review lens and retina regeneration: transdifferentiation ... · review lens and retina...

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, USAbDepartment 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

precious you should guard it as you do your eyes. Vision,

among all the other senses, provides the link to the outside

world which is extremely important for survival of species

and is much valued by humans. So it should not come as a

surprise that nature must have devised back-up strategies to

loss or damage of the eye tissues. Why are then, among

vertebrates, regenerative abilities of the lens and retina so

pronounced only in some amphibia? Why is regeneration of

the lens or retina an advantage to some salamanders and not

to the rest of the vertebrates? Thinking along these lines we

are dealing with an evolutionary paradox.

When it comes to evolution, regeneration of body parts

must have been an advantage, especially in asexually

reproduced animals (Tsonis, 2000; Brockes et al., 2001). In

many cases regeneration in asexual animals is very similar

to their mode of reproduction. As species became more

advanced and reproduction became sexual, regenerative

capabilities diminished. Several species, however, have

retained remarkable regenerative capabilities, some with

clear evolutionary advantage (tail regeneration in lizards)

and some with no obvious evolutionary advantage (i.e. lens

regeneration in newts). In recent years, however, intense

research, especially on stem cells, has shown that the body

has more remarkable reparative capabilities than previously

thought. The same we believe is true with repair of eye

tissues and in this review we intend to popularize this view.

Before we examine the regeneration process and mechan-

isms involved in lens and retina, let us take a note of the two

major strategies that animals use to repair damaged tissues.

Regeneration occurs by two strategies. One strategy uses

differentiated cells neighbouring the damaged site. These

cells restore the damaged tissue by proliferation or by

transdifferentiation. Transdifferentiation is the process by

which cells are able to dedifferentiate (lose the character-

istics of their origin) and subsequently redifferentiate. This

strategy is used in many cases, such as liver, pancreas and is

characteristic of epimorphic regeneration as well (Tsonis,

2000, 2002). As we will see transdifferentiation is the

strategy used in lens regeneration. The other strategy is by

stem cells. See later section for a discussion on the two

regeneration strategies. In retina regeneration, however,

both strategies can be used. As we will see depending on

species, transdifferentiation or progenitor cells can be

recruited to populate damaged retina.

0014-4835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

DOI:10.1016/j.exer.2003.10.022

Experimental Eye Research 78 (2004) 161–172

www.elsevier.com/locate/yexer

* Corresponding author. Dr Panagiotis A. Tsonis, University of Dayton,

Laboratory of Molecular Biology, Department of Biology, Dayton, OH

45469 2320, USA.

E-mail address: [email protected] (P.A. Tsonis).

http://www.elsevier.com/locate/yexer

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 regen-

eration 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

fiber differentiation. During days 15 –19, proliferation and

depigmentation of PECs slows down. In the internal layer,

the lens fibre complex is formed and in the margin of the

external layers non-dividing secondary lens fibres appear.

By 18–20 days the PECs have stopped proliferating, and the

lens fibres continue to accumulate crystallins (Fig. 1(C)).

Lens regeneration is considered complete by day 25–30

(Eguchi, 1963, 1964; Reyer, 1977; Yamada, 1977; Tsonis,

1999, 2000). Lens regeneration, therefore, is a clear case of

transdifferentiation. A very interesting restriction is that the

ventral iris, which is seemingly comprised by the same

PECs is not capable of regenerating a lens. The process of

transdifferentiation has been proven beyond any doubt in

this system. These processes can also be observed when

single PEC cells are placed in culture (Eguchi et al., 1974;

Kodama and Eguchi, 1995; Tsonis et al., 2001). The

restrictions that we see in the in vivo newt model do not

apply for the in vitro models. PECs from the whole eye

(including from the ventral iris) and from any species,

including aged humans are capable of transdifferentiating to

lens cells under certain conditions (Tsonis et al., 2001).

Among other amphibians frogs can regenerate their lost

lens, but in contrast to the newt, regeneration occurs via

transdifferentiation of the inner layer of the outer cornea.

Another important difference is that lens regeneration in

frogs is possible only during premetamorphic stages and

ceases after metamorphosis (Freeman, 1963; Filoni et al.,

1997; Henry and Elkins, 2001). Also, in Xenopus laevis, the

capacity seems to depend on factors that are provided by the

retina (Filoni et al., 1982). When a piece of outer cornea is

implanted in the vitreous chamber, even in the presence of

the host lens, transdifferentiation can occur. It is possible

that the rapid closure of the inner cornea after metamor-

phosis is an inhibitor to regeneration (Reeve and Wild,

1978; Filoni et al., 1997). The stages during lens

regeneration from the cornea are very similar to the ones

Fig. 1. Lens regeneration in newts via transdifferentiation of the PECs

from the dorsal iris (di). (A) Ten-days post-lentectomy. Note an early lens

vesicle (arrow) formed by dedifferentiation of the PECs from the dorsal

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-

lentectomy. A well differentiated lens with lens fibres (lf) covered by the

lens epithelium (le).

P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172162

seen from the dorsal iris in newts. A vesicle is first formed

and then gradually crystallins and lens fibres accumulate.

During the final stages the lens is positioned along the dorsal

and the ventral iris (Fig. 2). It is interesting to note here that

FGF-1 seems to be very important in the process of

transdifferentiation both in newts and in frogs. When newt

PECs or frog outer cornea is placed in vitro, FGF-1 is an

inducer of transdifferentiation (Hyuga et al., 1993; Bosco

et al., 1997).

In the past few years research has been concentrated in

the identification of key genes for the induction of

transdifferentiation. The strategy is to identify dorsal-

specific genes and examine their possible function during

lens regeneration. These genes can then become important

tools to probe why ventral iris (of the newt) or irises from

other animals are not capable for regenerating a lens. Some

of these genes are presented in Table 1. Table 1 clearly

indicates that genes that are normally expressed during lens

embryogenesis, and can induce lens morphogenesis, are also

activated during lens regeneration as well (Del Rio-Tsonis

et al., 1995, 1997, 1999; Mizuno et al., 1999; Schaefer et al.,

1999). It remains though to be seen whether or not these

genes are the real signals for initiation of lens regeneration

or their activation is a secondary step after the initiation of

lens regeneration by a yet unknown signal. Why related

salamanders show differences in the capacity of lens

regeneration remains a mystery. For example, the axolotl,

which is a urodele cannot regenerate the lens, even though

its ability to regenerate limbs or the tail parallels that of the

newt. Imokawa and Brockes (2003) have found that

thrombin activation could be a critical determinant. As

mentioned above, thrombin activation can be seen within

minutes after lentectomy in the dorsal iris of the newt, while

such an event is undetectable in the iris of the axolotl. These

findings stress the importance of comparative studies using

different species. Only then we will be able to understand

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).

P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 163

why some salamanders are endowed with such an advantage

and other species are not. Among other vertebrates only

some fish are capable of regenerating the lens in a manner

similar to the one seen in newts (Sato, 1961).

In mammals, lens regeneration studies have been

largely restricted to rabbits. It has been documented that

if someone performs an endocapsular lentectomy, in other

words removes the lens fibres, but leaves the lens capsule

behind, lens regeneration can occur (Stewart and Espi-

nase, 1959; Gwon et al., 1990, 1993a,b). The reconstruc-

tion of the lens depends largely on the presence of

adherent lens epithelial cells that remained on the capsule.

These cells follow their normal course and differentiate to

lens fibres, which in turn fill the capsule (Fig. 3) and

create a lens with many normal properties (Gwon et al.,

1993b). Such studies are very important because they

indicate that the lens has impressive reparative capabilities

if manipulated correctly. Also, such studies have impli-

cations in cataract therapy and surgery.

1.1. Clinical applications: towards strategies to materialize

lens regeneration in mammals

Cataracts are the main clinical manifestations of the lens.

Cataracts can be genetic or induced as a result of aging.

Also, cataracts can affect different regions of the lens, i.e.

the nucleus, or the cortex (Francis et al., 1999). In humans,

cataracts can be surgically corrected. The operation leaves

the capsule as intact as possible, which then is used to hold a

synthetic lens in the right place. The problem with this

operation is that lens epithelial cells that remain adhered to

the capsule transdifferentiate to mesenchymal cells and as

they migrate posteriorly they opacify the capsule. This is

called posterior capsule opacification (PCO) and is the

major complication of cataract surgery (Apple et al., 1992).

In most of the cases another surgery is necessary.

Obviously, if lens regeneration were to be successful in

humans, there would be no need for such an operation. But

how do we achieve lens regeneration in mammals? We

propose two major directions of research. In first, we must

identify dorsal iris-specific signals in the newt. These

factors can then be tested to examine if they are capable of

inducing lens regeneration from incompetent tissues.

Incompetent tissues should first include newt ventral iris

and then irises of other salamanders, such as the axolotl.

Once regeneration has been successfully induced, these

factors should be tested for their ability to induce

regeneration in mammals. Mice should be the best animal

model, because of the availability of genetic tools. If this is

successful, we should proceed in higher mammals. There is

compelling evidence that such strategy will succeed. When

PECs are cultured for a long time, they can transdifferentiate

to lens and this ability seems to have no species or age

barriers (Kodama and Eguchi, 1995). We just need to

identify the trigger that allows the newt to regenerate the

lens in vivo. The second research direction should deal with

the capacity of lens regeneration from the remaining lens

capsule. If rabbits and cats can reconstruct a lens, we see no

reason why this should not be the case in humans. Several

questions, though, must be answered. Is there an age factor?

Can lens capsules regenerate a functional lens? In regards to

the first question there is much research to be done and

unfortunately rabbits or cats are not favourable animal

models. If someone is to pursue research on factors involved

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).

Table 1

Expression of lens-specific genes during lens regeneration

Genes Newt/dorsal-ventral regulation Xenopus

Pax-6 þ /yes þ

Prox-1 þ /yes þ

FGFR-1 þ /yes ND

Otx-2 ND þ

Sox-3 ND þ

þ , expression; yes, dorsal–ventral regulation; ND ¼ not determined.


model is imperative. The answer to the second question

should be yes. If we could surgically manipulate the capsule

to remain in a spherical shape the reconstructed lens should

be functional. Some successful experiments have been

presented (Gwon et al., 1993b). In regard to PCO, animal

models should be established where the ability of the lens

epithelial cells to differentiate to lens fibres or to

transdifferentiate to mesenchymal cells will lead to the

identification of factors, whose use will help interfere with

the process. Again here we believe that these approaches are

realistic, we just need to pursue it more rigorously with the

right animal model.

2. Retina regeneration

Regenerating a retina or part of a retina has been

observed in a variety of organisms during either their

development or for some even during their adult life.

Among those with the ability to regenerate during adulthood

are fish, birds and amphibians (Del Rio-Tsonis and Tsonis,

2003). The modes of retina regeneration vary depending on

the organism (Fig. 4). The regenerative ability of some adult

forms can also be different from that present in some

embryos of the same species. For example, regeneration of

the retina in some fish, bird and amphibian embryos/larvae

has been observed to take place by transdifferentiation of the

retinal pigment epithelium (RPE). However, only certain

urodeles retain the capability to regenerate their retinas via

transdifferentiation as adults (Lopashov and Stroeva, 1964;

Mitashov, 1996, 1997; Raymond and Hitchcock, 2000;

Fischer and Reh, 2001a; Del Rio-Tsonis and Tsonis, 2003).

The process of transdifferentiations involves a cell

conversion not typically encountered in adult tissues. RPE

(Fig. 5(A)) lose their characteristics of origin and re-enter

the cell cycle to form a neuroepithelial cell layer (Fig.

5(B)) that eventually will differentiate into all the different

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,

1992; Mitashov, 1996, 1997; Raymond and Hitchcock,

2000; Del Rio-Tsonis and Tsonis, 2003). Embryonic

chicks, which can also regenerate their retinas via

transdifferentiation of the RPE with FGF, lose this layer

as it becomes the neuroepithelium and eventually differ-

entiates into all retinal layers (Fig. 6(D)). This neuroe-

pithelium seems to develop similar in sequence to that of

normal development, but with reverse polarity (Fig. 6F).

As a result, the rods and cones of the photoreceptor layer

are located in the inner most layer of the retina, which is

closest to the lens (Coulombre and Coulombre, 1965; Park

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 Muller 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 Muller 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).


Retina replacement in the embryonic chick eye has also

been observed to originate from the ciliary region (Fig. 4),

but only if a source of FGF is supplied (Fig. 6(C) and (E))

(Coulombre and Coulombre, 1965; Park and Hollenberg,

1991, 1993). This process appears to occur via the use of

neural precursors (Willbold and Layer, 1992). The CMZ in

adult chickens, on the other hand, is unable to replace

chemically damaged retina even though the cells of the

CMZ are mitotically active (Morris et al., 1976; Fischer and

Reh, 2000) and respond to growth factors by increasing their

proliferation and differentiation into different neural retina

cells (Fischer and Reh, 2000; Fischer et al., 2002a,b). Even

the cells from the pigmented ciliary margin are mitotically

active but are not conducive to produce neural retinal cells

(Fischer and Reh, 2001a). Recently Muller glia cells have

been identified as the cells responsible for responding to

local retinal damage and replacing neural retina in adult

birds (Fig. 4). The cells replaced depend on the type of cells

that were damaged originally and on the presence of growth

factors such as FGF-2 and insulin. Upon damage, Muller

glia cells re-enter the cell cycle, de-differentiate producing

neural precursors and eventually differentiate into neurons

and glia cells (Fig. 7) (Fischer and Reh, 2001b; Reh and

Fischer, 2001; Fischer et al., 2002a,b; Fischer and Reh,

2003).

In teleost fish, retina regeneration takes place via the use

of several cell sources including rod precursors (Raymond

et al., 1988; Braisted and Raymond, 1992; Hitchcock et al.,

1992), intrinsic stem cells in the INL (Raymond and

Hitchcock, 1997; Julian et al., 1998; Otteson et al., 2001;

Reh and Fischer, 2001; Wu et al., 2001; Otteson and

Hitchcock, 2003) and possibly Muller glia cells that

proliferate and migrate to the ONL (Braisted and Raymond,

1993; Braisted et al., 1994; Raymond and Hitchcock, 1997,

2000; Reh and Fischer, 2001; Wu et al., 2001; Fischer and

Reh, 2003) (Fig. 4). This replacement depends on the

damage elicited and has to include damage in the ONL cell

layer, otherwise no regeneration will take place (Negishi

et al., 1987, 1988; Raymond et al., 1988; Braisted and

Raymond, 1992; Hitchcock, 1992; Hitchcock and Ray-

mond, 1992; Otteson and Hitchcock, 2003).

The story is different for mammals where regeneration

has not even been observed in embryos, unless the tissue

was transplanted or manipulated in vitro (Stroeva, 1960;

Zhao et al., 1995; Ahmad et al., 1999; Chacko et al., 2000,

2003). Recent reports suggest the possibility that mammals

could regenerate their retina if properly induced. Adult

pigmented cells from the ciliary epithelium of rodents

(Ahmad et al., 2000; Tropepe et al., 2000) and humans

(Personal communication Arsenijevic, 2003, see Fig. 7)

have been induced to proliferate in vitro and eventually

differentiate into retinal specific cells including rod

photoreceptors, bipolar neurons and even Muller glia cells.

Other local sources for possible retinal progenitors in

mammals have been explored and include cells from the

iris, corneal limbus area, sclera and choroid (Haruta et al.,

2001; Zhao et al., 2002; Arsenijevic et al., 2003). Cultured

iris cells from rat have been induced to differentiate into

retinal cells, including photoreceptors when transfected

with Crx, a crucial photoreceptor developmental gene

(Haruta et al., 2001). On the other hand, rat limbal epithelial

cells cultured in vitro under certain growth conditions

express neural progenitor markers that eventually differen-

tiate towards the neural lineage (Zhao et al., 2002). When

these stem cells are transplanted unto eyes that have retinal

damage they migrate and integrate in different retinal layers

and start expressing retinal neural markers (Chacko et al.,

2003). Lastly, sclera and choroid cells isolated from adult

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.


human eyes have the potential to differentiate towards the

neural linage (Arsenijevic et al., 2003). Even though the last

sources described have not been exploited yet for the

production of retinal cells, the door is definitely open to

explore that option.

2.1. Clinical applications: toward repairing

diseased retinas

Dissecting the mechanisms underlying retina regener-

ation will contribute to the design of procedures that could

rescue eyes that had undergone retinal degeneration (Table

2). The different animal models available to study retina

regeneration and their corresponding modes of regeneration

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

and the newt could provide the necessary clues to induce

transdifferentiation of the RPE into neural retina in humans.

From these retina regeneration studies so far, we know that

growth factors are essential, specifically fibroblast growth

factors (FGFs). From in vitro transdifferentiation studies we

also know that transcriptional regulators are also essential.

For example, microphthalmia (Mitf), a retinal pigment

epithelium identity molecule, must be down-regulated

during transdifferentiation, while Pax-6, a master regulator

of eye development should be up-regulated (Mochii et al.,

1998).

On the other hand, if the damage is local, Muller glial

cells found in the inner nuclear layer of the retina could also

be used as source of new retina (Fig. 4). Muller glial cells

have been found to replace neural cells and glial cells in

adult damaged retina of birds and the possibility that these

cells could also do the same in mammals should be

considered. Again growth factors seem to be essential for

this process to take place as mentioned previously.

The use of other local sources of retina replacement

should be considered such as cells from the CMZ or the

ciliary epithelium (Fig. 4). Pigmented cells from these

regions could be induced to differentiate/transdifferentiate

in vivo to participate in retina repair. Also non-neuronal

stem cells within the eye such as cells from the corneal

limbal epithelium, sclera and choroid have been tested to

give rise to neurons and glial cells and could potentially

provide a source for replacing damaged retina.

It seems that we have plenty of possible sources for retina

replacement and studies at the basic cellular and molecular

level using the different animal models discussed should

provide the scaffold for clinical studies to take place.

Fig. 6. Retina regeneration in embryonic chick eyes. (A) Intact chick eye at

day 7 of development. (B) Retinectomized chick eye at day 4 of

development. The entire neural retina has been removed and the RPE

layer has thickened. When FGF-2 heparin beads were added to a

retinectomized eye, the retina regenerated from the CMZ/ciliary epithelium

region (C) and via transdifferentiation of the RPE at the posterior region of

the eye (D). The eyes were analyzed 5 days post-retinectomy (day 9 of

development). At this time a nice neuroepithelium has formed. (E)

Regeneration via the use of neural precursors from the CMZ/ciliary

epithelium at 11 days of development or 7 days post-retinectomy. Note all

the retinal layers are nicely formed by now: the outer nuclear layer (ONL),

the inner nuclear layer (INL) and the ganglion cell layer (GCL). (F)

Regenerating retina 7 days post-retinectomy (11 days of development)

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

layers in the original orientation while the retina regenerated via

transdifferentiation of the RPE has a reversed or mirrored orientation. l:

lens; RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner

nuclear layer and GCL: ganglion cell layer; NFL: nerve fiber layer. Sections

were stained with Hematoxylin and Eosin.


Fig. 7. Muller 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 Muller glia marker. (C) An overlay of images A and B. Scale bar ¼ 50 mm. Mitotically active cells

(100%) were Muller 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 Muller 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 Muller 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).


Transdifferentiation and stem cells

The term transdifferentiation was coined by Selman

and Kafatos (1974) to distinguish the switching of a

terminally differentiated cell type into another from the

neoplastic transformation seen during cancer formation.

The process of transdifferentiation, therefore, entails that a

terminally differentiated somatic cell dedifferentiates

(loses the characteristics of its tissue of origin) and then

differentiates into another cell type. This term was

adapted and literally became synonymous with the

process of regeneration, such as of limbs, tail, retina or

lens observed in adult salamanders. Transdifferentiation

has been shown to be the underlying mechanism whereby

regeneration in amphibia is achieved. Numerous studies

have clearly shown that during limb regeneration adult

myofibres can transdifferentiate to cartilage and vice

versa. Similarly, after lens removal the newt regenerates a

new one by transdifferentiation of the PECs of the dorsal

iris, a phenomenon that has been shown conclusively in

clonal cultures of PECs as well.

In recent years a new strategy involved in tissue

repair, marshalled by stem cells, has gained ground.

Stem cells can be totipotent (cells before the formation

of the blastocyst), or pluripotent, which can give rise to

different cell types upon selective activation. Once a

particular stem cell population has been activated and

committed to a lineage, these cells become progenitor

cells. Stem cells can be local, tissue-specific and reside

in adult tissues, such as brain, skeletal muscle, skin,

retina or liver. However, non-local stem cells can be

found as well. Such stem cells reside, for example, in

bone marrow and participate in repair of brain, heart,

liver or other tissues. Similarly, stem cells residing in

brain can become blood or muscle cells (Blau et al.,

2001). Accordingly, the term transdifferentiation was

adopted for these properties of non-local stem cells.

However, some of these studies have met with opposi-

tion. Additional experiments have shown that stem cells

could acquire characteristics of other cells by fusion,

which in turn might account for transdifferentiation of

non-local stem cells (Terada et al., 2002; Ying et al.,

2002; Wang et al., 2003; Vassilopoulos et al., 2003).

Soon thereafter, reviews and papers appeared indicating

the concept of transdifferentiation is in trouble (Wells,

2002). A clear distinction must be made here. The

process of transdifferentiation in non-local stem cells

might be in trouble, but not the process of transdiffer-

entiation in general. The process of transdifferentiation in

classical regenerative phenomenon such as the ones seen

in salamanders is not in trouble at all. We are not saying

that is unacceptable to use the term transdifferentiation

for stem cells, but it should not be used to address only

the properties of stem cells, because the classical

regenerative phenomena are accurately described by it.

Let us now see the other side of the coin. Can a

terminally differentiated cell be considered a stem cell?

For example, newt PECs can transdifferentiate to neural

retina cells and to lens cells and in addition they can

renew themselves (Del Rio-Tsonis and Tsonis, 2003).

This plasticity depends on their position within the eye as

well as the type of surgery performed. Therefore, because

PECs can give rise to different cell lineages, they can be

regarded as transdifferentiating stem cells. This estab-

lishes a common ground for the two regeneration

strategies. The approach we should take is to learn by

studying and comparing both of these strategies. In our

mind, the molecular mechanisms involved in transdiffer-

entiation of terminally differentiated cells and in acti-

vation and differentiation of local stem cells could be

remarkably similar, if not the same. For example, during

limb regeneration in the newt, blastema cells, the product

of dedifferentiation of the stump tissues, can re-differen-

tiate to muscle or cartilage. Likewise, in bone marrow

there are mesenchymal stem cells that they can differen-

tiate to muscle or cartilage cells. It is conceivable that a

blastema cell destined to become cartilage and a

mesenchymal stem cell destined to become cartilage

would have very similar molecular signatures at a certain

stage. At this stage both cells can be unified at the

molecular level. Along these ideas it is interesting to note

that different species use transdifferentiation of PECs or

stem cells to repair damaged retinas. Also, invertebrates,

animals with incredible regeneration deeds, make use of

both terminally differentiated cells and reserve cells to

restore lost parts of their bodies. A simple explanation for

this could be that both strategies lead to activation of

similar molecular programme to achieve their goals.

With this in mind, comparative studies can be designed

that could yield very important data on the mechanisms of

transdifferentiation and of stem cell biology. Indeed,

recent studies have shown that embryonic and adult

neural and hematopoietic stem cells do share a molecular

signature (or ‘stemness’) having some 200 genes

commonly transcribed (Ivanova et al., 2002; Ramalho--

Santos et al., 2002).

3. Concluding remarks

This review began with a wonder about the evolutionary

importance of lens and retina regeneration. It was argued that

if regeneration of eye tissues was an advantage it should be

more widespread than confined in only some salamanders.

We then reviewed current research and ideas that dominate

the fields of lens and retina regeneration. It was noted that

despite the favouritism that nature has shown to newts, in

reality the potential for regeneration of eye tissues is high.

Different species use different strategies to compensate for

damaged eye tissues. We also discussed the possibility that


regeneration research from different animal models will

eventually lead to therapies for diseases that affect eye

tissues. It is our firm belief that ‘regeneration therapies’ will

be a reality for diseased eyes in the future.

Acknowledgements

We thank Drs Stefano Cannata, Arlene Gwon, Thomas

Reh and Yvan Arsenijevic for contributing figures. We also

thank Mayur Madhavan and Natalia Vergara for contribut-

ing to the artwork. Supported by grants EY10540 to P.A.T.

and EY14197 to K.D.R.-T.

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