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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).
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.
P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172164
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).
P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 165
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.
P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172166
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.
P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 167
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).
P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172168
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
P.A. Tsonis, K. Del Rio-Tsonis / Experimental Eye Research 78 (2004) 161–172 169
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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