1997). The stages during lens lens cells under certain conditions (Tsonis et al., 2001). regeneration from the cornea are very similar to the ones P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 163

Fig. 2. Lens regeneration in pre-metamorphic Xenopus via transdifferentiation of the cornea. (A) Stage 2 (early). (B) Stage 2 (late, 3 days) representing the vesicle formation. (C) Stage 4, 6 days. Differentiation of lens fibres has started (red staining with anti-crystallin antibody). (D) Stage 4, 8 days. Definite differentiation of lens fiber. (E) Stage 5, 10 days. The lens has increased in size and has positioned by the dorsal and ventral iris. (Courtesy: Dr Stafano Cannata). seen from the dorsal iris in newts. A vesicle is first formed embryogenesis, and can induce lens morphogenesis, are also and then gradually crystallins and lens fibres accumulate. activated during lens regeneration as well (Del Rio-Tsonis During the final stages the lens is positioned along the dorsal et al., 1995, 1997, 1999; Mizuno et al., 1999; Schaefer et al., and the ventral iris (Fig. 2). It is interesting to note here that 1999). It remains though to be seen whether or not these FGF-1 seems to be very important in the process of genes are the real signals for initiation of lens regeneration transdifferentiation both in newts and in frogs. When newt or their activation is a secondary step after the initiation of PECs or frog outer cornea is placed in vitro, FGF-1 is an lens regeneration by a yet unknown signal. Why related inducer of transdifferentiation (Hyuga et al., 1993; Bosco salamanders show differences in the capacity of lens et al., 1997). regeneration remains a mystery. For example, the axolotl, In the past few years research has been concentrated in which is a urodele cannot regenerate the lens, even though the identification of key genes for the induction of its ability to regenerate limbs or the tail parallels that of the transdifferentiation. The strategy is to identify dorsal- newt. Imokawa and Brockes (2003) have found that specific genes and examine their possible function during thrombin activation could be a critical determinant. As lens regeneration. These genes can then become important mentioned above, thrombin activation can be seen within tools to probe why ventral iris (of the newt) or irises from minutes after lentectomy in the dorsal iris of the newt, while other animals are not capable for regenerating a lens. Some such an event is undetectable in the iris of the axolotl. These of these genes are presented in Table 1. Table 1 clearly findings stress the importance of comparative studies using indicates that genes that are normally expressed during lens different species. Only then we will be able to understand 164 P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172

Table 1 cataracts can be surgically corrected. The operation leaves Expression of lens-specific genes during lens regeneration the capsule as intact as possible, which then is used to hold a Genes Newt/dorsal-ventral regulation Xenopus synthetic lens in the right place. The problem with this operation is that lens epithelial cells that remain adhered to Pax-6 þ/yes þ the capsule transdifferentiate to mesenchymal cells and as Prox-1 þ/yes þ they migrate posteriorly they opacify the capsule. This is FGFR-1 þ/yes ND called posterior capsule opacification (PCO) and is the Otx-2 ND þ Sox-3 ND þ major complication of cataract surgery (Apple et al., 1992). In most of the cases another surgery is necessary. þ , expression; yes, dorsal–ventral regulation; ND ¼ not determined. Obviously, if lens regeneration were to be successful in humans, there would be no need for such an operation. But why some salamanders are endowed with such an advantage how do we achieve lens regeneration in mammals? We and other species are not. Among other vertebrates only propose two major directions of research. In first, we must some fish are capable of regenerating the lens in a manner identify dorsal iris-specific signals in the newt. These similar to the one seen in newts (Sato, 1961). factors can then be tested to examine if they are capable of In mammals, lens regeneration studies have been inducing lens regeneration from incompetent tissues. largely restricted to rabbits. It has been documented that Incompetent tissues should first include newt ventral iris if someone performs an endocapsular lentectomy, in other and then irises of other salamanders, such as the axolotl. words removes the lens fibres, but leaves the lens capsule Once regeneration has been successfully induced, these behind, lens regeneration can occur (Stewart and Espi- factors should be tested for their ability to induce nase, 1959; Gwon et al., 1990, 1993a,b). The reconstruc- regeneration in mammals. Mice should be the best animal tion of the lens depends largely on the presence of model, because of the availability of genetic tools. If this is adherent lens epithelial cells that remained on the capsule. successful, we should proceed in higher mammals. There is These cells follow their normal course and differentiate to compelling evidence that such strategy will succeed. When lens fibres, which in turn fill the capsule (Fig. 3) and PECs are cultured for a long time, they can transdifferentiate create a lens with many normal properties (Gwon et al., to lens and this ability seems to have no species or age 1993b). Such studies are very important because they barriers (Kodama and Eguchi, 1995). We just need to indicate that the lens has impressive reparative capabilities identify the trigger that allows the newt to regenerate the if manipulated correctly. Also, such studies have impli- lens in vivo. The second research direction should deal with cations in cataract therapy and surgery. the capacity of lens regeneration from the remaining lens capsule. If rabbits and cats can reconstruct a lens, we see no 1.1. Clinical applications: towards strategies to materialize reason why this should not be the case in humans. Several lens regeneration in mammals questions, though, must be answered. Is there an age factor? Can lens capsules regenerate a functional lens? In regards to Cataracts are the main clinical manifestations of the lens. the first question there is much research to be done and Cataracts can be genetic or induced as a result of aging. unfortunately rabbits or cats are not favourable animal Also, cataracts can affect different regions of the lens, i.e. models. If someone is to pursue research on factors involved the nucleus, or the cortex (Francis et al., 1999). In humans, in the differentiation of lens epithelial cells a better animal

Fig. 3. Lens regeneration in rabbits. (A) Six-days after endocapsular extraction of the lens. Note differentiation of lens fibres from the lens epithelial cells that remained attached to the lens capsule. (B) Slit lamp photo of 30 day regenerating lens. Arrows indicate fibres that have differentiated. (Courtesy: Dr Arlene Gwon). P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 165 model is imperative. The answer to the second question has been observed to take place by transdifferentiation of the should be yes. If we could surgically manipulate the capsule retinal pigment epithelium (RPE). However, only certain to remain in a spherical shape the reconstructed lens should urodeles retain the capability to regenerate their retinas via be functional. Some successful experiments have been transdifferentiation as adults (Lopashov and Stroeva, 1964; presented (Gwon et al., 1993b). In regard to PCO, animal Mitashov, 1996, 1997; Raymond and Hitchcock, 2000; models should be established where the ability of the lens Fischer and Reh, 2001a; Del Rio-Tsonis and Tsonis, 2003). epithelial cells to differentiate to lens fibres or to The process of transdifferentiations involves a cell transdifferentiate to mesenchymal cells will lead to the conversion not typically encountered in adult tissues. RPE identification of factors, whose use will help interfere with (Fig. 5(A)) lose their characteristics of origin and re-enter the process. Again here we believe that these approaches are the cell cycle to form a neuroepithelial cell layer (Fig. realistic, we just need to pursue it more rigorously with the 5(B)) that eventually will differentiate into all the different right animal model. cell types of the retina (Fig. 5(C)) recapitulating the appearance of retina during development (Fig. 5(D)) (reviewed in Reyer, 1977; Hitchcock and Raymond, 2. Retina regeneration 1992; Mitashov, 1996, 1997; Raymond and Hitchcock, 2000; Del Rio-Tsonis and Tsonis, 2003). Embryonic Regenerating a retina or part of a retina has been chicks, which can also regenerate their retinas via observed in a variety of organisms during either their transdifferentiation of the RPE with FGF, lose this layer development or for some even during their adult life. as it becomes the neuroepithelium and eventually differ- Among those with the ability to regenerate during adulthood entiates into all retinal layers (Fig. 6(D)). This neuroe- are fish, birds and amphibians (Del Rio-Tsonis and Tsonis, pithelium seems to develop similar in sequence to that of 2003). The modes of retina regeneration vary depending on normal development, but with reverse polarity (Fig. 6F). the organism (Fig. 4). The regenerative ability of some adult As a result, the rods and cones of the photoreceptor layer forms can also be different from that present in some are located in the inner most layer of the retina, which is embryos of the same species. For example, regeneration of closest to the lens (Coulombre and Coulombre, 1965; Park the retina in some fish, bird and amphibian embryos/larvae and Hollenberg, 1989, 1991, 1993).

Fig. 4. Representation of all the possible sources for retina replacement compiled from different animal models. All parts are colour coded for easy reference. The following have been reported as possible sources for retina regeneration. In amphibians: the RPE (retinal pigment epithelium) ¼ black and the CMZ (ciliary marginal zone) ¼ pink. In birds: the RPE ¼ black, the CMZ ¼ pink/ciliary epithelium ¼ dark green (embryonic) and Mu¨ller glial cells ¼ red (post- hatch). In ﬁsh: CGZ (circumferential germinal zone) ¼ pink (embryonic/larval), rod precursors in the ONL ¼ light green (embryonic/larval and adult); intrinsic stem cells and progenitors in the INL ¼ dark yellow (embryonic/larval and adult); and possibly Mu¨ller Glia Cells ¼ red. In mammals: RPE (embryonic), pigmented cells of the ciliary epithelium ¼ pigmented cells that accompany the dark green area, iris ¼ light brown, corneal limbus area ¼ light yellow, choroid ¼ dark brown and sclera ¼ aqua (adult). 166 P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172

Fig. 5. Retina regeneration in adult newts. (A) Retinectomized newt eye (5 days post-retinectomy). Here dedifferentiation and proliferation of the rPEC begin. Note that the cells shown by arrowheads have dedifferentiated or are in the process of dedifferentiation. (B) After about 2 weeks post-retinectomy a neuroepithelial (ne) cell layer forms that will eventually give rise to all the cells of the retina. (C) A month post-retinectomy, the regenerated differentiated retina stratiﬁes into the different retinal layers: the outer nuclear layer (o), the inner nuclear layer (i) and the ganglion cell layer (g). The RPE has also been renewed and the orientation of the newly formed retina is the same as the intact.After about 2 weeks post-retinectomy a neuroepithelial (ne) cell layer forms that will eventually give rise to all the cells of the retina. (D) Intact retina section showing all the layers of a mature retina. ne: neuroepithelium, RPE: retinal pigment epithelium o: the outer nuclear layer, i: the inner nuclear layer and g: the ganglion cell layer. Sections were stained with Hematoxylin and Eosin.

Retina replacement in the embryonic chick eye has also 2000; Reh and Fischer, 2001; Wu et al., 2001; Fischer and been observed to originate from the ciliary region (Fig. 4), Reh, 2003)(Fig. 4). This replacement depends on the but only if a source of FGF is supplied (Fig. 6(C) and (E)) damage elicited and has to include damage in the ONL cell (Coulombre and Coulombre, 1965; Park and Hollenberg, layer, otherwise no regeneration will take place (Negishi 1991, 1993). This process appears to occur via the use of et al., 1987, 1988; Raymond et al., 1988; Braisted and neural precursors (Willbold and Layer, 1992). The CMZ in Raymond, 1992; Hitchcock, 1992; Hitchcock and Ray- adult chickens, on the other hand, is unable to replace mond, 1992; Otteson and Hitchcock, 2003). chemically damaged retina even though the cells of the The story is different for mammals where regeneration CMZ are mitotically active (Morris et al., 1976; Fischer and has not even been observed in embryos, unless the tissue Reh, 2000) and respond to growth factors by increasing their was transplanted or manipulated in vitro (Stroeva, 1960; proliferation and differentiation into different neural retina Zhao et al., 1995; Ahmad et al., 1999; Chacko et al., 2000, cells (Fischer and Reh, 2000; Fischer et al., 2002a,b). Even 2003). Recent reports suggest the possibility that mammals the cells from the pigmented ciliary margin are mitotically could regenerate their retina if properly induced. Adult active but are not conducive to produce neural retinal cells pigmented cells from the ciliary epithelium of rodents (Fischer and Reh, 2001a). Recently Mu¨ller glia cells have (Ahmad et al., 2000; Tropepe et al., 2000) and humans been identified as the cells responsible for responding to (Personal communication Arsenijevic, 2003, see Fig. 7) local retinal damage and replacing neural retina in adult have been induced to proliferate in vitro and eventually birds (Fig. 4). The cells replaced depend on the type of cells differentiate into retinal specific cells including rod that were damaged originally and on the presence of growth photoreceptors, bipolar neurons and even Mu¨ller glia cells. factors such as FGF-2 and insulin. Upon damage, Mu¨ller Other local sources for possible retinal progenitors in glia cells re-enter the cell cycle, de-differentiate producing mammals have been explored and include cells from the neural precursors and eventually differentiate into neurons iris, corneal limbus area, sclera and choroid (Haruta et al., and glia cells (Fig. 7)(Fischer and Reh, 2001b; Reh and 2001; Zhao et al., 2002; Arsenijevic et al., 2003). Cultured Fischer, 2001; Fischer et al., 2002a,b; Fischer and Reh, iris cells from rat have been induced to differentiate into 2003). retinal cells, including photoreceptors when transfected In teleost fish, retina regeneration takes place via the use with Crx, a crucial photoreceptor developmental gene of several cell sources including rod precursors (Raymond (Haruta et al., 2001). On the other hand, rat limbal epithelial et al., 1988; Braisted and Raymond, 1992; Hitchcock et al., cells cultured in vitro under certain growth conditions 1992), intrinsic stem cells in the INL (Raymond and express neural progenitor markers that eventually differen- Hitchcock, 1997; Julian et al., 1998; Otteson et al., 2001; tiate towards the neural lineage (Zhao et al., 2002). When Reh and Fischer, 2001; Wu et al., 2001; Otteson and these stem cells are transplanted unto eyes that have retinal Hitchcock, 2003) and possibly Mu¨ller glia cells that damage they migrate and integrate in different retinal layers proliferate and migrate to the ONL (Braisted and Raymond, and start expressing retinal neural markers (Chacko et al., 1993; Braisted et al., 1994; Raymond and Hitchcock, 1997, 2003). Lastly, sclera and choroid cells isolated from adult P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 167

only increase the possibilities and hopes for treatment. There are many retinal degenerative diseases that affect the human eye. These conditions vary on their aetiology and inheritance patterns, but at the end visual loss is the common consequence in many of them (Fig. 8). Replacement of the lost retina becomes a priority for patients found in the last stages of a degenerative disease. In patients with cone-rod dystrophy, retinitis pigmentosa and Leber’s Congenital Amaurosis where photoreceptors have degenerated, provid- ing a source of new retina would be very beneficial. Retinal transplants are one option, but the possibility of rejection exists. Several studies have been reported where stem cells, either neural and non-neural or even embryonic cell sources have been used as possible sources for retina replacement (Takahashi et al., 1998; Nishida et al., 2000; Young et al., 2000; Chacko et al., 2000; Kurimoto et al., 2001; Pressmar et al., 2001; Warfvinge et al., 2001; Tomita et al., 2002; Chacko et al., 2003; Dong et al., 2003; Mizumoto et al., 2003). Even though in some cases stem cells integrated into damaged retina and differentiated into retinal cells, no evidence of functionality has been shown. Inducing regeneration from the existing tissues of the patient’s eye such as the RPE could provide another option. Knowledge obtained from studies on animal models such as the chick Fig. 6. Retina regeneration in embryonic chick eyes. (A) Intact chick eye at and the newt could provide the necessary clues to induce day 7 of development. (B) Retinectomized chick eye at day 4 of development. The entire neural retina has been removed and the RPE transdifferentiation of the RPE into neural retina in humans. layer has thickened. When FGF-2 heparin beads were added to a From these retina regeneration studies so far, we know that retinectomized eye, the retina regenerated from the CMZ/ciliary epithelium growth factors are essential, specifically fibroblast growth region (C) and via transdifferentiation of the RPE at the posterior region of factors (FGFs). From in vitro transdifferentiation studies we the eye (D). The eyes were analyzed 5 days post-retinectomy (day 9 of also know that transcriptional regulators are also essential. development). At this time a nice neuroepithelium has formed. (E) Regeneration via the use of neural precursors from the CMZ/ciliary For example, microphthalmia (Mitf), a retinal pigment epithelium at 11 days of development or 7 days post-retinectomy. Note all epithelium identity molecule, must be down-regulated the retinal layers are nicely formed by now: the outer nuclear layer (ONL), during transdifferentiation, while Pax-6, a master regulator the inner nuclear layer (INL) and the ganglion cell layer (GCL). (F) of eye development should be up-regulated (Mochii et al., Regenerating retina 7 days post-retinectomy (11 days of development) 1998). where new retina has been formed via the transdifferentiation of the RPE. Note that the inner loop of regenerated retina contains all the retinal cell On the other hand, if the damage is local, Mu¨ller glial layers in the original orientation while the retina regenerated via cells found in the inner nuclear layer of the retina could also transdifferentiation of the RPE has a reversed or mirrored orientation. l: be used as source of new retina (Fig. 4). Mu¨ller glial cells lens; RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner have been found to replace neural cells and glial cells in nuclear layer and GCL: ganglion cell layer; NFL: nerve fiber layer. Sections adult damaged retina of birds and the possibility that these were stained with Hematoxylin and Eosin. cells could also do the same in mammals should be considered. Again growth factors seem to be essential for human eyes have the potential to differentiate towards the this process to take place as mentioned previously. neural linage (Arsenijevic et al., 2003). Even though the last The use of other local sources of retina replacement sources described have not been exploited yet for the should be considered such as cells from the CMZ or the production of retinal cells, the door is definitely open to ciliary epithelium (Fig. 4). Pigmented cells from these explore that option. regions could be induced to differentiate/transdifferentiate in vivo to participate in retina repair. Also non-neuronal 2.1. Clinical applications: toward repairing stem cells within the eye such as cells from the corneal diseased retinas limbal epithelium, sclera and choroid have been tested to give rise to neurons and glial cells and could potentially Dissecting the mechanisms underlying retina regener- provide a source for replacing damaged retina. ation will contribute to the design of procedures that could It seems that we have plenty of possible sources for retina rescue eyes that had undergone retinal degeneration (Table replacement and studies at the basic cellular and molecular 2). The different animal models available to study retina level using the different animal models discussed should regeneration and their corresponding modes of regeneration provide the scaffold for clinical studies to take place. 168 P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172

Fig. 7. Mu¨ller glia cells respond to retinal damage in the post-hatched chick by re-entering the cell cycle and eventually differentiating into retinal neurons expressing neural retinal markers such as Hu. (A) Retinal damage was induced with N-methyl-D-aspartate (NMDA). Two days post-treatment, eyes were positive for BrdU and (B) for glutamate synthatase (GS), a Mu¨ller glia marker. (C) An overlay of images A and B. Scale bar ¼ 50 mm. Mitotically active cells (100%) were Mu¨ller glial cells (D) Fourteen-days later, cells were still positive for BrdU and were also (E) positive for Hu, a retinal neuron cell marker. (F) An overlay of images D and E showing that BrdU positive cells were also positive for Hu. (Courtesy: Dr Thomas Reh).

Table 2 Possible sources for retina regeneration/repair

Animal model Embryonic-larval stages Primary sources or potential sources in adults

Teleost ﬁsh CGZ ¼ CMZ Rod precursors Rod precursors in ONL Quiescent stem cells in INL Transdifferentiation of RPE Mu¨ller glial cells? Amphibians Urodeles: CMZ Transdifferentiation of RPE, i.e. newts Transdifferentiation of RPE, i.e. newts and axolotls CMZ-partial only, i.e. axolotls Anurans: CMZ, i.e. Rrana esculenta; Rana temporaria CMZ-partial only, i.e. Xenopus laevis Transdifferentiation of RPE, i.e. Rana catesbiana Birds CMZ/ciliary epithelium Mu¨ller glial cells Transdifferentiation of RPE Mammals Transdifferentiation of RPE in vitro and in association with transplantation PCE in vitro experiments Iris in vitro Corneal limbal epithelium in vitro/transplantations Choroid and sclera in vitro

CMZ, ciliary marginal zone; CGZ, circumferential germinal zone; PCE, pigmented ciliary epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; RPE, retinal pigment epithelium.

Fig. 8. Cultured cells from the pigmented region of the pars plana and plicata of humans. (A) A cell colony derived from pigmented cells of the pars plana and plicata of the adult human eye grown in the presence of EGF and then plated onto a coverslip coated with poly-ornithin and laminin. (B) An example of neuron- like cells derived from such colonies (immunolabelling with an antibody directed against the b-tubulin-III antigen). (Courtesy: Dr Yvan Arsenijevic). P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 169

Transdifferentiation and stem cells entiation in general. The process of transdifferentiation in classical regenerative phenomenon such as the ones seen The term transdifferentiation was coined by Selman in salamanders is not in trouble at all. We are not saying and Kafatos (1974) to distinguish the switching of a that is unacceptable to use the term transdifferentiation terminally differentiated cell type into another from the for stem cells, but it should not be used to address only neoplastic transformation seen during cancer formation. the properties of stem cells, because the classical The process of transdifferentiation, therefore, entails that a regenerative phenomena are accurately described by it. terminally differentiated somatic cell dedifferentiates Let us now see the other side of the coin. Can a (loses the characteristics of its tissue of origin) and then terminally differentiated cell be considered a stem cell? differentiates into another cell type. This term was For example, newt PECs can transdifferentiate to neural adapted and literally became synonymous with the retina cells and to lens cells and in addition they can process of regeneration, such as of limbs, tail, retina or renew themselves (Del Rio-Tsonis and Tsonis, 2003). lens observed in adult salamanders. Transdifferentiation This plasticity depends on their position within the eye as has been shown to be the underlying mechanism whereby well as the type of surgery performed. Therefore, because regeneration in amphibia is achieved. Numerous studies PECs can give rise to different cell lineages, they can be have clearly shown that during limb regeneration adult regarded as transdifferentiating stem cells. This estab- myofibres can transdifferentiate to cartilage and vice lishes a common ground for the two regeneration versa. Similarly, after lens removal the newt regenerates a strategies. The approach we should take is to learn by new one by transdifferentiation of the PECs of the dorsal studying and comparing both of these strategies. In our iris, a phenomenon that has been shown conclusively in mind, the molecular mechanisms involved in transdiffer- clonal cultures of PECs as well. entiation of terminally differentiated cells and in acti- In recent years a new strategy involved in tissue vation and differentiation of local stem cells could be repair, marshalled by stem cells, has gained ground. remarkably similar, if not the same. For example, during Stem cells can be totipotent (cells before the formation limb regeneration in the newt, blastema cells, the product of the blastocyst), or pluripotent, which can give rise to of dedifferentiation of the stump tissues, can re-differen- different cell types upon selective activation. Once a tiate to muscle or cartilage. Likewise, in bone marrow particular stem cell population has been activated and there are mesenchymal stem cells that they can differen- committed to a lineage, these cells become progenitor tiate to muscle or cartilage cells. It is conceivable that a cells. Stem cells can be local, tissue-specific and reside blastema cell destined to become cartilage and a in adult tissues, such as brain, skeletal muscle, skin, mesenchymal stem cell destined to become cartilage retina or liver. However, non-local stem cells can be would have very similar molecular signatures at a certain found as well. Such stem cells reside, for example, in stage. At this stage both cells can be unified at the bone marrow and participate in repair of brain, heart, molecular level. Along these ideas it is interesting to note liver or other tissues. Similarly, stem cells residing in that different species use transdifferentiation of PECs or brain can become blood or muscle cells (Blau et al., stem cells to repair damaged retinas. Also, invertebrates, 2001). Accordingly, the term transdifferentiation was animals with incredible regeneration deeds, make use of adopted for these properties of non-local stem cells. both terminally differentiated cells and reserve cells to However, some of these studies have met with opposi- restore lost parts of their bodies. A simple explanation for tion. Additional experiments have shown that stem cells this could be that both strategies lead to activation of could acquire characteristics of other cells by fusion, similar molecular programme to achieve their goals. which in turn might account for transdifferentiation of With this in mind, comparative studies can be designed non-local stem cells (Terada et al., 2002; Ying et al., that could yield very important data on the mechanisms of 2002; Wang et al., 2003; Vassilopoulos et al., 2003). transdifferentiation and of stem cell biology. Indeed, Soon thereafter, reviews and papers appeared indicating recent studies have shown that embryonic and adult the concept of transdifferentiation is in trouble (Wells, neural and hematopoietic stem cells do share a molecular 2002). A clear distinction must be made here. The signature (or ‘stemness’) having some 200 genes process of transdifferentiation in non-local stem cells commonly transcribed (Ivanova et al., 2002; Ramalho-- might be in trouble, but not the process of transdiffer- Santos et al., 2002).

3. Concluding remarks We then reviewed current research and ideas that dominate the fields of lens and retina regeneration. It was noted that This review began with a wonder about the evolutionary despite the favouritism that nature has shown to newts, in importance of lens and retina regeneration. It was argued that reality the potential for regeneration of eye tissues is high. if regeneration of eye tissues was an advantage it should be Different species use different strategies to compensate for more widespread than confined in only some salamanders. damaged eye tissues. We also discussed the possibility that 170 P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 regeneration research from different animal models will Del Rio-Tsonis, K., Tsonis, P.A., 2003. Eye regeneration at the molecular eventually lead to therapies for diseases that affect eye age. Dev. Dyn. 226, 211–224. tissues. 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