ARTICLE IN PRESS
Progress in Retinal and Eye Research 23 (2004) 149–181
Stem cells and retinal repair$
Henry Klassena, Donald S. Sakaguchib, Michael J. Youngc,*
a
Stem Cell Research, Children’s Hospital of Orange County, Orange, CA 92868, USA
Department of Genetics, Development and Cell Biology (GDCB) and Department of Biomedical Sciences, Iowa State University, Ames,
IA 50011, USA
c
Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA
b
Abstract
Retinal stem cells (RSCs) are multipotent central nervous system (CNS) precursors that give rise to the retina during the course of
development. RSCs are present in the embryonic eyecup of all vertebrate species and remain active in lower vertebrates throughout
life. Mammals, however, exhibit little RSC activity in adulthood and thus little capacity for retinal growth or regeneration. Because
CNS precursors can now be isolated from immature and mature mammals and expanded ex vivo, it is possible to study these cells in
culture as well as following transplantation to the diseased retina. Such experiments have revealed a wealth of unanticipated
findings, both in terms of the instructive cues present in the mature mammalian retina as well as the ability of grafted CNS
precursors to respond to them. This review examines current knowledge regarding RSCs, together with other CNS precursors, from
the perspective of investigators who wish to isolate, propagate, genetically modify, and transplant these cells as a regenerative
strategy with application to retinal disease.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Retina; Stem cell; Development; Transplantation; Progenitor cell
Contents
1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
150
2.
Retinal cellular development . . . . . . . . . . . . . . . . .
2.1. Cell birth and differentiation in the vertebrate retina .
2.1.1. Retinogenesis . . . . . . . . . . . . . . . . .
2.1.2. Control of retina cell-type specification . . . .
2.2. Transdifferentiation of the retinal pigment epithelium .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
151
151
151
153
154
3.
Developmental role of stem cells in the retina and CNS . . . . . . . .
3.1. Embryological origins of the vertebrate retina . . . . . . . . . .
3.2. Molecular mechanisms of retinal stem cell specification . . . . .
3.2.1. Molecular control of neural specification in Drosophila
3.2.2. Molecular control of neural specification in vertebrates .
3.2.3. Notch signaling and neuronal differentiation . . . . . .
3.2.4. Molecular control of eye specification in Drosophila . .
3.2.5. Molecular control of retinal specification in vertebrates .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
154
155
155
155
156
157
158
158
4.
Cultured CNS stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Multipotent cells from the mammalian CNS can be grown in culture .
4.1.1. Neural stem cells can be identified by marker expression . . .
4.1.2. Neural progenitor cells express cytokines . . . . . . . . . . .
4.2. Multipotent cells from the mammalian retina . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
160
160
160
161
162
$
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
The authors wish to dedicate this work to the memory of their friend and colleague Vadim V. Filatov, MD.
*Corresponding author. Tel.: +1-617-912-7419; fax: +1-617-912-0101.
E-mail address: mikey@vision.eri.harvard.edu (M.J. Young).
1350-9462/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.preteyeres.2004.01.002
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
150
5.
Immunology of CNS stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
164
6.
Transplantation of CNS stem cells . . . . . . . . . . . . . . . .
6.1. Clinical transplantation of fetal neural tissue . . . . . . .
6.2. Transplantation of NSCs in animal models of brain injury
6.3. Experimental retinal transplantation . . . . . . . . . . . .
6.4. Clinical retinal transplantation . . . . . . . . . . . . . . .
6.5. Retinal transplantation of CNS stem cells . . . . . . . . .
6.5.1. Transplantation of CNS stem cells to the Brazilian
investigating neural stem cell plasticity . . . . . .
6.5.2. Transplantation of retinal stem cells . . . . . . . .
.
.
.
.
.
.
168
168
168
169
169
169
. . .
. . .
171
174
7.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
175
8.
Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
175
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
176
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
176
1. Introduction
Stem cells have attracted considerable attention
recently, not only as a means of understanding
metazoan development but also as potential therapeutic
agents for a spectrum of currently untreatable diseases.
However, there remains a considerable degree of
latitude in what is meant by the term ‘‘stem cell’’ in a
given context. Any in-depth discussion of this area
therefore benefits from a clarification of the semantics to
be used, at the earliest opportunity. A basic definition of
‘‘stem cell’’ is a cell that both self-renews and gives rise
to multiple mature cell types. One corollary of this
definition is that an investigator who wishes to positively
identify a stem cell must demonstrate that both these
events are in fact occurring in the same clone of cells.
Proving this is not necessarily easy, but is required for
the unambiguous identification of true stem cells.
Nevertheless, we favor adherence to a precise definition
of a stem cell and, except for the case of embryonic stem
(ES) cells, prefer the term ‘‘progenitor cell’’, if only to
highlight the need for much additional research and
continued characterization of the cell populations under
discussion. Fortunately, researchers in the area have not
been shackled by semantic concerns. There is much
practical knowledge to be gained from models in which
the cells of interest are not stem cells per se. It is not
difficult to envision a large number of scenarios,
particularly in regenerative medicine, in which something other than a ‘‘true stem cell’’ will constitute the cell
of choice for transplantation. Furthermore, a precise
adherence to terminology is likely to be more meaningful at a time when our understanding of fate
determination is itself more mature. In this introduction,
we will briefly consider the implications of the basic
definition of a ‘‘stem cell’’ before turning our attention
to retinal development, advances in cell culture, and
transplantation experiments.
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
opossum: a novel model system
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. .
. .
. .
. .
. .
. .
for
. .
. .
.
.
.
.
.
.
.
.
.
.
.
.
Implicit in the basic concept of self-renewal is the
requirement for phenotypic plasticity before, and the
maintenance of phenotypic plasticity after, cell division.
Yet a subset of daughter cells must eventually abandon
phenotypic plasticity and differentiate, losing their
identity as stem cells. The basic definition of a stem cell
therefore contains an implicit requirement for asymmetric cell divisions in which one daughter cell remains
multipotent and ‘‘stem-like’’, while the other abandons
‘‘stemness’’ to differentiate into a mature cell. Furthermore, the very plasticity and multipotentiality that
makes these cells so interesting ultimately confronts us
with inherent logical paradoxes when trying to apply the
strict definition of stem cell to a given specimen. This is
not just theoretical, but practical. One cannot know
with certainty the potentiality of a cell until it
differentiates and, even then, all we definitively learn
from the exercise is something about the potential of a
cell at some point in the past. In other words, present
methods of assaying the phenotypic potential of
cultured cell populations lack predictive certainty with
respect to future results.
A look at tissue-specific stem cells of current clinical
significance provides ample evidence that the strict
definition of ‘stem cell’ does not apply. Hematopoietic
stem cells consistently give rise to every type of blood
cell (Appelbaum, 2001), yet recent blood shortages
highlight the fact that these cells have not been induced
to self-renew indefinitely ex vivo. In another example,
corneal limbal stem cells cannot be prospectively
identified and only give rise to a single-cell type, the
corneal epithelial cell. The message here is that in neither
case has the failure of biology to live up to ideology
thwarted the successful clinical application of these cells.
Practically, the biological facts are indeed likely to differ
between stem-like cell types from different sources and,
in the final analysis, the greatest relevance lies in a cell’s
capability in the setting of disease. Thus, common usage
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
has tended toward the association of the term ‘‘stem
cell’’ with any phenotypically plastic cell type that can be
induced to repair damaged tissue. While the strict
definition of a stem cell remains of understandable
interest to the developmental biologist, at the end of the
day, it is clearly the potential capacity to alleviate
human suffering that has brought so much attention to
this field and continues to drive it forward.
2. Retinal cellular development
The vertebrate retina provides an ideal model for
investigations of central nervous system (CNS) development and plasticity. It is organized into distinct, welldefined layers and, because of its location in the eye, is
readily accessible for experimental manipulation and
analysis. The cytoarchitectural organization of the
mature retina results from a complex series of developmental processes involving both intrinsic and extrinsic
cues. A large body of literature exists in which the
processes of neurogenesis and differentiation within the
retina have been described in considerable detail (Sidman, 1961; Hinds and Hinds, 1974; Cepko et al., 1996).
In addition, a wide range of species have been used as
model systems for studying retinal and visual system
development, each system being of comparative interest
because of its own unique attributes.
2.1. Cell birth and differentiation in the vertebrate retina
All vertebrate retinas consist of seven major classes of
cells. Closer examination, however, reveals numerous
subtypes within each major class bringing the total to
more than 50 unique cell types (Masland, 2001). The
genesis, determination, and differentiation of these
different cell types are under active investigation in
multiple vertebrate species. Issues of significant interest
include the control of phenotypic plasticity in retinal
precursors, the role of extrinsic factors in cell fate
decisions, and the role of transcription factors in celltype specification and differentiation. Factors regulating
interactions between retinal cells during neurogenesis
are also under active investigation.
2.1.1. Retinogenesis
As previously noted, the eye fields give rise to bilateral
evaginations of the diencephalon in the early neurula
stage of the vertebrate embryo. In fish and amphibians
this process is marked by bulging of the optic primordia,
whereas in mammals it is characterized by formation of
the optic pit. Continued evagination of the optic
primordia results in the formation of the optic vesicles,
and these extend toward the overlying surface ectoderm
that will give rise to the cornea and lens. The eye begins
forming as a result of the coordinated invagination of
151
the lens placode and the primary optic vesicle resulting
in the formation of the lens vesicle and the two-layered
structure, the secondary optic vesicle, or optic cup. The
innermost layer, closest to the lens vesicle, ultimately
forms the multilayered neural retina while the outer,
single layer remains an epithelial monolayer and gives
rise to the retinal pigment epithelium (RPE). Although
sharing a common embryological origin, the presumptive RPE and retinal neuroepithelium express different
transcription factor profiles during development, and
exhibit quite distinct properties following differentiation. As invagination of the optic cup progresses, a
groove forms in the ventral aspect of the optic vesicle.
This groove is referred to as the optic (or choroidal)
fissure. The structural organization of the optic fissure
provides an exit route for axons of the retinal ganglion
cells (RGCs), as well as a conduit for blood vessels
supplying the eye. The peripheral margin of the optic
cup, where the inner and outer layers meet, gives rise to
the ciliary epithelium and iris (Beebe, 1986). As the optic
cup continues to form, the cells of the developing retinal
epithelium are multipotential retinal progenitor cells
(RPCs).
The cellular organization and gross morphogenesis of
the eye and retina are similar across vertebrate species
(Cepko et al., 1996) and the basic pattern of retinal
histogenesis has been evident for some time, primarily
from the Golgi studies of Ramon y Cajal. More recent
studies have extended the original observations utilizing
a variety of methodologies including electron microscopy, birthdating analysis with [H3] thymidine or
bromodeoxyuridine (BrdU), single-cell labeling techniques, molecular techniques, as well as additional
histological techniques. Cell lineage analyses in fish,
amphibians, chick, and mammals have revealed that
RPCs are multipotent, proliferative, and give rise to
postmitotic progeny that ultimately differentiate into the
various cell types that comprise the retina. These cells
exit the mitotic cycle in a characteristic, stereotyped
sequence that is highly conserved across vertebrates
(Young, 1985; Holt et al., 1988; Spence and Robson,
1989) (Fig. 1).
Coupled with the use of cell-type specific antibodies,
birthdating studies using [H3] thymidine or bromodeoxyuridine and lineage analyses using intracellular injection of tracers or retroviruses have contributed much to
our understanding of cell specification in the developing
retina. Studies in Xenopus embryos and larvae have
revealed that the multipotent retinal progenitors can
give rise to all major cell types of the retina in a lineageindependent fashion (Wetts and Fraser, 1989). In
addition, retroviral tracing in the postnatal rodent
retina revealed a common retinal progenitor for both
neurons and glia (Turner and Cepko, 1987).
The first cells produced in the developing retina are
the RGCs followed by cones and amacrine cells. The
ARTICLE IN PRESS
152
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Fig. 1. Retinogenesis.
horizontal cells are born shortly thereafter in some
species (chick and mammals) while in others (fish and
amphibians) they are born a bit later. Rods appear in
the next wave of cell birth, with most bipolar cells and
Muller
.
glia being generated during the last phase of
retinal neurogenesis (Holt et al., 1988; Wetts and Fraser,
1989; Prada et al., 1991; Hu and Easter, 1999; Das et al.,
2003). Although this birth sequence occurs in a
stereotyped pattern, there is considerable overlap among
the different classes of cell types. For example, at almost
any given time point during the period of retinogenesis
multiple retinal cell types are being produced. As the
various cell types are generated, they migrate to one of
three cellular layers in the developing retina, i.e., the
outer nuclear layer (ONL), inner nuclear layer (INL), or
ganglion cell layer (GCL), to occupy their final position.
Retinal neurons differentiate in a central to peripheral
sequence, such that cells closest to central retina are the
first to be born and differentiate, while those at the
periphery generally lag behind.
In the cold-blooded vertebrates the retina continues
to grow throughout the animal’s life by the addition of
new neurons at the rim of the retina from a germinal
zone at the ciliary margin (Otteson and Hitchcock,
2003). A gradient is thus created whereby the first born
ganglion cells are clustered in the central region
surrounding the optic nerve head near the center of
the growing retina. In the teleost retina, all neurons
(except for rod photoreceptors) are continually added
by the progenitors positioned at the margin of the
retina. However, rod photoreceptors are added from a
special class of dedicated rod progenitor cells. Interestingly, when the retina is damaged, rod progenitors
lose this restriction and become capable of regenerating the full range of retinal cell types (Otteson and
Hitchcock, 2003).
Contrary to previous theories in which the decision to
commit to neuronal or glial lineage occurs relatively
early in development, experimental studies have now
consistently demonstrated that CNS progenitor cells
remain multipotent well into development. For example,
retinal progenitors remain capable of giving rise to most
all retinal cell classes, as well as self-renewing to generate
more retinal progenitors (Adler and Hatlee, 1989; Wetts
and Fraser, 1989; Cepko et al., 1996). An exception is
the formation of the astrocytes which populate the inner
surface of the retina. These cells are generated outside
the eye and subsequently migrate into the retina via the
optic nerve.
In studies of the fate of RPCs in mammals, Cepko
and colleagues used retroviral vectors to mark clones in
embryonic and postnatal rodent retinas. Analysis of
marked clones following retinal maturation revealed
that retinal progenitors were multipotent and that a
single progenitor could generate most classes of retinal
cell. Retroviral mediated gene transfer to mark clones in
postnatal retina revealed these progenitors to be more
restricted than those in the embryo, yet nevertheless
capable of generating three types of neurons or two
types of neurons and a Muller
.
glial cell. Thus, although
more restricted, postnatal retinal progenitors were still
generating diverse cell types at a time point near the end
of the developmental period (Turner and Cepko, 1987).
From these studies, as well as others, a model has
emerged for the generation of retinal cells in which the
cessation of mitosis and cell fate determination are
independent events, regulated by microenvironmental
interactions within the developing retina.
Cell fate determination in the retina is thought to be
regulated, in part, by a series of local cell–cell interactions, together with intrinsic mechanisms. A growing
body of evidence suggests that retinal precursors utilize
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Delta–Notch-mediated signaling to regulate cell fate
determination (Ahmad et al., 1997; Rapaport and
Dorsky, 1998). In neurogenic regions, including the
retina, Delta–Notch signaling seems to be involved in
prohibiting neural stem cells (NSCs) from entering into
a neuronal differentiation pathway (Ahmad et al., 1997;
Rapaport and Dorsky, 1998). However, introduction of
the mouse Notch gene into rat retinal progenitors
stimulated cell division and promoted formation of
Muller
.
glial cells (Furukawa et al., 2000). Delta and
Notch are structurally related proteins that act as ligand
and receptor, respectively. In the developing chick
retina, Delta is co-expressed spatially and temporally
with Notch and their expression is associated with
temporal aspects of cell birth. These results support the
view and provide evidence that Delta–Notch signaling is
used in progenitors to maintain themselves in an
uncommitted state. Thus, fluctuations in Delta–Notch
signaling might be instrumental to sorting out competent cells during successive cell fate determination
events.
2.1.2. Control of retina cell-type specification
As previously discussed, the major retinal cell types
arise from a pool of multipotential retinal progenitors. It
is likely that both the retinal microenvironment and the
progenitors themselves change over time in order to
regulate the developmental process. Numerous studies
have implicated multiple genes, including secreted
factors, in the guidance of retinal progenitors toward
different specific cell fates. Extrinsic factors shown to
influence the cell fate decisions of retinal progenitors
include growth factors, secreted transcription factors,
extracellular matrix molecules (ECM), and retinoids.
In a simple model one might propose that extrinsic
signaling mechanisms regulate generation of specific
retinal cell types from a uniform or homogeneous
population of multipotent RPCs. If this were the case,
one would simply predict that late (postnatal) retinal
progenitors would adopt cell fates normally generated
during early retinogenesis if placed among an embryonic
retinal environment. However, late retinal progenitors
show intrinsic limits in the production of cell types when
cultured with an excess of embryonic cells (Belliveau
et al., 2000). Under these conditions, postnatal cells
exhibited an apparent suppression of rod cell fate,
together with an increase in the percentage of bipolar
cells, suggesting a possible respecification of fate.
However, the postnatal cells did not adopt cell fates
normally produced by embryonic cells. In a similar
fashion, embryonic retinal cells in general fail to acquire
cell fates associated with postnatal retinal progenitors
when cultured with an excess of postnatal retinal cells
(Belliveau and Cepko, 1999). Furthermore, in response
to extrinsic secreted factors, retinal progenitors can
change their responsiveness to mitogenic factors and
153
such changes are likely to contribute to the regulation of
proliferation by these cells (Lillien and Cepko, 1992).
As discussed previously, the basic helix–loop–helix
(bHLH) genes code for an important family of
transcription factors involved in cell-type specification.
In Xenopus, targeted expression of Xath5 biased retinal
progenitors toward RGC differentiation (Kanekar et al.,
1997), while neuroD diverted cells toward amacrine or
bipolar cell fates at the expense of photoreceptors and
Muller
.
cells. However, in chick retina, neuroD is found
principally within developing photoreceptors (Toy et al.,
1998). In addition, Math5 activity in retinal progenitors
can lead to activation of Brn3b, a POU domain
transcription factor, thereby driving the progenitors
toward an RGC fate (Davis et al., 2000; Liu et al., 2001;
Wang et al., 2001). Furthermore, Math5 null mutants
fail to generate RGCs and this is accompanied by an
increase in amacrine cells and cone photoreceptors
(Brown et al., 2001). Results of this nature suggest that,
in addition to directing cell fate decisions, bHLH
transcription factors are likely to have roles in controlling retinal cell number and may regulate successive
stages of neuronal differentiation (Marquardt and
Gruss, 2002).
Rod photoreceptor development is promoted by
bFGF (FGF-2), sonic hedgehog, taurine, and laminin
beta 2 (Hicks and Courtois, 1992; Hunter et al., 1992;
Altshuler et al., 1993; Libby et al., 1996; Levine et al.,
1997), while ciliary neurotrophic factor (CNTF) and
leukemia inhibitory factor (LIF) appear to inhibit rod
differentiation by driving cells destined to be rods
toward a bipolar neuron phenotype (Ezzeddine et al.,
1997). In vitro studies in chick retina suggest that CNTF
may play a transient role in photoreceptor development
(Fuhrmann et al., 1998) by increasing the number of
opsin-expressing cells. The neurotrophic factor glial-cellline-derived neurotrophic factor (GDNF) may also play
a role in regulating photoreceptor development, at least
in vitro (Rothermel and Layer, 2003). Depending on the
stage of development, GDNF can influence proliferation, differentiation, and survival of photoreceptors.
Hedgehog proteins are also candidate factors for
promoting photoreceptor differentiation. Both Indian
and sonic hedgehog promote rod photoreceptor differentiation in cultured mammalian retinal cells (Levine
et al., 1997). Retinoic acid has also been shown to
promote photoreceptor differentiation in vitro in zebrafish, chick, rodent, and human retinas (Stenkamp et al.,
1993; Kelley et al., 1994, 1995, 1999; Hyatt et al., 1996).
Activin A, a TGF beta-like protein, also appears to be
an important regulator of photoreceptor differentiation
in the developing retina, both in vitro and in vivo (Davis
et al., 2000). Mash1 and Ngn2 also appear to influence
photoreceptor differentiation. When activated in nonoverlapping subpopulations of progenitor cells, they
appear to produce bipolar cells and photoreceptors
ARTICLE IN PRESS
154
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
(Perron et al., 1999; Marquardt et al., 2001). In addition,
photoreceptors and bipolar cells appear to be molecularly related. In Xenopus, the orthodenticle homolog
XOtx5b is expressed in photoreceptors and bipolar cells,
while XOtx2 (the Xenopus homolog of Otx2) is
expressed only in bipolar cells (Viczian et al., 2003).
Furthermore, transfection of retinal progenitors with
XOtx5b directs them toward a photoreceptor cell fate,
whereas transfection with XOtx2 promotes bipolar cell
specification. Other studies, involving the analysis of
mutant mice, have suggested that the homeodomain
gene Chx10 and bHLH genes MASH1 and Math3 are
required for generation of bipolar cells (Hatakeyama
et al., 2001).
Prox1, a homeodomain protein, regulates the exit of
retinal progenitors from the cell cycle (Dyer et al., 2003).
Retinal cells lacking Prox1 are less likely to stop
dividing, and ectopic expression of Prox1 forces retinal
progenitors to exit the cell cycle. In addition, Prox1 has
been observed in horizontal, bipolar, and AII amacrine
cells during retinogenesis (Dyer et al., 2003). Moreover,
horizontal cells are absent in Prox1 knockout mice and
misexpression of Prox1 in late progenitors promotes
horizontal cell differentiation (Dyer et al., 2003). The
bHLH genes Math3 and neuroD also appear to regulate
amacrine cell fate specification, with the Math3–neuroD
double mutant retina exhibiting a severe reduction in
amacrine cells accompanied by an increase in ganglion
and Muller
.
cells (Morrow et al., 1999; Inoue et al.,
2002). In recent years, experiments that alter normal
gene expression have revealed many of the molecular
mechanisms involved in cell fate decisions in the
developing vertebrate retina. Despite these findings, a
conclusive model describing how the orchestrated
expression of such factors acts to generate a functional
retina remains elusive.
Zhao et al., 1995; Sakaguchi et al., 1997). Regeneration of new neural retina via transdifferentiation
from the RPE occurs in a number of amphibians
as well as in embryonic chick and rat (Park and
Hollenberg, 1993).
Members of the fibroblast growth factor family may
play integral roles in signaling neural retina formation
during normal development as well in the process of
transdifferentiation from the RPE. Much of this
evidence has come from studies demonstrating that the
RPE of amphibians and chick can transdifferentiate into
neural retina when cultured in the presence of FGFs
(Park and Hollenberg, 1989; Pittack et al., 1991;
Guillemot and Cepko, 1992; Opas and Dziak, 1994;
Sakaguchi et al., 1997; Vogel-Hopker et al., 2000). The
expression of the ligands and receptors of the FGF
family of growth factors within the visual system is
consistent with the idea that FGFs play an important
role in retinal development, and thus, may play an
equally important role during retinal regeneration (Gao
and Hollyfield, 1992; Bugra et al., 1993; Ishigooka et al.,
1993; McFarlane et al., 1995). Indeed, FGFs have been
shown to promote retinal regeneration in amphibians,
embryonic chick and rat RPE in vitro (Pittack et al.,
1991; Guillemot and Cepko, 1992; Opas and Dziak,
1994; Zhao et al., 1995; Sakaguchi et al., 1997) and from
embryonic chick RPE in vivo (Park and Hollenberg,
1989). These studies have clearly demonstrated an
important role of FGFs in inducing the RPE to
regenerate neural retina, including RPCs that were
capable of producing a variety of neuronal phenotypes
such as ganglion-like cells, amacrine-like cells, and
photoreceptor-like cells (Pittack et al., 1991; Guillemot
and Cepko, 1992; Opas and Dziak, 1994; Zhao et al.,
1995; Sakaguchi et al., 1997).
2.2. Transdifferentiation of the retinal pigment
epithelium
3. Developmental role of stem cells in the retina and CNS
Although the RPE represents a phenotypically mature
and relatively stable cell population, RPE cells nevertheless retain the ability to proliferate and, in addition,
possesses a remarkable growth potential when exposed
to pathological or culture conditions (Okada, 1980).
Studies in a number of vertebrates have demonstrated
transdifferentiation of the RPE, the process of phenotypic switching whereby differentiated cells alter their
identity to become unique cell types of a different
lineage (Coulombre and Coulombre, 1965; Reyer, 1977).
In certain species, under the appropriate conditions,
transdifferentiation of the RPE into neural retina
has been demonstrated to occur both in vivo (Stone,
1950; Tsunematsu and Coulombre, 1981; Yasuda et al.,
1981; Klein et al., 1990; Kaneko and Saito, 1992) and
in vitro (Pittack et al., 1991; Opas and Dziak, 1994;
The presence of highly plastic, multipotent cells
within the developing organism has long been appreciated by experimental embryologists. Indeed, the
‘‘stem-like’’ properties of embryonic cells are essential
to metazoan development. The totipotent fertilized
ovum must be capable of generating all three embryonic
germ layers and the placenta as well. The germ layers
must in turn give rise to all the organ systems of the
body. The potential for generating an entire organism is
clearly inherent in the first cell of the sequence. Then, as
development progresses, the mature organism arises out
of a process in which successive daughter cells undergo
progressive fate determination. This process is extremely
complex and can vary tremendously between cells of
different phenotype. Overall, the process of development involves a movement away from stemness and
toward increased cellular specification. This restrictive
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
tendency is increasingly apparent across phylogeny,
particularly in terms of regenerative capacity.
Many metazoan species exhibit a high capacity for
tissue regeneration in the mature organism. For
instance, it has long been known that planaria, a type
of helminth, can regenerate an entire body-half when
bisected, thereby resulting in two complete organisms.
This degree of regeneration is not seen in vertebrates
although a considerable regenerative capacity is retained
in many cold-blooded species. For instance, some
teleosts can regenerate half their brain and urodeles
can regenerate their limbs. Regeneration in reptiles is
less robust, although the ability of lizards to grow a
new tail is common knowledge. In mammals, regenerative capacity has been further restricted. Mice cannot
regenerate digits, let alone limbs or tails. Although
humans can regenerate substantial amounts of liver
tissue, the corneal and intestinal epithelia, and every
type of blood cell, the human capacity for endogenous repair is severely limited in critical tissues such
as cardiac muscle, the corneal endothelium, and the
CNS.
The loss of regenerative capacity in humans can be
viewed as part of the phylogenetic tendency toward
increasing restriction on stem cell potential and appears
to result from an active process, not a permanent loss of
information, in that it is selectively applied. For
instance, even developmentally mature humans contain
gametes with the potential to generate a new organism.
Reduction of regenerative capacity may represent an
evolutionary strategy for minimizing the risk of uncontrolled cell division in long-lived, slowly reproducing
species. Hence, phenotypic stability appears to be
superimposed upon an underlying, more fundamental,
cellular plasticity over the course of both development
and evolution. It appears there is much to gain from the
comparative study of stem cell biology, particularly with
respect to the molecular genetics of development. In this
section, we will consider the origins of retinal stem and
progenitor cells by stepping back and placing the retina
within its developmental context as part of both the
CNS and the eye.
3.1. Embryological origins of the vertebrate retina
Eye development involves a series of complex interactions between neural ectoderm, surface ectoderm,
intervening mesoderm, and migrating neural crest.
The cellular origins of the retina, however, can be
traced to neural ectoderm exclusively. It has long
been postulated that the region of neural ectoderm
giving rise to the eyes is determined prior to formation
of the forebrain or optic vesicles and recent molecular
work has elegantly confirmed the existence of ‘‘eye
fields’’ in the anterior neural plate (Seo et al., 1998).
Morphologically, the first correlates of eye development
155
are the optic pits, paired dimples appearing in the
anterior aspect of the neural plate (Mann, 1950).
At this stage the rostral neuropore has not yet
closed, therefore the optic pits represent indentations
in the surface of the embryo. Due to their location
within the head fold of the neural plate, the floor
of each pit is in direct contact with surface ectoderm
on the opposite side of the embryo, providing the
basis for potential inductive interactions between these
layers. The pits continue to deepen with time and
balloon out, forming the optic vesicles. The most distal
end of each optic vesicle subsequently invaginates back
toward the neural tube, eventually obliterating the
vesicular lumen and resulting in the optic cup. The
outer layer of the optic cup gives rise to the RPE,
while the inner becomes the neural retina. The first
morphological correlate of the future retina is thus
at the optic cup stage. However, in terms of retinal
specification, and hence retinal stem cells (RSCs), the
prior existence of ocular primordia indicates that
important specification events have already occurred at
the molecular level. The nature of this specification has
been the subject of much recent interest, one reason
being that such knowledge is fundamental to an
understanding of the role of stem cells in generating
the retina.
3.2. Molecular mechanisms of retinal stem cell
specification
The molecular genetic events underlying the commitment of neural ectoderm to a retinal fate are crucial to
an understanding of the origin, prospective identification, and phenotypic potential of RSCs. Much that
pertains to this subject has been elucidated in the fruit
fly. In addition, recent work in vertebrates has identified
important molecular correlates of the embryological
events described above. Together, these studies have
revealed a remarkable similarity in the molecular genetic
programs underlying eye development across a wide
spectrum of vertebrate and invertebrate species. This
includes a high degree of sequence homology between
key nuclear transcription factors (NTFs), from fly to
human, as well as frequent parallels in the role of these
factors during development. The work in Drosophila will
now be briefly summarized, as a prelude to consideration of the molecular pathways important to retinal
development in vertebrates.
3.2.1. Molecular control of neural specification in
Drosophila
In flies, as in vertebrates, development of the CNS
depends upon a commitment to neural lineage of the
cells comprising the embryonic neuroectoderm. Central
to the formation, specification, and differentiation of
neuroblasts is the coordinated expression of numerous
ARTICLE IN PRESS
156
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
NTFs including homeodomain proteins, proneural
NTFs of the bHLH family, as well as members of
the zinc finger family. Also important are signaling
molecules, their receptors, and intermediary gene
products of these pathways.
Early neural specification in Drosophila has long been
intimately associated with genes involved in pattern
formation. Loss-of-function mutation in these genes
results in defective or incomplete neurogenesis within
specific segments or columns of the developing embryo.
For example, genes important to establishing neural
structures along the dorsal–ventral axis include ventral
nervous system defective (vnd), intermediate neuroblasts
defective (ind), and muscle segment homeobox (msh).
Function of these genes is important to formation of the
ventral, intermediate, and dorsal columns, respectively
(Mellerick and Modica, 2002). Neural specification
along the anterior–posterior axis, on the other hand, is
dependent upon the segment polarity genes, such as
gooseberry, hedgehog (hh), patched, and wingless (Patel
et al., 1989).
In work more analogous to studies of mammalian
neural progenitors, isolated Drosophila neuroblasts have
been cultured and patterns of gene expression studied.
The temporal development of neuroblasts has been
associated with the sequential expression of the proteins
Hunchback (Hb), Kruppel, Pou-homeodomain proteins
1 and 2 (Pdm), followed by Castor (Cas) (Kambadur
et al., 1998; Isshiki et al., 2001). Hb and Cas are zincfinger proteins that repress, and thus temporally restrict,
Pdm expression. Later in development, the terminal
progeny of these neuroblasts can be identified by
expression of the bHLH transcription factor Grainyhead (Gh) (Brody and Odenwald, 2000). Additional
examples of genes involved in neuroblast development
in Drosophila, and important to mention because of
their relevance to mammalian neurogenesis, are the
homeodomain genes engrailed (en), orthodenticle (otd),
and prospero (pros) (Doe et al., 1991; Acampora et al.,
2001; Ryter et al., 2002). Furthermore, in addition to the
hedgehog and wingless signaling pathways already
mentioned, there is the pathway mediated by the
Drosophila EGF receptor (DER). Also known as faint
little ball or torpedo, this gene is associated with the
ligand-expressing genes spitz (spi), vein (vn), and keren
(krn) (Skeath, 1998; Reich and Shilo, 2002).
Following the early events of neural specification,
proneural NTFs of the bHLH family are expressed, in
concert with negative regulators of proneural gene
expression. The proneural gene group is comprised of
the achaete–scute (AC-S) complex, as well as the genes
atonal (ato) and daughterless (da) (Jarman et al., 1993;
Skeath and Carroll, 1994; Brown et al., 1996). Negative
regulators of these bHLH genes include hairy (h) and
the Enhancer of split complex, as well as extramacrochaete (emc) (Modolell, 1997). The importance of these
genes extends to the many vertebrate homologs which
retain crucial roles in neuronal development such as the
mammalian proneural homologs of the Mash and Math
gene families, the daughterless homolog Thing, and the
Hes genes (Anderson, 1994).
3.2.2. Molecular control of neural specification in
vertebrates
Given the complexity of the vertebrate CNS, it should
not be surprising that the molecular genetics underlying
neural development are neither simple nor completely
understood. Nevertheless, a number of important genes
and signaling pathways have been recently identified
and, once again, the groundbreaking work in Drosophila
has proven invaluable. Despite vast morphological
disparities in terms of neural organization, the molecular similarities between insects and vertebrates are
frequent and striking. These similarities are particularly
evident for homeobox genes, genes encoding bHLH
transcription factors of the AC-S and atonal families,
and genes involved in a number of signaling pathways,
notably the notch pathway. It is important to bear in
mind that, while the molecules involved in vertebrate
neural development frequently exhibit high degrees of
homology with their counterparts in the fly, this is
not invariably the case. Furthermore, the functional role
of vertebrate genes often differs markedly at the level of
the organism. In this context, it should be appreciated
that the enormous differences in body plan vis-a" -vis
insects make complete functional congruity essentially
impossible.
Two examples of Drosophila genes encoding homeodomain transcription factors with relevance to vertebrate neural development are orthodenticle (otd) and
iroquois (iro). The orthodenticle orthologs Otx1 and
Otx2 play important roles in anterior neural patterning
across a range of vertebrate species (Boyl et al., 2001). In
the mouse, expression of Otx2 corresponds to the
presumptive forebrain and midbrain, and falls off
abruptly at the midbrain–hindbrain junction (Wassarman et al., 1997). Disruption of Otx gene function is
associated with gastrulation defects and embryological
anomalies, including severe craniofacial malformations
(Hide et al., 2002) such as the agnathia-holoprosencephaly complex in humans (Wallis and Muenke, 2000)
and a headless phenotype in mouse embryos (MartinezBarbera and Beddington, 2001). The sum of the
evidence suggests that the highly conserved Otx genes
play a crucial role in specification and morphogenesis of
the rostral CNS, including the telencephalon and optic
vesicles.
The vertebrate Iroquios genes (Irx) are also important
in neural development and associated with CNS pattern
formation (Gomez-Skarmeta and Modolell, 2002). For
instance, Iroquois genes have been associated with
specification of the neural plate in Xenopus and
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
specification of forebrain subdivisions in the chick
embryo. Whereas the boundary delineated by Otx2 in
the presumptive brainstem is generated in opposition to
more caudal expression of Gbx2, a completely separate
boundary is defined by rostral expression of Six3 in
opposition to the more caudal Irx3 expression domain.
This Six3/Irx boundary occurs within the presumptive
diencephalon (Kobayashi et al., 2002). Here it is worth
noting that the above discussion illustrates the complexity of the relationship between neural specification and
eye specification, as exemplified by the fact that Otx2
and Six3 have been implicated in both. This overlap
highlights the mechanistic similarities of the two
processes while also exposing the danger of attributing
too much phenotypic causality to any one particular
transcription factor.
Following a preliminary phase of regional specification within the vertebrate CNS that is intimately
associated with homeodomain transcription factors, a
secondary phase of cellular differentiation occurs in
which transcription factors of the bHLH family play a
prominent role. Once again, conserved counterparts of
Drosophila genes are critical to the process, notably the
mammalian AC-S homolog (Mash1), as well as atonalrelated genes of the Math, neurogenin, and neuroD
families (Kageyama et al., 1997). These bHLH gene
families appear to be critical to the differentiation of
neural precursors throughout the neuraxis, including the
retina, although the particular genes required for a given
mature phenotype frequently vary. For instance, neurogenin1 is critical to the determination of specific CNS
neuronal subpopulations (Ma et al., 1999) and, in the
PNS, neurogenins specify sensory neurons while Mash1
specifies autonomic neurons (Lo et al., 2002). In
addition, bHLH genes are frequently expressed sequentially during neural development, with expression of
Mash1 preceding that of neurogenin and neuroD. For
instance, proliferating neural progenitors in the olfactory epithelium of the mouse express Mash1 prior to
becoming neurogenin1+ intermediate neuronal precursors (Calof et al., 1998).
In addition to transcription factors, signaling pathways known to be involved in vertebrate CNS development are frequently related to important Drosophila
genes including notch, wingless, and hedgehog, as well
as the epidermal growth factor pathway. Even bone
morphogenic protein (BMP) signaling shows striking
parallels with ‘‘BMP-like’’ activity in the fruit fly,
mediated by the ligands Decapentaplegic (Dpp) and
Glass Bottom Boat (Gbb) (Conley et al., 2000). In
vertebrates, Notch and BMP signaling are known to
play pivotal roles in aspects of telencephalic development (Schuurmans and Guillemot, 2002), as well as in
the retina. Because of its importance to CNS and, in
particular, retinal development, the Notch signaling
pathway will now be discussed in greater detail, before
157
turning to an examination of the molecular genetics of
retinal specification in vertebrates.
3.2.3. Notch signaling and neuronal differentiation
Numerous molecular pathways have been shown to
be important to development of the neural retina. Here
we will consider the pathway mediated by the receptor
Notch. A considerable literature now exists detailing
aspects of this crucial and complex signaling pathway.
Much of the work done in the fruit fly has now been
extended to vertebrates, including humans and other
mammalian species. Notch is a highly conserved gene
encoding a surface receptor which, in the fly, is activated
by ligands corresponding to the genes delta, jagged, and
serrate. In mammals, genes of the Notch family are
known to show similar tissue expression patterns as
ligands of the Delta and Jagged families (Hayes et al.,
2003) and, in addition, ligands of the Serrate variety
have also been identified (Schroder and Gossler, 2002).
An interesting aspect of Notch signaling is its dependence on release of the Notch intracellular domain
(NICD) by intramembranous proteolytic cleavage. This
constitutive endoproteolysis is presenilin-dependent and
shares interesting similarities with cleavage of the betaamyloid precursor protein (Okochi et al., 2002).
Following cleavage, NICD acts through translocation
to the nucleus and formation of a transcriptional
complex with DNA binding proteins of the Suppressor
of Hairless, or Su(H), family (Iso et al., 2003). There are
multiple influences regulating function of the Notch/
Su(H) complex. For instance, Notch also interacts with
the transcriptional co-activator Mastermind (Mam)
(Wu et al., 2000) and, in the absence of Notch signaling,
Su(H) acts as a transcriptional repressor. The mammalian Su(H) homolog is known as RBP-J kappa.
Mastermind homologs belong to the MAML family
(Wu et al., 2002).
Ultimately, the Notch/Su(H) complex initiates transcription of the Hes (hairy/Enhancer of split) family of
bHLH transcription factors. Hes1 and Hes5 work
together to exert negative transcriptional control over
proneural genes, particularly those of the AC-S/Mash
and atonal/Math families of bHLH transcription factors
(Ohtsuka et al., 1999, 2001). Thus, the overall effect of
Notch signaling in the CNS is to delay the onset of
neural differentiation during development. Consequently, loss of effective Notch signaling results in
premature differentiation and inadequate growth of
neural structures. In addition, activity of the eye
specification genes in Drosophila is under the antagonistic control of the Notch and EGF-R signaling
pathways (Kumar and Moses, 2001). It is now clear that
Notch signaling profoundly impacts the proliferative
and phenotypic potential of mammalian retinal and
other CNS progenitor cells (Ahmad et al., 1995, 1997).
The potential for active or inadvertent manipulation of
ARTICLE IN PRESS
158
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
cultured populations via this pathway deserves further
consideration.
3.2.4. Molecular control of eye specification in
Drosophila
The compound eye of insects bears little morphological resemblance to the camera obscura design of
vertebrates, yet it is now appreciated that the molecular
genetic template underlying the development of both
these structures has been conserved to a remarkable
degree. It has been established that the combined
activities of seven genes, from four gene families, are
necessary for eye specification in the fruit fly. These
genes are eyeless (ey), twin of eyeless (toy), and eyegone
(eyg) of the PAX6 gene family; sine oculis (so) and optix
(optx) of the SIX gene family; eyes absent (eya) of the
EYA gene family; and dachshund (dac) of the DACH
gene family (Table 1). All encode nuclear proteins
involved in transcription. Knocking out any one of these
genes results in failure of eye development, while ectopic
Table 1
Eye specification genes
Drosophila eye specification genes (and abbreviations)
PAX6 family:
Eyeless (ey)
Twin of eyeless (toy)
Eyegone (eyg)
so/SIX family:
Sine oculis (so)
Optix (optx)
EYA family:
Eyes absent (eya)
DACH family:
Dachshund (dac)
Xenopus eye specification genes (and related Drosophila genes)
T-BOX family:
ET (optomotorblind)
LIM family:
Lhx2 (apterous)
PAX family:
Pax6 (eyeless, twin of eyeless, eyegone)
RX family:
Rx1 (DRx)
so/SIX family:
Six3 (optix, sine oculis)
Six6/Optx2 (optix, sine oculis)
GAP family:
tll (tailless)
Note: A comparison of invertebrate and vertebrate species, by gene
family.
expression of any one gene is sufficient to induce eye
formation, even in abnormal locations (Halder et al.,
1995; Shen and Mardon, 1997; Gehring and Ikeo, 1999;
Kumar and Moses, 2001). The mechanisms underlying
this behavior are not entirely understood but appear to
relate to the observation that the proteins coded by these
genes interact intimately with one another, either as part
of an NTF cascade or as components of the same
transcriptional complex (Chen et al., 1997; Pignoni et al.,
1997). Furthermore, a given eye specification gene
frequently demonstrates the capacity to induce expression of its fellows. This applies not only for downstream
genes in an induction cascade but also via positive
feedback networks capable of activating earlier genes
(Pan and Rubin, 1998). For example, expression of ey
induces expression of the downstream genes eya and so,
which in turn induce expression of dac. These later genes
then form part of a positive feedback network activating
expression of ey (Bonini et al., 1997). The factors dac,
eya, and so come together to form a functional
transcriptional complex.
It is important to note that the functional coexpression of the seven eye specification genes takes
place in the regulatory context of signaling pathways,
including those already discussed because of their
importance to neural differentiation. These include
Notch and EGF-R (Kumar and Moses, 2001), as well
as wingless (Baonza and Freeman, 2002), hedgehog, and
the BMP-like pathway via Dpp (Kango-Singh et al.,
2003). Interactions between signaling pathways and
transcription factor networks occur throughout the
course of eye specification and retinal differentiation in
the fly and are involved in regulation of the bHLH
transcription factor atonal. Expression of atonal is
intimately associated with neural differentiation and the
initiation of photoreceptor differentiation (Baonza and
Freeman, 2001). There is no question that the study of
eye specification in the fruit fly has proven invaluable to
work in vertebrates, from fish to humans, as will be
made evident in the following section.
3.2.5. Molecular control of retinal specification in
vertebrates
The preceding sections have highlighted a relatively
small number of genes based on their importance to
neural and retinal specification in the fly, as well as
neural specification in vertebrates. In the following
discussion of retinal specification in vertebrates, many of
these same genes will be revisited, thereby substantiating
the view that the molecular genetic substrates of neural
and retinal development are highly conserved. Looked
at slightly differently, many of the genes important to
the generation and control of RSCs have already been
described in the eye of the fruit fly or elsewhere in neural
development. Nevertheless, these genes show characteristic expression patterns during vertebrate retinal
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
development, as illustrated by a large body of recent
work in the area.
As previously discussed, the orthodenticle homolog
Otx2 is essential to ocular development and is expressed
in the anterior neuroectoderm of the mouse where it
defines the territory that gives rise to the prosencephalon, mesencephalon, and optic vesicles. Furthermore,
Otx2 continues to be expressed in the optic vesicle and
optic cup, although expression becomes more restricted
over the course of development (Zhang et al., 2002).
While the distribution of Otx2 expression does not
appear to be eye specific, it would appear to be eye
permissive, i.e., important to establishing the competence of anterior neural plate cells to form the
embryonic eye fields (Chuang and Raymond, 2002).
The role of actually establishing the eye fields is more
consistent with transcription factor genes expressed
specifically within the presumptive retinal territory of
the neural plate. Genes consistently associated with this
distribution are Pax6, Rx, and Six3 (Bernier et al., 2000).
Strikingly, all of these early genes are homologs of
Drosophila genes involved in neural development
(Eggert et al., 1998), with Pax6 and Six3 belonging to
two of the four families of Drosophila eye specification
genes. Given their early expression, it would appear that
the core group of Pax6, Rx, and Six3 work together with
Otx2 at the beginning of the developmental transcription factor network controlling the conversion of
anterior neural ectoderm to retina (Table 1). Of course
other genes are also likely to be involved.
Recent work, largely in the mouse and Xenopus, has
expanded the number of eye-field transcription factors
(EFTFs) from three to seven (Zuber et al., 2003). In
addition to Pax6, Rx, and Six3, the list now includes ET,
Lhx2, Six6, and tll. Of these additional genes, ET, Lhx2
(Lim family), appear to be expressed early in the
presumptive eye field, while Six6 is expressed somewhat
later and thus unlikely to participate in initial specification, (Li et al., 1997; Mathers et al., 1997). Six6 (also
known as Optx2) is a homolog of the Drosophila eye
specification gene optix and, like the closely related Six3,
a member of the sine oculis (SIX) family. While Six6
would not appear to be essential for eye-field specification, it is of great interest because of its association with
retinal fate specification, as well as proliferation of the
presumptive retina during development. Injections of
mouse Six6 into the eye, or midbrain, of Xenopus
embryos result in an enlarged retina, or the transformation of midbrain into retina, respectively (Bernier et al.,
2000). In addition, injection of Six6 is capable of
inducing not only neural retinal genes such as Chx10
(Toy et al., 1998) but also upstream genes such as Pax6
and Rx, apparently ‘‘locking in’’ the initial commitment
to a neural retinal fate.
The potential for induction of upstream, as well as
lateral and downstream genes, illustrates how the
159
program for retinal specification is both self-reinforcing
and resilient. While the sequential NTF expression
patterns seen during the course of normal development
do resemble a molecular cascade, experimental studies
show that a network model including complex feedback
loops is more accurate (Bernier et al., 2000; Zuber et al.,
2003). Naturally, this feedback network operates in the
context of signaling pathways. In addition to those
pathways previously discussed, the neural inducer
noggin should also be mentioned in the context of
vertebrate development. In a model of vertebrate eye
formation recently proposed by Harris and colleagues
(Zuber et al., 2003), noggin sets the stage for eye
development through induction of Otx2, and thus
specification of the anterior brain, thereby giving rise
to expression of EFTFs and specification of the eye
fields. In their model, ET initiates eye-field specification.
ET in turn induces the expression of Rx1, Lhx2, and tll.
In the next stage the situation becomes more complicated with these latter three NTFs inducing Pax6,
together with various combinations of the remaining
EFTFs, as well as up-regulating each other. Finally,
Pax6 induces Optx2 (Six6).
There is now an increasingly large body of work
investigating the role of transcription factors in the
specification and differentiation of specific retinal cell
types as eye development progresses. As previously
observed in the brain and eye of the fly, as well as in the
vertebrate brain, bHLH transcription factor genes
of the AC-S and atonal variety feature prominently.
These include Mash1, Math5, neurogenin2, and neuroD. With the exception of neuroD, which is required
for the generation of amacrine cells, these genes are
activated by Pax6 (Ashery-Padan and Gruss, 2001;
Marquardt and Gruss, 2002). Mash1 is required for
bipolar cell differentiation, Math5 for ganglion cell
differentiation, and neurogenin2 has also been implicated in retinal neurogenesis. Consistent with this, yet
still quite remarkable, the retina of Pax6-deficient mice
appears to contain only amacrine cells (Marquardt
et al., 2001).
In summary, it is now clear that vertebrate eye
development owes much to a common molecular genetic
program, elements of which are shared between most, if
not all, sighted animals. The remarkable level of
conservation observed undoubtedly owes much to the
advantages conferred by vision upon survival but also
likely reflects the organizational complexity necessary to
achieve these advantages, in terms of both the developmental regulatory network and the final biological
structure. Given the weight of the evidence, it seems
highly probable that the specification, proliferation, and
differentiation of cultured retinal stem and progenitor
cells relies on these same pathways. Furthermore, there
can be little doubt that application of current molecular
genetic knowledge will result in a greater understanding
ARTICLE IN PRESS
160
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
of the identity, behavior, and phenotypic potential of
these cells. With the work reviewed above serving as a
foundation, the next several years are likely to witness
considerable progress in the molecular characterization
of retinal precursors, along with stem and progenitor
cells of all types.
4. Cultured CNS stem cells
The term ‘‘CNS stem cell’’ has been used to
inclusively describe all self-renewing multipotent cellular
populations propagated in culture from any location in
the neuraxis, including the brain, spinal cord, and retina.
As such, CNS stem cells are basically the same as NSCs,
with the caveat that not all of them fit the more
restrictive definition applied to NSCs. There is widespread agreement that NSCs must be capable of
generating neurons, astrocytes, and oligodendrocytes.
The importance of this point relates to the observation
that stem cells from the neural retina should not be
required to generate oligodendrocytes, since none are
normally generated within that tissue. Nevertheless,
stem-like cells from the retina are very similar to brainderived NSCs, and thus the collective term ‘‘CNS stem
cell’’ is a convenient way to highlight the commonalities,
above and beyond the very real differences that do exist
between these cell types. Again, the use of this term does
not necessarily imply that all criteria for ‘‘stemness’’
have been satisfied.
4.1. Multipotent cells from the mammalian CNS can be
grown in culture
A major recent breakthrough in the field of neurobiology was the demonstration that immature progenitor cells with multiphenotypic potential can be isolated
from the CNS of both developing and adult rodents and
propagated for long periods in culture (Reynolds and
Weiss, 1992; Richards et al., 1992). As discussed above,
these cells are frequently referred to as NSCs and are
multipotent cells which, by definition, can differentiate
into any type of neural cell, including neurons, astrocytes, and oligodendrocytes. Standard cell culture
methods employ serum, however, prolonged exposure
to serum results in loss of multipotency and differentiation of these cells; therefore, a critical methodological
modification was the development of serum-free defined
medium. The availability of recombinant growth factors, particularly EGF and bFGF, was also crucial,
since addition of these factors, alone or in combination,
is necessary to both induce proliferation and maintain
an undifferentiated state.
The extension of these findings to the human nervous
system has been another important development,
particularly from the perspective of potential therapeu-
tic applications (Svendsen et al., 1997; Yandava et al.,
1999). Although fetal human NSCs can be grown with
little difficulty, significant challenges facing this work
have centered on the availability and political acceptability of work with human fetal material. As it turns
out, NSCs can be harvested from postmortem human
material of different ages, including the postnatal
period, therefore such considerations should not prove
insurmountable to work in the area (Palmer et al., 2001).
Furthermore, there is evidence of active proliferation in
the brain of even elderly people, thus potentially
increasing the potential sources for these cells (Eriksson
et al., 1998). Such optimism must be tempered by the
evidence that cells derived from older (postnatal) human
donors propagate less well and exhibit less multipotency, with a general tendency to differentiate into
astrocytes (Palmer et al., 2001).
4.1.1. Neural stem cells can be identified by marker
expression
The now classic marker for NSCs is the intermediate
filament nestin, a component of the cytoskeleton of
immature neuroepithelial cells (Lendahl et al., 1990)
(Fig. 2). While expression of this marker is not a perfect
indicator of NSCs, researchers find it useful for
confirming the presence in vitro of highly plastic neural
progenitors, at least to a first approximation. The
cellular role of nestin is not well-understood, but could
possibly be involved in conferring morphological
plasticity to developing neural precursors. Additional
markers of interest include Notch, Numb, Musashi-1,
and Presenilin, to name a few. Recently, the nuclear
markers FGFR4, Fz9, and Sox2 have also been
added to the list of putative NSC markers (Cai et al.,
Fig. 2. Expression of stem cell markers by human neural progenitor
cells. The cells shown were cultured from human cerebral tissue
obtained postmortem from a premature infant. These cells grow as
clusters (upper left corner of figure) or as a monolayer (center) and
express the intermediate filament nestin (green) and the NTF Sox2
(red), both of which are putative NSC markers. Image taken by
Boback Ziaeian and kindly provided by Dr. Philip H. Schwartz.
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
161
2002), along with Nucleostemin (Tsai and McKay,
2002) (Fig. 2).
Surface markers are of particular interest as potential
targets for enrichment strategies and also as potential
drug targets. Enrichment is directed toward enhancing
yield during CNS stem cell isolation, while ligand
binding could be used to influence key processes such
as proliferation, migration and differentiation. In
addition to Notch, CD133 is one such marker (Uchida
et al., 2000), while others are the carbohydrate moiety
CD15 (Klassen et al., 2001) and the membrane
glycolipid GD2 ganglioside (Klassen et al., 2001)
(Fig. 3). Of note, GD2 ganglioside and CD15 are nonprotein epitopes. In addition, some surface molecules
can be used indirectly for cell sorting, for instance by
taking advantage of the capacity of the ABCG2
transporter to extrude the vital dye Hoechst 33342 from
a distinct cellular subpopulation (Scharenberg et al.,
2002; Bhattacharya et al., 2003).
In addition, there are many less specific markers
expressed by CNS stem cells. These include the
intermediate filament protein vimentin, the proliferation
markers Ki-67 and Cyclin D1, as well as various forms
of the surface adhesion molecule NCAM together the
surface markers CD9 and CD81, both members of the
tetraspanin family (Klassen et al., 2001). While it seems
increasingly likely that no single perfect marker for
NSCs will be agreed upon, the use of multiple positive
and negative markers in combination can be quite
instructive. Ultimately, a more comprehensive understanding of cellular transcriptional activity should
provide a better means of assessing ontogenetic status.
4.1.2. Neural progenitor cells express cytokines
Examining brain-derived neural progenitor cells
grown under standard proliferation conditions, we
found among the genes expressed those encoding a
number of cytokines (Klassen et al., 2003a). This work
compared NPCs derived from fetal human brain to
GFP-transfected cells from adult rat brain, as well as
GFP-transgenic cells from neonatal mouse brain.
Cytokine genes expressed by human NPCs included
IL-1a, Il-1b, IL-6, TGF-b1, TGF-b2, and TNF-a, but
not IL-2, IL-4, or IFN-l. Interestingly, the cytokines
expressed included both pro-inflammatory (IL-1a, Il-1b,
IL-6, and TNF-a) as well as anti-inflammatory (TGFb1, TGF-b2) cytokines (Fig. 4). Cytokine expression by
rodent NPCs was more restricted, with rat cells
expressing IL-6, TNF-a, and TGF-b1, and mouse cells
expressing only the last of these.
Cytokines are potent intercellular signaling agents
involved in a variety of processes including development
and wound repair. The significance of the expression of
a particular cytokine can be difficult to assess in
isolation because, under physiological conditions, cytokine pathways interact as complex homeostatic regula-
Fig. 3. Flow cytometric analysis of surface marker expression by
multipotent human and GFP-transgenic mouse NPCs. Both cell lines
express GD2 ganglioside, the Lewis antigen CD15, and the tetraspanins CD9 and CD81 (TAPA-1). Markers detected on human, but
not mouse, NPCs include the hematopoietic stem cell marker CD34,
CD95 (Fas), and MHC class I. For each plot the shaded profile shows
mAb-associated labeling and the black line the isotype control. Each
vertical axis represents cell count and horizontal axis log relative
fluorescence. Reprinted from Klassen et al. (2001) with kind
permission.
tory networks. The significance of the expression
patterns exhibited by NPCs is not yet clear, but seems
unlikely to be related to direct generation of immune
and inflammatory responses. Nevertheless, the tropism
shown by a variety of stem cells for areas of injury has
been repeatedly demonstrated and suggests that they
ARTICLE IN PRESS
162
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Fig. 4. Expression of cytokines transcripts by NPCs from human, rat, and mouse. RT-PCR revealed the expression of both the b1 and b2 isoforms of
TGF-b by human neural progenitor cells, whether hNPCs were grown as neurospheres (sph) or an adherent monolayer (mon). Bands corresponding
to IL-1a and IL-1b were also present. There was no evidence for expression of IL-2, IL-4, IL-10, or IFN-g by hNPCs grown under either condition.
In addition to cytokines, spheres expressed ATM and LYN. Rodent cells expressed a subset of the cytokines produced by human cells: rat NPCs
expressed IL-6, TNF-a, and TGF-b1, while mouse NPCs expressed TGF-b1. Lanes for positive (+, supplied with primers), and negative (, no
DNA) controls are indicated. Product size (bp) is indicated adjacent to bands. Reprinted from Klassen et al. (2003a, b) with kind permission.
may be receptive to inflammatory mediators. For
example, bFGF, EFG, and TGF-b are known to
strongly influence both NPC proliferation and maintenance of multipotentiality. Together with our data
showing cytokine expression, this raises the interesting
possibility of autocrine or paracrine signaling. Interestingly, the TGF-b signaling pathway in particular has
been repeatedly implicated in stem cell behavior,
including maintaining ‘‘stemness’’ as well as the
quiescence of hematopoietic progenitors, the development of germ cells, and the proliferation of RPCs
(Anchan and Reh, 1995; Lawson et al., 1999; RamalhoSantos et al., 2002).
4.2. Multipotent cells from the mammalian retina
Compared to brain-derived NPCs, there are fewer
reports on stem or progenitor cell cultures from the
mammalian retina. As discussed previously, however,
much work has been directed toward analysis of retinal
development, either in vivo or in dissociated cultures of
embryonic rodent retina. Some interesting studies to
mention at this point include the effect of growth factors
on cultured rat retinal progenitors (Lillien and Cepko,
1992), work with cultured human retinal progenitors
(Kelley et al., 1995), work investigating the timing of
fate determination in embryonic chick retinal cells
(Adler, 1996) and the potential for re-specification of
cell fate (James et al., 2003). Work in these and other
laboratories represents the intellectual context for our
own contributions to this field.
We have developed a modified protocol that allows us
to isolate RPCs from the early postnatal mouse neural
retina (Shatos et al., 2001). Most of the modifications we
made to established brain progenitor cell techniques
relate to increased delicacy in the generation of singlecell suspensions, without which the majority of the cells
die during dissociation. The reason for the delicacy of
developing retinal tissue may reflect the tightly packed
and highly laminated retinal architecture, the many
intercellular junctions between cells, and the fragile
nature of developing photoreceptors which constitute a
large component of the early postnatal mouse retina.
The use of GFP-transgenic mouse eyes as donors
allowed us to generate a population of self-renewing,
multipotent RPCs ubiquitously expressing the GFP
transgene (Fig. 5). Reliable reporter gene expression is
an important prerequisite to the transplantation studies
arising from this work.
RPCs can be expanded greatly in culture. We have
expanded these cells up to 60 passages without evident
changes in cell cycle behavior. At greater than approximately 40 passages, however, we have encountered a
greater tendency to differentiate along glial lineage.
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Fig. 5. Green mouse CNS stem cells. (a) BSCs form self-renewing
neurospheres that show uniform green fluorescence under FITC
illumination. (b) BSCs are labeled by anti-Ki-67, a marker for
mitotically active cells, as well as anti-nestin, (c) a marker for neural
progenitors ((a)–(c) cultured under proliferating conditions of EGF
and bFGF, 20 ng/ml). BSCs differentiate into cells of the neural
lineage, including neurons (d), astrocytes (e), and oligodendrocytes (f)
((d)–(f) cultured under differentiating conditions of 10% FCS). BSCs
are easily identified after transplantation to the adult mouse brain (g)
as are RSCs, which differentiate into cells of retinal lineage when
grafted to the diseased or injured adult mouse retina (recoverin and
GFP co-expression in mechanically injured B6 mouse ONL (h)–(j)).
Reprinted from Shatos et al. (2001) with kind permission.
Therefore, lower passage cells are to be preferred for
most studies. RPCs can be frozen at 150 C for at least
12 months and thawed with 95–97% survival. While
RPCs meet the established stem cell criteria of selfrenewal and multipotency, they survive very poorly as
isolated single cells thus making clonal analysis of this
population difficult. Moreover, the strict interpretation
of multipotency as applied to the retina implies the
ability to generate all cellular lineages of the retina. As
we have only conclusively demonstrated neural and
astrocytic lineages, together with rod photoreceptor and
bipolar cell differentiation, we term this cultured retinal
population ‘‘progenitor’’ rather than ‘‘stem’’ cells.
In the context of RSCs, there is another cell type that
has generated considerable interest. These cells are not
obtained from the neural retina, but from the pigmented
epithelium (PE) of the ciliary body. They represent a
small fraction of ciliary PE cells and are capable of
generating multiple retinal cell types, including glia,
bipolar cells, and rod photoreceptors. They also posses a
number of unusual properties for CNS stem cells
including pigmentation and spontaneous proliferation
in the absence of exogenous growth factors or any added
protein (Tropepe et al., 2000). Furthermore, the cells
reside in a tissue other than the one they would
theoretically be suited to repair in the setting of wound
163
healing. These observations are novel as well as
puzzling.
The analogy has been made between the PE of the
ciliary body and the ciliary marginal zone (CMZ) of
lower vertebrates, a well-known hotbed of RSC activity.
On closer inspection, however, the CMZ is an undifferentiated neuroepithelium along the peripheral-most
margin of the developing retina (Wetts and Fraser,
1989), whereas the PE of the ciliary body is a mature,
differentiated epithelial monolayer. The ciliary PE is
physically distinct from the retina and lies anterior to
the anatomical retinal margin known clinically as the
ora serrata. Mature mammals do not have a clear CMZ
per se. Based on morphology and embryology, the ora
serrata of the neural retina, or perhaps the adjacent
RPE, would arguably be most homologous to the CMZ
of fish and amphibians. It is conceivable that a
population of RSCs has been displaced evolutionarily
to the ciliary body, where they retain their phenotypic
potential but no longer serve to generate retina,
although it must be admitted that such an arrangement
would be rather unusual. Further investigation is needed
to clarify this issue.
Apart from stem cells, evidence is accumulating that
non-neural retinal cells do retain the potential for
expressing retinal genes and even generating retinal cell
types in some instances. For example, cells of the ciliary
body have been found to express genes involved in the
phototransduction cascade under normal conditions
(Escribano and Coca-Prados, 2002). As discussed
previously, in lower vertebrates the RPE can be induced
to transdifferentiate into retinal neurons (Opas and
Dziak, 1994; Fischer and Reh, 2001) and there is
evidence that bFGF signaling, the transcription factor
Mitf, and the MAPK pathway influence this process
(Pittack et al., 1991; Zhao et al., 1995; Sakaguchi et al.,
1997). Whether these phenomena have a functional role
in mammals or represents a vestigial artifact of eye
specification remains unclear at present. In humans, it
has long been appreciated that certain tumors of the
ciliary body contain rosettes as well as areas that
recapitulate aspects of retinal development. These
neoplasms, generally classified as medulloepitheliomas
or diktyomas, appear to originate in the developing
neuroepithelium of the eye. Along with location and
developmental potential, medulloepitheliomas share
with ciliary PE stem cells the propensity to proliferate,
with or without exogenous growth factors. Further
analysis of ciliary PE stem cells may cast light on the
origins of this tumor or, perhaps, vice versa. From a
clinical perspective, a particularly interesting possibility
evoked by the preceding discussion is the ex vivo
transformation of iris pigment epithelium (IPE) into
RPE or neural retina (Haruta et al., 2001). The potential
use of autologous cells carries a number of obvious
advantages, although immune rejection may not present
ARTICLE IN PRESS
164
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
the threat to transplanted CNS stem cells that it does to
many other tissues and cell types, as will now be
discussed.
5. Immunology of CNS stem cells
The transplantation of neural cells or tissue to the
retina represents a promising, albeit challenging,
approach to replacement of retinal neurons lost to
injury or disease. While the prospect of using stem cells
to repair the diseased CNS has generated unprecedented
interest in the field of neuroscience, the idea of
transplanting living tissue to replace organs that are
no longer functional is a very old one. In early attempts
at transplantation, however, transplants exchanged
between two members of a species routinely failed and
the reasons for that failure were unknown until the
1940s. At that time, Sir Peter Medawar, working on the
problem of providing skin grafts for soldiers wounded in
the 2nd World War, discovered that the destruction of
transplanted tissues was dependent upon an immune
response made by the recipient and directed against socalled transplantation antigens expressed by the graft.
These seminal observations laid the groundwork for the
field of transplantation immunology. It is no accident
that within 20 years of Medawar’s discoveries, the first
successful solid organ transplants were conducted in
human beings. By the 1980s, kidney, heart, liver and
bone marrow transplants were being routinely performed in humans with a significant rate of success.
However, transplantation is not yet a clinical option
for many other organs and tissues. Neural tissues such
as brain and retina present formidable biological and
technical barriers to successful transplantation. In
addition, the immunologic issues surrounding brain
and retinal transplants are complex and perplexing.
Medawar (1948) himself pointed to this matter when he
defined the phenomenon of immunologic privilege. In
fact, until Medawar and his colleagues worked out the
principles of transplantation immunology, it was not
possible to understand why foreign grafts survived in the
anterior chamber of the eye but not elsewhere in the
body. Medawar correctly understood that the anterior
chamber and the brain represented unique sites in the
body where the rules of transplantation immunology did
not apply. At the time (1950s), it was believed that the
brain and the eye lacked lymphatic drainage pathways,
and resided behind blood/tissue barriers. Therefore, he
proposed that immune privilege resulted from ‘‘immunologic ignorance’’ (Barker and Billingham, 1977).
While elements of Medawar’s logic remain intact today,
the information base concerning immune privileged
tissues and sites is considerably greater. It is now
abundantly clear that immune privilege in the eye
(Niederkorn, 1990; Ksander and Streilein, 1994; Strei-
lein, 1995, 1996) and the brain (Wenkel et al., 2000)
results from a number of active regulatory processes in
which immunologic ignorance plays only a minor role.
There is now rather widespread acceptance of the
concept that the systemic immune response to grafts
placed in immune privileged sites acts to promote graft
survival. Moreover, it is also known that immune
privilege exists in two forms: privileged sites (such as
the eye and brain) and privileged tissues or cells (such as
the testis, cornea and as we shall demonstrate, neural
progenitor cells). The cellular and molecular mechanisms responsible for the existence of privileged tissues
and sites are similar, but not identical, and the strategies
used by one privileged tissue may be different from
another. The demonstration that retinal grafts placed in
the brain of neonatal hosts survive, develop, and make
functional connections with the host brainstem (Klassen
and Lund, 1987) prompted investigators to attempt
orthotopic retinal transplantation as well (Lund et al.,
2000). Since ectopic retinal transplants made functional
connections, it was anticipated that achieving local
connectivity in the retina would be relatively straightforward. Unfortunately, this has clearly not been the
case. The enthusiastic wave of retinal transplant
experiments performed in the 1980s and 1990s resulted
in a welter of data that, from both neurobiological and
immunologic standpoints, has been difficult to interpret.
Most studies revealed that histo-incompatible grafts of
neural retina or RPE were initially ‘‘accepted’’ but,
frequently, the grafts succumbed to a process that was
interpreted as immune rejection. In retrospect, the
ability of investigators to distinguish graft failure due
to immune rejection from that due to neurobiologic
failure was inadequate.
Over the past decade much work has been done to
separate immune influences on retinal graft survival
from neurobiologic considerations, such as appropriate
growth and survival factors, and capacity to achieve
integration with the recipient retina. This effort is not
yet complete, but much has been learned about
immunologic features of neuronal retina and RPE as
allografts. For instance, it is now known that the RPE
expresses factors that render it ‘‘immune privileged’’,
and that the subretinal space functions as an immune
privileged site. More importantly, investigators have
begun to understand the factors that maintain immune
privilege in the subretinal space—knowledge that will
work to the advantage of retinal transplants in the
future. In addition, we have shown that microglia within
immature neural grafts are central players in promoting
graft viability, on the one hand, and in rendering the
graft vulnerable to immune rejection, on the other hand.
Increased knowledge of the roles played by microglia in
neuronal retina grafts, should make possible the
generation of strategies for improving the immunologic
fate of these grafts.
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Immunogenicity is by no means the only, or even the
major, barrier to successful retinal transplantation.
Nevertheless, CNS stem cells could potentially be ideal
transplantable cells from an immunological perspective,
in that they are highly homogeneous populations devoid
of professional antigen presenting cells and do not
express MHC class II antigens under normal conditions
(Klassen et al., 2001). However, recent reports suggest
that CNS stem cells have the capacity to differentiate
into hematopoietic cells (Bjornson et al., 1999), and
perhaps even derivatives of all three embryonic germ
layers (Clarke et al., 2000). Such reports have been
difficult to replicate, yet serve as reminders of the need
for vigilance when working with these highly plastic
cells. Moreover, a clear understanding of the basic
immunological characteristics of CNS stem cells is
needed before considering the clinical application of
these cells.
In a series of in vitro and in vivo experiments we have
examined the relative immunogenicity of brain-derived
neural progenitor cells from rodents. In the first of these
studies (Klassen et al., 2003b), we examined the
immunogenicity of NPCs previously derived from the
hippocampal formation of adult Fischer 344 rats (Sah
et al., 1997). These AHPCs had been transfected with
the gene for enhanced green fluorescent protein using a
retroviral construct. AHPCs were co-cultured with
human peripheral blood mononuclear cells (PBMCs),
half the AHPCs and PMBCs being treated with
mitomycin. The final mixed co-cultures contained
multiple wells representing every cell type, alone or in
combination, as well as every treatment condition, and
were assayed for thymidine uptake (Fig. 6). The analysis
was repeated for multiple time points in culture, with
Fischer 344 spleen cells plus PBMCs serving as positive
controls, and AHPCs plus Fischer 344 spleen cells
serving as negative controls (Fig. 7). In addition, cocultures containing FBS were examined to rule out
cellular viability and differentiation as confounding
variables. These experiments consistently demonstrated
a lack of response of PBMCs to xenogeneic AHPCs, as
compared to positive controls. AHPCs, on the other
hand, clearly proliferated in response to PBMCs. We
interpreted the first result as a lack of immunogenicity
on the part of AHPCs, and the second as the result of a
mitogenic influence exerted by PBMCs on AHPCs,
possibly by way of a diffusible factor. Additional
experiments were performed to investigate both these
hypotheses.
With respect to progenitor cell immunogenicity, the
absence of a Th1-type cytokine response on the part of
PBMCs in reaction to AHPCs was documented by
ELISA, consistent with our initial interpretation of the
co-culture data. MHC expression by AHPCs was
evaluated by flow cytometry and low level class I was
found to be present, whereas no class II antigen was
165
Fig. 6. Proliferation study of Fischer 344 rat AHPCs and human
PBMCs in mixed co-cultures, with comparison between different
PBMC donors. Note the lack of response for co-culture combination
7, indicating that hPBMCs do not proliferate in response to
mitomycin-treated rAHPCs. In contrast, combinations 2 and 3 show
proliferation of rAHPCs in response to treated or untreated hPBMCs.
The co-culture profile shown here was highly reproducible and was not
dependent on PBMC donor. Shown are mean and SEM of pooled data
from two experiments, each with triplicate determinations at each
position for each donor. [3H]TdR was added 24 h prior to harvest on
day 6. The suffix ‘m’ indicates cells treated with mitomycin C.
Reprinted from Klassen et al. (2003b) with kind permission.
Fig. 7. Summary of co-culture data. Filled circles connected by solid
line illustrate the inability of rAHPCs to induce hPBMC proliferation
across a range of time points. Similar results were obtained from cocultures containing FBS (filled triangles, broken line) as well as from
allogeneic co-cultures using Sprague–Dawley rat spleen cells instead of
hPBMCs (filled square). Positive control verifies proliferation of
hPBMCs in response to Fischer 344 rat spleen cells syngeneic to
rAHPCs (open diamond). Negative control shows no response of
syngeneic spleen cells to rAHPCs (open square). In each case, [3H]TdR
was added 24 h prior to harvest. Mean, SEM. Reprinted from Klassen
et al. (2003b) with kind permission.
detectable (Fig. 8). AHPCs did express the antiinflammatory cytokine TGF-b1; however, there was no
evidence for active PBMC suppression by AHPCs
in vitro (Fig. 9). Thus, it would appear that a lack
of AHPC-associated immunogenicity, specifically, the
ARTICLE IN PRESS
166
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Fig. 8. Cultured AHPCs were examined for expression of MHC molecules and Fas Ligand (FasL) by flow cytometry. Low level class I expression
was evident whereas labeling for class II (Ia) and FasL was similar to isotype controls. Bold line=antigen, fine broken line=isotype. Reprinted from
Klassen et al. (2003b) with kind permission.
Table 2
Fate of EGFP+ neural progenitor cells grafts placed under the kidney
capsule of normal mice
Donor/host
Graft survival
Progenitor cells/syngeneic
Progenitor cells/allogeneic
Neonatal cerebellum/syngeneic
Neonatal cerebellum/allogeneic
0
7 days
14 days
28 days
5/5
n.d.
n.d
3/3
5/5
5/5
n.d.
2/2
5/5
n.d.
5/5
5/5
5/5
5/5
5/5
1/4
Note: The survival of grafted progenitor or neonatal cerebellum grafts,
placed in either syngeneic or allogeneic kidney capsule, demonstrates
that progenitor grafts, but not neonatal cerebellum grafts, survive for
28 days in allogeneic hosts. n.d.=not done. Reprinted from Hori et al.
(2003) with kind permission.
Fig. 9. PBMC stimulation study. To investigate whether AHPCs exert
an inhibitory effect on PBMC proliferation, mitomycin-treated
AHPCs were co-cultured with PBMCs in the presence of OKT3conditioned supernatant or PHA. Both mitogens induced PBMC
proliferation at both the 2- and 4-day time points. The addition of
AHPCs did not impede this proliferation at either time point, for either
mitogen. [3H]TdR added 24 h prior to harvest. Reprinted from Klassen
et al. (2003b) with kind permission.
absence of MHC class II expression, is sufficient to
explain the in vitro results. With respect to the mitogenic
influence of PBMCs on AHPCs, this effect was
reproduced by the addition of cell-free conditioned
media conditioned by PBMCs, supporting the hypothesis of a diffusible factor. Taken together, these results
suggested that transplanted NPCs would be better
tolerated in vivo than solid tissue grafts. This hypothesis
was tested in a subsequent in vivo study.
Our in vivo immunology work was restricted to the
well-proven mouse model and involved both syngeneic
and allogeneic comparisons. In this instance NPCs were
derived from the brains of early postnatal GFPtransgenic mice on a C57BL/6 genetic background.
These cells survived for 28 days following transplantation to a non-immune privileged cite, in this case under
the kidney capsule of allogeneic BALB/c hosts. Survival
Table 3
Fate of EGFP+ neural progenitor cells grafts placed under the kidney
capsule of pre- or post-sensitized mice
Recipient (BALB/c)
Sensitizeda
(days)
Evaluated
(days)
# Accepted
grafts/# grafts
Pre-sensitized
Post-sensitized
Na.ıve
7
25+32
None
13
42
28
0/5
0/5
5/5
Note: Animals that are injected with spleen cells from the same strain
as the donor progenitor cells reject the progenitor cell grafts, and do so
whether they received the spleen cell injections before (pre-sensitized)
or after (post-sensitized) the progenitor cell grafts. Reprinted from
Hori et al. (2003) with kind permission.
a
Sensitization with respect to graft placement beneath kidney
capsule, at time=0.
at this time point (the longest tested) was equal (5/5) for
both syngeneic and allogeneic NPCs and sufficient to
demonstrate an absence of graft rejection. In contrast,
although grafts of syngeneic neonatal cerebellar tissue
also survived well (5/5), allogeneic grafts survived poorly
(1/4), suggesting that CNS solid tissue grafts are rejected
and thus immunogenic (Table 2). Furthermore, we
acquired evidence that allografted NPCs did not
sensitize their hosts, nor express detectable MHC class
I or class II antigens, although they could be rejected by
hosts who had been previously sensitized (Table 3).
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Flow cytometry confirmed that mouse NPCs did not
express either class I or class II MHC antigens in
culture, although both could be induced by treatment
with the cytokine IFN-g (Fig. 10).
These results reveal mouse NPCs to be immune
privileged cells which survive allografting without
immune suppression and are more likely to be immunologically tolerated than CNS solid tissue grafts,
which contain microglia. Although the situation in
large mammals, including humans, may prove more
complex, we already know that human NPCs do not
express class II as well (Klassen et al., 2001). Together
with the fact that NPC transplantation is directed
toward immune privileged cites such as the retina,
brain, or spinal cord, it therefore seems likely that
any future transplantation of these cells will be less
167
challenging from an immunological perspective than has
been the clinical experience with class II-expressing
hematopoietic stem cells, let alone solid organ transplants. In this regard, it is important that transplantation immunologists and neurobiologists who are
interested in the intriguing yet formidable challenge of
making retinal transplantation successful set modest,
precise goals for their experiments in order to add
knowledge to this young field in a deliberate, incremental manner. If RSC transplantation is to be
successfully employed in humans, many problems must
still be solved and the effort must be multidisciplinary,
ranging from basic science to the clinical specialties. The
results obtained from such studies will define the
strategies central to making retinal transplantation a
therapeutic success.
Fig. 10. Cultured CNS progenitor cells did not express MHC antigens when grown under proliferation conditions (EGF) and the absence of
detectable MHC expression was not altered by differentiation conditions (FCS 10% 8 days). In contrast, both class I (H2Kb, b2 microglobulin) and
class II (I-Ab) MHC antigens were induced by the addition of murine recombinant IFN-g, appearing by day 3 and reaching high levels on day 4,
before returning to negative baseline expression following subsequent IFN-g withdrawal. Dark profiles=molecule of interest, unfilled lines=isotype
control. Reprinted from Hori et al. (2003) with kind permission.
ARTICLE IN PRESS
168
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
6. Transplantation of CNS stem cells
The diseased brain, retina, and spinal cord present
special challenges to medical therapy, in large part
reflecting the biological similarities among these structures as parts of the CNS. Neuronal loss anywhere in the
mammalian CNS results in irreversible functional
deficits due to the severely restricted capacity for
effective endogenous neural regeneration. Furthermore,
attempts at exogenous reconstruction are hampered by
the nervous system’s extreme complexity. Until recently,
no plausible strategies for CNS repair could be
envisioned. Extensive studies of neural plasticity have
provided the basis for rethinking this problem. It is now
believed that the lack of neural regeneration seen
clinically is not simply due to an absence of the cellular
machinery necessary for regrowth, but relates more
closely to inhibitory properties added over the course of
vertebrate evolution. For instance, numerous studies
have demonstrated that the retinas of teleosts, urodeles
and anurans effectively regenerate in response to a range
of experimental perturbations. This regeneration includes the addition of new retinal neurons, regrowth of
the optic nerve and the return of visual function.
Although such is not the case in mammals, a large
number of studies have examined each of these aspects
of regeneration, often with encouraging results.
6.1. Clinical transplantation of fetal neural tissue
Altman (Altman and Das, 1965) showed that,
contrary to general expectations, new neurons are
continuously generated in the hippocampal formation
.
of mature rats. Bjorklund
and colleagues (Bjorklund
and Stenevi, 1977, 1979) showed that grafts of fetal
neural tissue are capable of normalizing motor function
in mature rats with Parkinson-like symptoms. Schwab
and colleagues (1988) discovered that mammalian
neurons will extend neurites when removed from the
CNS and grown in vitro, but that this outgrowth is
inhibited by contact with oligodendrocytes. Aguayo and
others showed that mature CNS axons can regenerate if
provided a permissive substrate such as a sciatic nerve
graft, even leading to functional connections (Sasaki
et al., 1996). Merzenich et al. (1983, 1987) showed that
interruption of peripheral conduction results in rapid
functional reorganization of the cerebral cortex in
mature monkeys. On the strength of a large body of
findings such as these, numerous clinical trials have been
carried out, for the most part directed toward neurodegenerative diseases.
Cell transplantation has emerged over the past 25
years as a promising experimental approach for the
restoration of neural function in degenerative brain
diseases, particularly Parkinson’s and Huntington’s
Diseases (Bjorklund et al., 2003; Rosser and Dunnett,
2003). Since 1987, upward of 250 patients with advanced
Parkinson’s Disease have received transplants at several
centers in Europe and America. Clinical trials have thus
far emphasized the use of human mesencephalic tissue
obtained from 6- to 9-week-old fetuses, the rational
being that this tissue contains fate-committed dopaminergic neuroblasts. These cells should possess a substantial degree of plasticity associated with their
developmental immaturity, together with the capacity
to develop into fully mature dopamine neurons following transplantation to the host brain.
Much effort has been expended in the attempt to
demonstrate that fetal human nigral neural precursors,
taken at a stage of development when they have started
to express their dopaminergic phenotype, will survive,
integrate and function over an extended period in the
human brain. Specifically, it is imperative to show that
neural grafts will integrate functionally in a tissue
environment with an ongoing disease process. The
results to date have been suggestive of some benefits,
but certainly less than hoped for, particularly given that
an intracranial procedure is involved (Park, 2000; Park
et al., 2002; Piccini, 2002; Check, 2003).
A major limitation of the fetal cell transplantation
procedure is the low survival rate of the grafted neurons
(in the range of 5–20%) making it difficult to obtain
sufficient cells for grafting in patients. Currently,
mesencephalic fragments from at least 6 to 8 embryos
are needed for transplantation in one hemisphere of a
Parkinsonian patient. Moreover, the many political,
ethical, and practical issues associated with the use of
tissue from aborted human fetuses have proven to be
problematic and would likely restrict the application of
such procedures, even were they clearly efficacious, to a
few highly specialized centers. In contrast, the successful
propagation of mammalian NSCs in culture, first
reported by Reynolds and Weiss, and separately by
Bartlett in 1992, has opened up hitherto unforeseen
opportunities in the field of neural transplantation. CNS
stem cells represent prime candidates for restorative cell
replacement and gene transfer therapies, and might
eventually offer a powerful alternative to primary fetal
CNS tissue in the setting of clinical transplantation.
Therefore, studies of the behavior of these cells
following transplantation are of considerable current
interest.
6.2. Transplantation of NSCs in animal models of brain
injury
There is now considerable evidence from animal
models that NSCs can be used in place of fetal tissue,
often with strikingly better results. For example, NSCs
have shown promising results in a mouse model of
hypoxic–ischemic (HI) injury. NSCs injected into brains
with focal HI injury appeared to integrate extensively
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
within the lesioned CNS, with robust engraftment and
reporter gene expression within the region of HI injury
(Park, 2000; Park et al., 2002). They also appeared to
have migrated preferentially to the site of ischemia,
exhibited limited proliferation, and differentiated into
neural cells in an apparent attempt to repopulate
damaged areas of the brain. This appears to apply not
only to neuronal replacement but also to glia, including
oligodendrocytes, where grafted multipotential neural
precursors preferentially differentiated along the oligodendrocyte lineage in weaver mice with a defect in that
cell type (Brustle et al., 1998). In addition, there is
evidence that various marrow-derived stem cell fractions
can seed the brain with new neurons and glia (Eglitis
and Mezey, 1997), and replace vascular endothelial cells
and augment CNS capillary networks (Otani et al.,
2002). However, such conclusions are not without
controversy (Castro et al., 2002; Wehner et al., 2003)
and may need to be re-evaluated in light of possible cell
fusion events (Weimann et al., 2003a, b).
The transplantation of exogenous NSCs may, in fact,
represent the augmentation of a natural self-repair
process in which the injured CNS attempts to mobilize
its own pool of stem cells (Fallon et al., 2000). This
mechanism, which is quite proficient in lower vertebrates, appears to have been down-regulated over the
course of vertebrate evolution, possibility as a strategy
to protect against neoplastic transformation within
active stem cell pools. Providing additional NSCs and
trophic factors may unmask the full restorative potential
of this otherwise muted response. Stem cell transplantation therefore provides a new and powerful approach to
the reconstitution of the injured CNS in the context of a
variety of pathological processes, including HI injury,
Parkinson’s Disease, and other neurodegenerative disorders. In addition, there is evidence for CNS stem cellmediated repair in not only the brain but also in the
spinal cord (McDonald et al., 1999) and retina as well.
169
of this response reflected the strength of the underlying
graft-host projection (Klassen and Lund, 1987, 1990).
This work also demonstrated the functional efficacy of
retinal xenografts as part of the first systematic studies
of neural transplant immunology (Young et al., 1989).
The intracranial work was mirrored by work with
orthotopic retinal and RPE transplantation which
showed survival and differentiation of grafts to the
retina, subretinal space, and vitreous (Lopez et al., 1989;
Sheedlo et al., 1989; Silverman and Hughes, 1989a, b;
Adolph et al., 1994; Aramant and Seiler, 1994, 1995;
Little et al., 1998).
In contrast to the experience with intracranial grafts,
solid tissue retinal transplants to the eye proved to be
more problematic in terms of functional integration with
the mature host. This crucial problem of orthotopic
integration now appears to have a solution, namely, the
transplantation of CNS stem cells. Before this was
evident, however, the initial scientific investigations into
fetal tissue and RPE grafts were followed rapidly by
clinical trials, much as was the case with neural
transplantation for Parkinson’s Disease.
6.4. Clinical retinal transplantation
Retinal cells and tissue of various types has now been
allografted to patients with a number of retinal
degenerative diseases, using a number of different
protocols. These include the transplantation of RPE
cells to patients with macular degeneration (Algvere
et al., 1994, 1997; Weisz et al., 1999) and neural retinal
cells to patients with retinitis pigmentosa or macular
degeneration (Kaplan et al., 1997). Overall, these
procedures appear to pass the criteria for patient safety
and, in some cases, graft survival was evident. In terms
of visual benefit, however, the results have been less
gratifying (Berson and Jakobiec, 1999).
6.5. Retinal transplantation of CNS stem cells
6.3. Experimental retinal transplantation
Similar to neural transplantation for Parkinson’s
Disease, mammalian retinal transplantation was set in
motion by unexpected and exciting experimental findings using fetal tissue grafts, progressed rapidly to
clinical trials with less than gratifying results, and has
been more recently reinvigorated by experimental results
using NSCs. Evidence for hitherto unappreciated neural
plasticity in the mammalian retina was revealed by
McLoon et al. (1982), who demonstrated that embryonic retinas grafted to the brain of newborn rats establish
a projection to the superior colliculus. Also working in
Lund’s laboratory, we subsequently demonstrated function in this model by showing that retinal transplants
can drive a pupillary reflex in the host eye by way of the
pretectum, even in mature rats, and that the magnitude
The problem with intraocular grafts of fetal retina is
not so much survival or differentiation, but rather a lack
of widespread functional integration with the host. As a
prerequisite to functional success, donor cells must be
capable of widespread integration in the mature
degenerating mammalian retina. Furthermore, they
must exhibit this capability in the face of active disease.
These criteria were first met by hippocampal progenitor
cells isolated and grown in the lab of Fred H. Gage.
Hippocampal progenitor cells are a type of NSC
derived from the region of the dentate gyrus, a region
notable for active neurogenesis in adult rats. Although
the presence of NSCs within the developing mammalian
CNS and dentate gyrus of the adult rat has been
appreciated for some time, the successful isolation and
propagation of these cells using recombinant growth
ARTICLE IN PRESS
170
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
factors has served to reinvigorate the field of neural
transplantation. In the first of two seminal papers on the
transplantation of this type of cell to the eye, Takahashi
et al. (1998) demonstrated that intravitreal injections of
hippocampal progenitors in neonatal rats resulted in a
spectacular degree of morphological integration within
the neural retina. Progenitor cells injected into the eyes
of normal adult rats, however, failed to integrate.
In the second paper, we reported that hippocampal
progenitor cells, again simply injected into the vitreous,
were capable of widespread migration and morphological integration into the degenerating retina of mature
RCS rats with active retinal degeneration (Young et al.,
2000). The grafted cells differentiated into neurons and
exhibited a notable tendency to respect the retinal
cytoarchitecture of the host (Fig. 11). Somata of grafted
calls were found predominantly within the cellular
layers, while bouton-rich processes extended into the
plexiform layers, typically at an appropriate orientation
and frequently arborizing within specific sublaminae
(Fig. 12). In addition, GFP+ donor cells within the
GCL expressed NF-200 and extended large numbers
of growth cone-tipped processes into the optic nerve
(Fig. 13). We have also found that xenografted neural
progenitors can exhibit widespread retinal integration,
although this is not invariably the case (Mizumoto et al.,
2001).
The factors responsible for stem cell integration into
the CNS are likely to be multifaceted. In addition to
fundamental metabolic and size considerations, the
degree of genetic disparity between graft and host will
likely impact on the efficacy of intercellular signaling.
Furthermore, the relative plasticity of the donor cell line
as well as the developmental state of the host will
influence outcome, as will any ongoing degeneration or
inflammation. Finally, there is the complex issue of
immune tolerance. Recent studies are providing a
baseline from which to predict the immunological
consequences of grafting multipotent neural stem and
progenitor cells to various sites within the CNS.
Transplantation studies have provided much encouragement by demonstrating a high capacity for migration
and cytoarchitectual integration of various types of
neural progenitor cells. Indeed, the ultimate plasticity of
NPCs may be even greater than initially anticipated
(Suhonen et al., 1996; Bjornson et al., 1999; Clarke et al.,
Fig. 11. Localization of grafted AHPCs to specific retinal layers. Cells (green) were grafted into the vitreous of (a–d) 4-, (e) 10-, and (f) 18-week-old
rats, and examined 4 weeks later. Sections were stained with anti-synaptophysin/Cy3 antibody (red) and viewed under FITC and Cy3 fluorescent
illumination. Arrow in (a) indicates cell seen in (b) at higher power; arrow in (c) indicates cell seen in (d) at higher power vit, vitreous; gcl, ganglion
cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; srs, subretinal debris and degenerating
photoreceptor elements. Reprinted from Young et al. (2000) with kind permission.
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
171
Fig. 13. Confocal images of GFP+ neurites projecting, via the host
optic fiber layer, into the optic nerve head 4 weeks after grafting
(a+b). These fibers have large growth cones (arrows in (a), and in
higher power in (b)), which approach, but do not cross, the scleral
outlet (sc) at 4 weeks post-grafting into 1-week-old hosts. When
animals were examined 8 weeks after grafting, numerous growth conetipped processes were found to have entered the optic nerve, extending
over 300 mm beyond the scleral outlet (c). Reprinted from Young et al.
(2000) with kind permission.
Fig. 12. Confocal images of anti-synaptophysin/Cy3 (red) antibody,
showing grafted AHPCs (green) sending processes into the IPL (a–d),
or the OPL (e–h) (grafted at 4 weeks, examined 4 weeks after grafting).
In (a+b), a cell is shown merged (a), and reconstructed to show entire
neuritic arbor (b). In (c–h), AHPCs send neurites into the IPL ((c),
higher power in (d)), and OPLs ((e+g), higher power in (f+h),
respectively). These processes intermingle with, and appear to contact
synaptophysin positive profiles of the host. Reprinted from Young
et al. (2000) with kind permission.
2000). Analogous to the epigenetic respecification
hypothesized to occur following nuclear transfer into
oocytes, multipotent NSCs exposed to certain environments may undergo an expansion in phenotypic
plasticity such that they behave more like pluripotent
ES cells. At this time, however, the limits of stem cell
plasticity remain ill-defined and highly contentious. The
biology of NSCs must be examined systematically, both
in terms of basic science and as a prerequisite to
responsible application of such cells in the clinical
setting.
6.5.1. Transplantation of CNS stem cells to the Brazilian
opossum: a novel model system for investigating neural
stem cell plasticity
The visual system of the Brazilian opossum (Monodelphis domestica) represents a novel in vivo model
preparation for investigation of CNS stem cell development and plasticity. Over the years the vertebrate retina
has emerged as an important and tractable model
system for studying how a complex CNS structure is
patterned and organized during neural development. In
all vertebrates the major retinal cell types are in general
produced during the late stages of embryogenesis. The
current thinking is that retinal progenitors exit the cell
cycle and commit to specific fates over time as a
consequence of changes in their extrinsic environment.
As cell migration and differentiation proceed, the soma
of individual retinal cell types assume distinct positions
and form three distinct nuclear layers and these layers
eventually connect to each other through synapses in the
adjoining plexiform layers. The molecular events leading
to the establishment of the precise architectural organization of the retina are under intense investigation. In
response to the changing environment, the intrinsic
genetic program of the retinal progenitor population is
continuously modulated. The dynamic interplay between the extracellular environment and intrinsic genetic
programming is essential to the formation of the retina.
ARTICLE IN PRESS
172
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
The essential genes and complex genetic pathways
responsible for cell fate determination and phenotypic
differentiation are now being identified using a number
of experimental model systems and methods.
Studies using lower vertebrates such as fish (goldfish
and zebrafish), amphibians (Xenopus laevis), and avian
(the chick) species (Rager, 1980; Sakaguchi, 1989;
Raymond, 1991; Chien and Harris, 1994; Hu and
Easter, 1999; Mey and Thanos, 2000) as model
organisms have provided a great deal toward our
understanding into the cellular and molecular basis of
neural development, including the visual system. Amphibians, especially Xenopus laevis, were among the first
experimental organisms employed for investigations of
eye development. These lower vertebrate models have
proved especially useful from an embryological, as well
as molecular and cell biological perspective. For
example, in vivo embryological manipulations can be
routinely carried out in lower vertebrates, and the use of
viral vectors for gene transfer and lineage analysis have
proved extremely useful for studies of neural development in these species. Using the zebrafish, a number of
investigators have described methods for addressing
retinal development using genetic and cell biological
approaches. The chick retina has also been, and
remains, a powerful system in which to probe mechanisms of retinal development in vivo.
Until recently, most studies investigating mammalian
retinal development have relied principally on rodents,
cats, ferrets, and primates. While the mouse is an
especially powerful genetic model system since the
genome can readily be altered by adding, changing, or
deleting genes, this species is born with relatively welldeveloped nervous system since a major portion of
neural development occurs in utero. In the case of cats,
ferrets, or primates, the economics of maintaining a
colony prevents their routine use in developmental
studies. Again, in all these placental mammals, a
significant proportion of retinal development occurs
during fetal life. The transition between lower vertebrates and conventional mammalian systems can,
however, be appropriately bridged by the use of
marsupials. Marsupials are metatherian mammals and
are phylogenetically distinct from conventional mammalian biomedical models, all of which are eutherian
(placental) species. However, marsupials and eutherians
are more closely related to one another than to other
vertebrate model species such as fishes, amphibians, and
birds (Ji et al., 2002). As such, the marsupial/eutherian
relationship represents a unique transition midpoint in
age relative to existing mammalian and non-mammalian
vertebrate models. Marsupials and eutherians share
basic genetic mechanisms and molecular processes that
represent fundamental mammalian characteristics.
The Brazilian gray short-tailed opossum, Monodelphis
domestica (also referred to as the laboratory opossum),
is a small pouchless marsupial native to South America
and is widely used as a model organism for comparative
research on a broad range of topics relevant to human
development, physiology, and disease susceptibility
(VandeBerg and Robinson, 1997). The Brazilian opossum breeds well under laboratory conditions and their
young are born in an extremely immature, fetal-like
state after a 14-day gestation (compared to 19 and 21
days gestation for mice and rats, respectively) (KuehlKovarik et al., 1995; West Greenlee et al., 1996). Litters
vary in size from 2 to 13 pups (Fig. 14). The retina of the
Brazilian opossum possesses several advantages over
other mammalian retinas and has served as a unique
host CNS environment for recent transplant studies
Fig. 14. The Brazilian opossum as an in vivo experimental model
system. (a) 3 PN Monodelphis pup is approximately 1 cm in length. The
eye is clearly visible beneath the skin (arrow). (b) Ventral view of a
female with 10, 7 PN pups attached to her nipples. (c) Adult female
opossum (about the size of a hamster) with 3, 65 PN pups grasping
onto their mother. (d) and (e) Cytogenesis within the developing
Monodelphis retina. Images showing Bromodeoxyuridine-IR within
the postnatal retina. Pups received subcutaneous injections of BrdU at
5 and 10 PN, and were sacrificed 2 h later. Extensive BrdU incorporation was observed during early postnatal development. Abbreviations:
CB, cytoblast layer; g and GCL, ganglion cell layer; IPL, inner
plexiform layer; RPE, retinal pigment epithelium. Scale bars for (d)
and (e)¼ 20 mm. Reprinted from Sakaguchi et al. (2003) in Annals
from the New York Academy of Science with kind permission.
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
(Van Hoffelen et al., 2003). At birth (1 day postnatal:
1 PN) the retina is at an embryonic state developmentally and undergoes a protracted period of neurogenesis
(West Greenlee et al., 1996). Their lack of a pouch
and fetal-like nature at birth circumvents the need for
in utero surgical procedures, such as are required
when working on the early visual system of more conventional mammalian model systems such as mice, rats,
rabbits, ferrets, cats, or primates. Eye opening in the
Brazilian opossum occurs around 35 PN and the pups
are weaned at 60–65 PN. The animals reach reproductive maturity at about 6 months of age (Kuehl-Kovarik
et al., 1995).
Birthdating studies using the thymidine analog,
bromodeoxyuridine (BrdU), have revealed that the vast
majority of retinal cytogenesis occurs postnatally in
Monodelphis. Fig. 14 illustrates the extensive BrdU
incorporation, indicative of DNA synthesis, occurring
during early postnatal life. At birth, the Monodelphis
retina is a relatively undifferentiated neuroepithelium
with the first postmitotic cells, early differentiating
ganglion cells, located in the dorso-central aspect of
the retina. At 5 PN the outer cytoblast region displays
extensive BrdU incorporation and the GCL has formed,
although the other retinal layers are not yet obvious
(Fig. 14d). By 10 PN the nascent inner plexiform layer
(IPL) is present, separating the GCL from the mitotically active cytoblast layer (Fig. 14e). Retinal neurogenesis continues to at least 25 PN in Monodelphis. The first
12–15 days of postnatal life in Monodelphis development
correspond roughly with the late embryonic period in
rodents. For example, at 12–15 PN, CNS development
in Monodelphis is approximately equivalent to that of a
postnatal day 1 rat (Van Hoffelen et al., 2003). This
immaturity at birth, accompanied by a protracted
period of postnatal development, makes marsupials like
Monodelphis excellent models for the study of development and plasticity in the visual system.
As discussed previously, in vivo lineage studies
suggest that the various cell types in the retina arise
from multipotential progenitors whose fate is guided by
the surrounding environment (Altshuler et al., 1991).
Recent studies have demonstrated that transplanted
neural progenitor cells are able to differentiate and
integrate morphologically into developing host retinal
microenvironments (Suzuki et al., 2003; Van Hoffelen
et al., 2003). Is the ability to differentiate and the ability
to undergo structural integration into the CNS microenvironment unique to specific CNS stem cells, or is this
plasticity a function of host age, or both? To address
these issues we have taken advantage of the developing
retinal of the Brazilian opossum and have performed a
systematic comparison of the structural integration of
neural stem/progenitor cells into hosts of different ages.
Brain-derived NPCs (BPCs) from GFP-transgenic
mice were transplanted by intraocular injection into
173
the vitreal chamber of the eye through the caudal pole
of the globe. In all, 1–2 ml of cell suspension containing
approximately 50,000 cells/ml was pressure injected
using a beveled glass micropipette. Hosts were permitted
to survive for 1, 2, or 4 weeks post-transplant at
which time the eye tissue was prepared for analysis.
By using ‘‘fetal-like’’ hosts (10 PN and younger),
maturing (30–35 PN), and mature (older than 35 PN)
host animals we have been able to investigate the
influence of the host cellular microenvironment on
donor BPCs in vivo. The xenografted BPCs survived
and were easily identified following transplantation due
to their endogenous GFP fluorescence (Fig. 15).
Furthermore, the host tissue appeared relatively normal
without obvious disruption of the laminar organization
of the developing retina (Suzuki et al., 2003; Van
Hoffelen et al., 2003). Although transplanted cells
survived, differentiated morphologically, and integrated
into host tissue, dramatic differences were encountered
when the BPCs were grafted into host retinal environments of different ages. Extensive morphological differentiation and integration were only observed after
transplantation into the youngest (5–10 PN) host
opossum retinas. While grafted cells were capable of
surviving in the older host retinas, little integration was
observed. Transplanted cells were often found in the
posterior segment of the eye along the inner limiting
membrane (ILM), around the lens, as well as in the
vitreous. We focused our efforts on characterizing the
integration of the transplanted cells into the developing
environment of the eye since few transplanted cells were
Fig. 15. Structural integration of BPCs into a 10 PN host retina 4
weeks post-transplant. GFP expressing cells appeared intermixed
between host cells in an organized extensive network. GFP expressing
cells were found throughout the host retina, with somata located
principally within nuclear layers (INL and GCL) and processes
extending throughout the plexiform layers (OPL and IPL). Moreover,
GFP processes in the IPL were segregated into OFF and ON
sublamina of the IPL. Thus, the transplanted cells respected the
architectural organization of the host retina.
ARTICLE IN PRESS
174
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
observed to have integrated in the older host eyes.
Transplanted cells in the youngest hosts often displayed
differentiated morphologies characteristic of specific
retinal cell types. For example, some GFP+ cells
possessed morphologies similar to horizontal, bipolar,
amacrine, and ganglion cells (Van Hoffelen et al., 2003).
One of the most interesting observations was the ability
of the grafted cells to respect the architectural organization of the host retina. In general, transplanted GFPexpressing cell bodies were localized in the nuclear layers
of the retina, while their GFP+ processes were
elaborated principally within the plexiform layers.
Moreover, GFP+ processes often appeared to segregate
within specific sublaminae of the IPL. The IPL can be
subdivided into OFF and ON sublaminae both anatomically, neurochemically, and physiologically (Marc,
1986). In many cases GFP+ processes were observed to
ramify specifically within one sublamina. Together,
these results suggest that BPCs transplanted into a
developing retina were capable of migrating into specific
layers and that the transplanted cells were capable of
recognizing specific molecular cues located within the
host microenvironment.
In addition to the morphological differentiation and
integration of transplanted BPCs, a subset of GFP+
cells were observed to differentiate and expressed the
molecular markers MAP2, calretinin, and recoverin,
markers associated with neurons, retinal interneurons,
and photoreceptors, respectively. The expression of
these particular proteins by transplanted cells appears
to be specific since not all GFP+ cells expressed these
markers. This suggests that subpopulations of BPCs
may exist that possess particular cell fate potentials with
respect to their differentiation capabilities. Alternatively, the position of the transplanted cells within the
host retina may strongly influence their fate. Results of
this nature suggest that there may be regional differences within the host CNS microenvironment capable of
influencing the phenotypic specification of BPCs following transplantation.
Using the Brazilian opossum as a model system, we
have thus far focused our attention on transplant studies
of murine BPCs in order to study the influence of the age
of the host environment upon the differentiation and
integration potential of the transplanted neural progenitor cells. However, recently we have also successfully transplanted murine RPCs, as well as rat adult
hippocampal progenitor cells, and preliminary results
clearly demonstrate that these cells are also capable of
survival following xenotransplantation, even in the
absence of immunosuppression. As discussed previously, this may be due to the relative purity of
these CNS progenitors, which lack antigen presenting
cells and passenger leukocytes that would be present
in conventional grafts of neural tissue, as well as
the low level of MHC expression generally exhibited
by CNS stem cells (Klassen et al., 2001, 2003; Hori
et al., 2003).
Recent studies suggest that grafted cells are capable of
responding to cues in the developing CNS (Takahashi
et al., 1998; Van Hoffelen et al., 2003; Suzuki et al.,
2003). Other studies have indicated that grafted stem
cells are also able to respond to cues within the diseased
CNS. Indeed, several studies have shown that the
transplanted cells integrate and morphologically differentiate much better in dystrophic retinas than in normal
control retinas (Young et al., 2000; Wojciechowski et al.,
2002). While there are many possible interpretations for
these types of results, one could hypothesize that the
diseased CNS in some manner recapitulates a developing environment, expressing factors normally only
present during fetal growth. The identification of these
factors, and the understanding of their impact upon
stem cell differentiation, is an important area of study in
stem cell biology. The use of recipients such as the
Brazilian opossum provides one with an excellent model
system to investigate the role of these factors for neural
transplantation.
In summary, neural stem and progenitor cells clearly
possess a remarkable degree of plasticity. Our recent
studies demonstrate that BPCs can survive intravitreal
transplantation and incorporate within the intact,
developing retina. It seems that the developing microenvironment may be critical in determining the fate of
transplanted progenitor cells.
6.5.2. Transplantation of retinal stem cells
We and others have shown that a profound degree of
engraftment can be achieved through the use of brainderived progenitor cells for retinal transplantation. It
has become apparent, however, that brain-derived cells
do not differentiate into authentic retinal neurons in the
microenvironment of the mature, diseased retina.
Several possible strategies can be employed in an effort
to overcome this obstacle. For example, one could either
attempt to modify a brain-derived cell to induce
transdifferentiation along a retinal lineage, or induce
the differentiation of a more plastic, less differentiated
cell type such as an ES cell into such a fate. We and
several other groups have chosen a third strategy,
namely, the isolation of progenitor cells from the neural
retina of the developing mammalian eye (Shatos et al.,
2001). While these studies are in the early stages, one
important discovery is that these cells, upon transplantation to the retina of adult mammals with retinal
disease, possess both the integrative plasticity that is a
hallmark of CNS stem cells, as well as the ability to
differentiate into retinal neurons, including photoreceptors (Fig. 5). We are hopeful that further studies of RPC
grafts will point the way forward to the development of
clinical strategies aimed at restoring vision to the
blinded eye.
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
7. Conclusions
The nascent field of stem cell biology has much to
offer those interested in the study of the mammalian
retina. From a developmental perspective, the retinal
specification and maturation events that occur during
embryogenesis bear directly on changes in transcription
factor expression and phenotypic potential observed in
cultured stem and progenitor cell populations. From a
pathophysiological perspective, the response of transplanted CNS stem cells to various forms of retinal
injury, including photoreceptor degeneration, provides a
powerful means of assaying the status of local homeostatic mechanisms operating within the diseased microenvironment. From a clinical perspective, stem and
progenitor cell transplantation has shown that the
actively degenerating adult retina can be repopulated
with donor-derived neurons, including photoreceptors,
that these new cells survive without exogenous immune
suppression as well as exhibiting morphological evidence of integration with host circuitry. While much
remains to be demonstrated, particularly in terms of
host visual benefits, retinal stem cell transplantation
provides an exciting new strategy for the treatment of
retinal disease and offers the hope that effective
treatments may be within reach.
8. Future directions
A major issue facing stem cell research, and retinal
transplantation of stem cells in particular, is the
question of functional integration of grafted cells. This
issue incorporates the related question of phenotypic
maturation of stem cells, particularly into functional
neurons and especially photoreceptors, as well as the
question of how surviving host circuits react to the
presence of newly integrated donor cells. For instance,
the possibility has frequently been raised that such cells
might integrate incorrectly. On the other hand, donor
cells might successfully create new functional circuits,
improve host signaling, or induce rescue effects. These
possibilities should all be investigated in multiple
settings since results tend to vary considerably between
models.
Findings generated with retinal stem cell transplantation need to be extended to a large animal model to
demonstrate efficacy in a setting more closely approximating the challenges faced clinically. Such work will
provide a more homologous biological context, allow
more accurate assessment of functional correlates, and
facilitate the development of applicable cell delivery
technology. Species of potential interest include the pig,
dog, and cat, all of which have disease models available.
The monkey is also of potential interest, particularly for
strategies directed at macular repair. Although experi-
175
mental xenotransplantation is certainly of interest, in
each case it would be advantageous to derive speciesspecific RPCs as well.
The first step toward application of this strategy to
humans will be the isolation and characterization of
human RPCs. It will be important to know whether
these cells can be obtained from donors other than
aborted fetuses, how well they can be propagated ex
vivo, what their phenotypic potential is, how immunogenic they are, and how well they survive, integrate,
differentiate, and function following transplantation. As
this information becomes available, it will provide the
basis for further considerations such as which patient
populations would most likely benefit from future
clinical application of these cells.
In terms of molecular characterization of stem cells,
we anticipate a shift away from single marker phenotyping and toward multiple marker profiling, with particular emphasis on transcription factors. Because of the
role of coordinated transcription factor expression in
fate specification, such profiles should ultimately have
predictive significance in terms of phenotypic potential.
As a caveat to this approach, it must be appreciated that
CNS stem cell cultures are inherently heterogeneous and
therefore additional measures will have to be taken if the
individual profiles of various cell types present are to be
separated from the population data. That being said, it
can be envisioned that our ability to control stem cell
differentiation will be vastly improved when transitional
ontogenetic states can be resolved in sufficient molecular
genetic detail.
Apart from neuronal replacement, another interesting
potential application for stem cells in the retina is gene
delivery. There is a sizable animal literature indicating
that cytokines and related gene products have potential
benefits in a variety of ocular diseases, notably as a means
of inducing photoreceptor rescue and thereby ameliorating retinal degeneration. While depot release of agents
can be problematic and direct injection of vector into
patients has proven to be hazardous, ex vivo transgenic
modification of stem cells followed by transplantation
may ultimately provide a reliable means of long-term
gene delivery. Of note, functional neuronal integration of
donor cells is not required with this strategy; a glial posttransplantation phenotype should suffice.
Looking beyond simple subretinal injection of RPCs,
it is clear that tissue engineering will have an important
role to play in the development of strategies directed
toward more complex retinal pathologies, particularly
those in which multiple cytoarchitectural layers must be
reconstructed such as macular degeneration. For instance, it may prove advantageous to combine tissue
and stem cell transplants as discrete layers within a
single composite graft. We envision that engineered
retinal grafts will eventually become sufficiently sophisticated so as to restore function to the blinded eye.
ARTICLE IN PRESS
176
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Acknowledgements
We wish to thank the many members of our
laboratories, past and present, who have contributed
to this work: Simon Whiteley, Yasuo Kurimoto, Marie
Shatos, Tat Fong Ng, Aziza Mwidau, and Tasneem
Zahir from the Young Lab; M. Heather West Greenlee
and Samantha Van Hoffelen from the Sakaguchi lab,
and Michael Schwartz, Karen Imfeld, Ivan Kirov, and
Boback Ziaeian from the Klassen lab, as well as our
CHOC colleagues Philip Schwartz and Monique Berman. We also thank our collaborators around the
world: Fred H. Gage and Jaso Ray from the Salk
Institute, Erin Lavik and Robert Langer from MIT,
Karin Warfvinge from the Univ. of Lund, and Jens
Kiilgaard and Erik Scherfig from the Panum Institute in
Copenhagen. We are also indebted to the mentors who
have guided our work in this field, especially Raymond
D. Lund, J. Wayne Streilein, and William Harris.
We lastly acknowledge the generous support of the
NINDS and NEI, as well as the Minda de Gunzburg
Research Center for Retinal Transplantation, Richard
and Gail Seigal, the Lions Eye Research Fund, the
Stem Cell Research Foundation, the Glaucoma Foundation, N.Y., and the CHOC Foundation, Guilds, and
Padrinos.
References
Acampora, D., Gulisano, M., Broccoli, V., Simeone, A., 2001.
Otx genes in brain morphogenesis. Prog. Neurobiol. 64 (1),
69–95.
Adler, R., 1996. Mechanisms of photoreceptor death in retinal
degenerations. From the cell biology of the 1990s to the
ophthalmology of the 21st century? Arch. Ophthalmol. 114 (1),
79–83.
Adler, R., Hatlee, M., 1989. Plasticity and differentiation of embryonic
retinal cells after terminal mitosis. Science 243 (4889), 391–393.
Adolph, A.R., Zucker, C.L., Ehinger, B., Bergstrom, A., 1994.
Function and structure in retinal transplants. J. Neural. Transplant. Plast. 5 (3), 147–161.
Ahmad, I., Zaqouras, P., Artavanis-Tsakonas, S., 1995. Involvement
of Notch-1 in mammalian retinal neurogenesis: association of
Notch-1 activity with both immature and terminally differentiated
cells. Mech. Dev. 53 (1), 73–85.
Ahmad, I., Dooley, C.M., Polk, D.L., 1997. Delta-1 is a regulator of
neurogenesis in the vertebrate retina. Dev. Biol. 185 (1), 92–103.
Algvere, P.V., Berglin, L., Gouras, P., Sheng, Y., 1994. Transplantation of fetal retinal pigment epithelium in age-related macular
degeneration with subfoveal neovascularization. Graef. Arch. Clin.
Exp. Ophthalmol. 232 (12), 707–716 (see comments).
Algvere, P.V., Berglin, L., Gouras, P., Sheng, Y., Kopp, E.D., 1997.
Transplantation of RPE in age-related macular degeneration:
observations in disciform lesions and dry RPE atrophy. Graef.
Arch. Clin. Exp. Ophthalmol. 235 (3), 149–158.
Altman, J., Das, G.D., 1965. Autoradiographic and histological
evidence of postnatal hippocampal neurogenesis in rats. J. Comp.
Neurol. 124, 319–335.
Altshuler, D.M., Turner, D.L., Cepko, C.L., 1991. Specification of cell
type in the vertebrate retina. In: Man-Kit Lam, D., Shatz, C.J.
(Eds.), Development of the Visual System. Massachusetts Institute
of Technology, Cambridge, MA, pp. 37–58.
Altshuler, D., Lo, T.J., Rush, J., Cepko, C., 1993. Taurine promotes
the differentiation of a vertebrate retinal cell type in vitro.
Development 119 (4), 1317–1328.
Anchan, R.M., Reh, T.A., 1995. Transforming growth factor-beta-3 is
mitogenic for rat retinal progenitor cells in vitro. J. Neurobiol. 28
(2), 133–145.
Anderson, D.J., 1994. Stem cells and transcription factors in
the development of the mammalian neural crest. FASEB J. 8
(10), 707–713.
Appelbaum, F.R., 2001. Haematopoietic cell transplantation as
immunotherapy. Nature 411 (6835), 385–389.
Aramant, R.B., Seiler, M.J., 1994. Human embryonic retinal cell
transplants in athymic immunodeficient rat hosts. Cell Transplant.
3 (6), 461–474.
Aramant, R.B., Seiler, M.J., 1995. Fiber and synaptic connections
between embryonic retinal transplants and host retina. Exp.
Neurol. 133 (2), 244–255.
Ashery-Padan, R., Gruss, P., 2001. Pax6 lights-up the way for eye
development. Curr. Opin. Cell Biol. 13 (6), 706–714.
Baonza, A., Freeman, M., 2001. Notch signalling and the initiation of
neural development in the Drosophila eye. Development 128 (20),
3889–3898.
Baonza, A., Freeman, M., 2002. Control of Drosophila eye specification by Wingless signalling. Development 129 (23), 5313–5322.
Barker, C., Billingham, R., 1977. Immunologically privileged sites.
Adv. Immunol. 24, 1–54.
Beebe, D.C., 1986. Development of the ciliary body: a brief review.
Trans. Ophthalmol. Soc. UK 105 (Pt 2), 123–130.
Belliveau, M.J., Cepko, C.L., 1999. Extrinsic and intrinsic factors
control the genesis of amacrine and cone cells in the rat retina.
Development 126 (3), 555–566.
Belliveau, M.J., Young, T.L., Cepko, C.L., 2000. Late retinal
progenitor cells show intrinsic limitations in the production of
cell types and the kinetics of opsin synthesis. J. Neurosci. 20 (6),
2247–2254.
Bernier, G., Panitz, F., Zhou, X., Hollemann, T., Gruss, P., Pieler, T.,
2000. Expanded retina territory by midbrain transformation upon
overexpression of Six6 (Optx2) in Xenopus embryos. Mech. Dev. 93
(1–2), 59–69.
Berson, E.L., Jakobiec, F.A., 1999. Neural retinal cell transplantation:
ideal versus reality. Ophthalmology 106 (3), 445–446.
Bhattacharya, S., Jackson, J.D., Das, A.V., Thoreson, W.B.,
Kuszynski, C., James, J., Joshi, S., Ahmad, I., 2003. Direct
identification and enrichment of retinal stem cells/progenitors by
Hoechst dye efflux assay. Invest. Ophthalmol. Vis. Sci. 44 (6),
2764–2773.
Bjorklund, A., Stenevi, U., 1977. Reformation of the severed
septohippocampal cholinergic pathway in the adult rat by
transplanted septal neurons. Cell Tissue Res. 185 (3), 289–302.
Bjorklund, A., Stenevi, U., 1979. Reconstruction of the nigrostriatal
dopamine pathway by intracerebral nigral transplants. Brain Res.
177 (3), 555–560.
Bjorklund, A., Dunnett, S.B., Brundin, P., Stoessl, A.J., Freed, C.R.,
Breeze, R.E., Levivier, M., Peschanski, M., Studer, L., Barker, R.,
2003. Neural transplantation for the treatment of Parkinson’s
disease. Lancet Neurol. 2 (7), 437–445.
Bjornson, C.R., Rietze, R.L., Reynolds, B.A., Magli, M.C., Vescovi,
A.L., 1999. Turning brain into blood: a hematopoietic fate
adopted by adult neural stem cells in vivo. Science 283 (5401),
534–537.
Bonini, N.M., Bui, Q.T., Gray-Board, G.L., Warrick, J.M., 1997. The
Drosophila eyes absent gene directs ectopic eye formation in a
pathway conserved between flies and vertebrates. Development 124
(23), 4819–4826.
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Boyl, P.P., Signore, M., Annino, A., Barbera, J.P., Acampora, D.,
Simeone, A., 2001. Otx genes in the development and evolution of
the vertebrate brain. Int. J. Dev. Neurosci. 19 (4), 353–363.
Brody, T., Odenwald, W.F., 2000. Programmed transformations in
neuroblast gene expression during Drosophila CNS lineage
development. Dev. Biol. 226 (1), 34–44.
Brown, N.L., Paddock, S.W., Sattler, C.A., Cronmiller, C., Thomas,
B.J., Carroll, S.B., 1996. Daughterless is required for Drosophila
photoreceptor cell determination, eye morphogenesis, and cell cycle
progression. Dev. Biol. 179 (1), 65–78.
Brown, N.L., Patel, S., Brzezinski, J., Glaser, T., 2001. Math5 is
required for retinal ganglion cell and optic nerve formation.
Development 128 (13), 2497–2508.
Brustle, O., Choudhary, K., Karram, K., Huttner, A., Murray, K.,
Dubois-Dalcq, M., McKay, R.D., 1998. Chimeric brains generated
by intraventricular transplantation of fetal human brain cells into
embryonic rats. Nat. Biotechnol. 16 (11), 1040–1044.
Bugra, K., Oliver, L., Jacquemin, E., Laurent, M., Courtois, Y., Hicks,
D., 1993. Acidic fibroblast growth factor is expressed abundantly
by photoreceptors within the developing and mature rat retina.
Eur. J. Neurosci. 5 (12), 1586–1595 (published erratum appears in
Eur. J. Neurosci. 1994 Jun 1;6(6):1062).
Cai, J., Wu, Y., Mirua, T., Pierce, J.L., Lucero, M.T., Albertine, K.H.,
Spangrude, G.J., Rao, M.S., 2002. Properties of a fetal multipotent
neural stem cell (NEP cell). Dev. Biol. 251 (2), 221–240.
Calof, A.L., Mumm, J.S., Rim, P.C., Shou, J., 1998. The neuronal
stem cell of the olfactory epithelium. J. Neurobiol. 36 (2), 190–205.
Castro, R.F., Jackson, K.A., Goodell, M.A., Robertson, C.S., Liu, H.,
Shine, H.D., 2002. Failure of bone marrow cells to transdifferentiate into neural cells in vivo. Science 297 (5585), 1299.
Cepko, C.L., Austin, C.P., Yang, X., Alexiades, M., Ezzeddine, D.,
1996. Cell fate determination in the vertebrate retina. Proc. Natl.
Acad. Sci. USA 93 (2), 589–595.
Check, E., 2003. Parkinson’s transplant therapy faces setback. Nature
424 (6952), 987.
Chen, R., Amoui, M., Zhang, Z., Mardon, G., 1997. Dachshund
and eyes absent proteins form a complex and function synergistically to induce ectopic eye development in Drosophila. Cell 91 (7),
893–903.
Chien, C.B., Harris, W.A., 1994. Axonal guidance from retina to
tectum in embryonic Xenopus. Curr. Top Dev. Biol. 29, 135–169.
Chuang, J.C., Raymond, P.A., 2002. Embryonic origin of the eyes in
teleost fish. Bioessays 24 (6), 519–529.
Clarke, D.L., Johansson, C.B., Wilbertz, J., Veress, B., Nilsson, E.,
Karlstrom, H., Lendahl, U., Frisen, J., 2000. Generalized potential
of adult neural stem cells. Science 288 (5471), 1660–1663.
Conley, C.A., Silburn, R., Singer, M.A., Ralston, A., Rohwer-Nutter,
D., Olson, D.J., Gelbart, W., Blair, S.S., 2000. Crossveinless 2
contains cysteine-rich domains and is required for high levels of
BMP-like activity during the formation of the cross veins in
Drosophila. Development 127 (18), 3947–3959.
Coulombre, J.L., Coulombre, A.J., 1965. Regeneration of neural
retina from the pigmented epithelium in the chick embryo. Dev.
Biol. 12, 79–92.
Das, T., Payer, B., Cayouette, M., Harris, W.A., 2003. In vivo timelapse imaging of cell divisions during neurogenesis in the
developing zebrafish retina. Neuron 37 (4), 597–609.
Davis, A.A., Matzuk, M.M., Reh, T.A., 2000. Activin A promotes
progenitor differentiation into photoreceptors in rodent retina.
Mol. Cell Neurosci. 15 (1), 11–21.
Doe, C.Q., Chu-LaGraff, Q., Wright, D.M., Scott, M.P., 1991. The
prospero gene specifies cell fates in the Drosophila central nervous
system. Cell 65 (3), 451–464.
Dyer, M.A., Livesey, F.J., Cepko, C.L., Oliver, G., 2003. Prox1
function controls progenitor cell proliferation and horizontal cell
genesis in the mammalian retina. Nat. Genet. 34 (1), 53–58.
177
Eggert, T., Hauck, B., Hildebrandt, N., Gehring, W.J., Walldorf, U.,
1998. Isolation of a Drosophila homolog of the vertebrate
homeobox gene Rx and its possible role in brain and eye
development. Proc. Natl. Acad. Sci. USA 95 (5), 2343–2348.
Eglitis, M.A., Mezey, E., 1997. Hematopoietic cells differentiate into
both microglia and macroglia in the brains of adult mice. Proc.
Natl. Acad. Sci. USA 94 (8), 4080–4085.
Eriksson, P.S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A.M.,
Nordborg, C., Peterson, D.A., Gage, F.H., 1998. Neurogenesis
in the adult human hippocampus. Nat. Med. 4 (11), 1313–1317
(see comments).
Escribano, J., Coca-Prados, M., 2002. Bioinformatics and reanalysis
of subtracted expressed sequence tags from the human ciliary
body: identification of novel biological functions. Mol. Vis. 8,
315–332.
Ezzeddine, Z.D., Yang, X., DeChiara, T., Yancopoulos, G., Cepko,
C.L., 1997. Postmitotic cells fated to become rod photoreceptors
can be respecified by CNTF treatment of the retina. Development
124 (5), 1055–1067.
Fallon, J., Reid, S., Kinyamu, R., Opole, I., Opole, R., Baratta, J.,
Korc, M., Endo, T.L., Duong, A., Nguyen, G., Karkehabadhi, M.,
Twardzik, D., Patel, S., Loughlin, S., 2000. In vivo induction of
massive proliferation, directed migration, and differentiation of
neural cells in the adult mammalian brain. Proc. Natl. Acad. Sci.
USA 97 (26), 14686–14691.
Fischer, A.J., Reh, T.A., 2001. Transdifferentiation of pigmented
epithelial cells: a source of retinal stem cells? Dev. Neurosci. 23
(4–5), 268–276.
Fuhrmann, S., Heller, S., Rohrer, H., Hofmann, H.D., 1998. A
transient role for ciliary neurotrophic factor in chick photoreceptor
development. J. Neurobiol. 37 (4), 672–683.
Furukawa, T., Mukherjee, S., Bao, Z.Z., Morrow, E.M., Cepko, C.L.,
2000. Rax, Hes1, and notch1 promote the formation of Muller glia
by postnatal retinal progenitor cells. Neuron 26 (2), 383–394.
Gao, H., Hollyfield, J.G., 1992. Basic fibroblast growth factor (bFGF)
immunolocalization in the rodent outer retina demonstrated
with an anti-rodent bFGF antibody. Brain Res. 585 (1–2),
355–360.
Gehring, W.J., Ikeo, K., 1999. Pax 6: mastering eye morphogenesis
and eye evolution. Trends Genet. 15 (9), 371–377.
Gomez-Skarmeta, J.L., Modolell, J., 2002. Iroquois genes: genomic
organization and function in vertebrate neural development. Curr.
Opin. Genet. Dev. 12 (4), 403–408.
Guillemot, F., Cepko, C.L., 1992. Retinal fate and ganglion cell
differentiation are potentiated by acidic FGF in an in vitro assay of
early retinal development. Development 114 (3), 743–754.
Halder, G., Callaerts, P., Gehring, W.J., 1995. Induction of ectopic
eyes by targeted expression of the eyeless gene in Drosophila.
Science 267 (5205), 1788–1792.
Haruta, M., Kosaka, M., Kanegae, Y., Saito, I., Inoue, T., Kageyama,
R., Nishida, A., Honda, Y., Takahashi, M., 2001. Induction of
photoreceptor-specific phenotypes in adult mammalian iris tissue.
Nat. Neurosci. 4 (12), 1163–1164.
Hatakeyama, J., Tomita, K., Inoue, T., Kageyama, R., 2001. Roles of
homeobox and bHLH genes in specification of a retinal cell type.
Development 128 (8), 1313–1322.
Hayes, A.J., Dowthwaite, G.P., Webster, S.V., Archer, C.W., 2003.
The distribution of Notch receptors and their ligands during
articular cartilage development. J. Anat. 202 (6), 495–502.
Hicks, D., Courtois, Y., 1992. Fibroblast growth factor stimulates
photoreceptor differentiation in vitro. J. Neurosci. 12 (6),
2022–2033.
Hide, T., Hatakeyama, J., Kimura-Yoshida, C., Tian, E., Takeda, N.,
Ushio, Y., Shiroishi, T., Aizawa, S., Matsuo, I., 2002. Genetic
modifiers of otocephalic phenotypes in Otx2 heterozygous mutant
mice. Development 129 (18), 4347–4357.
ARTICLE IN PRESS
178
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Hinds, J.W., Hinds, P.L., 1974. Early ganglion cell differentiation in
the mouse retina: an electron microscope analysis utilizing serial
sections. Dev. Biol. 37, 381–416.
Holt, C.E., Bertsh, T.W., Ellis, H.M., Harris, W.A., 1988. Cellular
determination in the Xenopus retina is independent of lineage and
birthdate. Neuron 1, 15–26.
Hori, J., Ng, T.F., Shatos, M., Klassen, H., Streilein, J.W., Young,
M.J., 2003. Neural progenitor cells lack immunogenicity and resist
destruction as allografts. Stem Cells 21 (4), 405–416.
Hu, M., Easter, S.S., 1999. Retinal neurogenesis: the formation of
the initial central patch of postmitotic cells. Dev. Biol. 207 (2),
309–321.
Hunter, D.D., Murphy, M.D., Olsson, C.V., Brunken, W.J., 1992.
S-laminin expression in adult and developing retinae: a potential
cue for photoreceptor morphogenesis. Neuron 8 (3), 399–413.
Hyatt, G.A., Schmitt, E.A., Fadool, J.M., Dowling, J.E., 1996.
Retinoic acid alters photoreceptor development in vivo. Proc. Natl.
Acad. Sci. USA 93 (23), 13298–13303.
Inoue, T., Hojo, M., Bessho, Y., Tano, Y., Lee, J.E., Kageyama, R.,
2002. Math3 and NeuroD regulate amacrine cell fate specification
in the retina. Development 129 (4), 831–842.
Ishigooka, H., Kitaoka, T., Boutilier, S.B., Bost, L.M., Aotaki-Keen,
A.E., Tablin, F., Hjelmeland, L.M., 1993. Developmental expression of bFGF in the bovine retina. Invest. Ophthalmol. Vis. Sci. 34
(9), 2813–2823.
Iso, T., Kedes, L., Hamamori, Y., 2003. HES and HERP families:
multiple effectors of the notch signaling pathway. J. Cell Physiol.
194 (3), 237–255.
Isshiki, T., Pearson, B., Holbrook, S., Doe, C.Q., 2001. Drosophila
neuroblasts sequentially express transcription factors which
specify the temporal identity of their neuronal progeny. Cell 106
(4), 511–521.
James, J., Das, A.V., Bhattacharya, S., Chacko, D.M., Zhao, X.,
Ahmad, I., 2003. In vitro generation of early-born neurons from
late retinal progenitors. J. Neurosci. 23 (23), 8193–8203.
Jarman, A.P., Grau, Y., Jan, L.Y., Jan, Y.N., 1993. Atonal
is a proneural gene that directs chordotonal organ formation
in the Drosophila peripheral nervous system. Cell 73 (7),
1307–1321.
Ji, Q., Luo, Z.X., Yuan, C.X., Wible, J.R., Zhang, J.P., Georgi, J.A.,
2002. The earliest known eutherian mammal. Nature 416 (6883),
816–822.
Kageyama, R., Ishibashi, M., Takebayashi, K., Tomita, K., 1997.
BHLH transcription factors and mammalian neuronal differentiation. Int. J. Biochem. Cell Biol. 29 (12), 1389–1399.
Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S.J.,
Odenwald, W.F., 1998. Regulation of POU genes by castor and
hunchback establishes layered compartments in the Drosophila
CNS. Genes Dev. 12 (2), 246–260.
Kanekar, S., Perron, M., Dorsky, R., Harris, W.A., Jan, L.Y., Jan,
Y.N., Vetter, M.L., 1997. Xath5 participates in a network
of bHLH genes in the developing Xenopus retina. Neuron 19 (5),
981–994.
Kaneko, Y., Saito, T., 1992. Appearance and maturation of voltagedependent conductances in solitary spiking cells during retinal
regeneration in the adult newt. J. Comp. Physiol. A 170 (4),
411–425.
Kango-Singh, M., Singh, A., Henry Sun, Y., 2003. Eyeless collaborates
with Hedgehog and Decapentaplegic signaling in Drosophila eye
induction. Dev. Biol. 256 (1), 49–60.
Kaplan, H.J., Tezel, T.H., Berger, A.S., Wolf, M.L., Del Priore, L.V.,
1997. Human photoreceptor transplantation in retinitis pigmentosa. A safety study. Arch. Ophthalmol. 115 (9), 1168–1172.
Kelley, M.W., Turner, J.K., Reh, T.A., 1994. Retinoic acid promotes
differentiation of photoreceptors in vitro. Development 120 (8),
2091–2102.
Kelley, M.W., Turner, J.K., Reh, T.A., 1995. Regulation of proliferation and photoreceptor differentiation in fetal human retinal cell
cultures. Invest. Ophthalmol. Vis. Sci. 36 (7), 1280–1289.
Kelley, M.W., Williams, R.C., Turner, J.K., Creech-Kraft, J.M., Reh,
T.A., 1999. Retinoic acid promotes rod photoreceptor differentiation in rat retina in vivo. Neuroreport 10 (11), 2389–2394.
Klassen, H., Lund, R.D., 1987. Retinal transplants can drive a
pupillary reflex in host rat brains. Proc. Natl. Acad. Sci. USA 84
(19), 6958–6960.
Klassen, H., Lund, R.D., 1990. Parameters of retinal graft-mediated
responses are related to underlying target innervation. Brain Res.
533 (2), 181–191.
Klassen, H., Schwartz, M.R., Bailey, A.H., Young, M.J., 2001. Surface
markers expressed by multipotent human and mouse neural
progenitor cells include tetraspanins and non-protein epitopes.
Neurosci Lett. 312 (3), 180–182.
Klassen, H.J., Imfeld, K.L., Kirov I.I., Tai, L., Gage, F.H., Young,
M.J., Berman, M.A., 2003a. Expression of cytokines by multipotent neural progenitor cells. Cytokine 22(3–4), 101–106.
Klassen, H., Imfeld, K.L., Ray, J., Young, M.J., Gage, F.H., Berman,
M.A., 2003b. The immunological properties of adult hippocampal
progenitor cells. Vision Res. 43 (8), 947–956.
Klein, L.R., MacLeish, P.R., Wiesel, T.N., 1990. Immunolabelling by
a newt retinal pigment epithelium antibody during retinal
development and regeneration. J. Comp. Neurol. 293 (3), 331–339.
Kobayashi, D., Kobayashi, M., Matsumoto, K., Ogura, T., Nakafuku,
M., Shimamura, K., 2002. Early subdivisions in the neural plate
define distinct competence for inductive signals. Development 129
(1), 83–93.
Ksander, B.R., Streilein, J.W., 1994. Regulation of the immune
response within privileged sites. Chem. Immunol. 58, 117–145.
Kuehl-Kovarik, M.C., Sakaguchi, D.S., Iqbal, J., Sonea, I., Jacobson,
C.D., 1995. The gray short-tailed opossum: a novel model for
mammalian development. Lab. Animal. 24, 24–29.
Kumar, J.P., Moses, K., 2001. EGF receptor and Notch signaling act
upstream of Eyeless/Pax6 to control eye specification. Cell 104 (5),
687–697.
Lawson, K.A., Dunn, N.R., Roelen, B.A., Zeinstra, L.M., Davis,
A.M., Wright, C.V., Korving, J.P., Hogan, B.L., 1999. Bmp4 is
required for the generation of primordial germ cells in the mouse
embryo. Genes Dev. 13 (4), 424–436.
Lendahl, U., Zimmerman, L.B., McKay, R.D., 1990. CNS stem cells
express a new class of intermediate filament protein. Cell 60 (4),
585–595.
Levine, E.M., Roelink, H., Turner, J., Reh, T.A., 1997. Sonic
hedgehog promotes rod photoreceptor differentiation in mammalian retinal cells in vitro. J. Neurosci. 17 (16), 6277–6288.
Li, H., Tierney, C., Wen, L., Wu, J.Y., Rao, Y., 1997. A single
morphogenetic field gives rise to two retina primordia under the
influence of the prechordal plate. Development 124 (3), 603–615.
Libby, R.T., Hunter, D.D., Brunken, W.J., 1996. Developmental
expression of laminin beta 2 in rat retina. Further support for a
role in rod morphogenesis. Invest. Ophthalmol. Vis. Sci. 37 (8),
1651–1661.
Lillien, L., Cepko, C., 1992. Control of proliferation in the retina:
temporal changes in responsiveness to FGF and TGF alpha.
Development 115 (1), 253–266.
Little, C.W., Cox, C., Wyatt, J., del Cerro, C., del Cerro, M., 1998.
Correlates of photoreceptor rescue by transplantation of human
fetal RPE in the RCS rat. Exp. Neurol. 149 (1), 151–160.
Liu, W., Mo, Z., Xiang, M., 2001. The Ath5 proneural genes function
upstream of Brn3 POU domain transcription factor genes to
promote retinal ganglion cell development. Proc. Natl. Acad. Sci.
USA 98 (4), 1649–1654.
Lo, L., Dormand, E., Greenwood, A., Anderson, D.J., 2002.
Comparison of the generic neuronal differentiation and neuron
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
subtype specification functions of mammalian achaete–scute and
atonal homologs in cultured neural progenitor cells. Development
129 (7), 1553–1567.
Lopez, R., Gouras, P., Kjeldbye, H., Sullivan, B., Reppucci, V., Brittis,
M., Wapner, F., Goluboff, E., 1989. Transplanted retinal pigment
epithelium modifies the retinal degeneration in the RCS rat. Invest.
Ophthalmol. Vis. Sci. 30 (3), 586–588.
Lund, R., Klassen, H., Young, M., 2000. Retinal transplantation and
the pupillary light reflex. In: Birnstock, G., Sillito, A. (Eds.),
Nervous Control of the Eye. Harwood Academic Publishers,
Amsterdam.
Ma, Q., Fode, C., Guillemot, F., Anderson, D.J., 1999. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev. 13 (13),
1717–1728.
Mann, I., 1950. The development of the human eye. Grune & Stratton,
New York.
Marc, R.E., 1986. Neurochemical stratification in the inner plexiform
layer of the vertebrate retina. Vision Res. 26 (2), 223–238.
Marquardt, T., Gruss, P., 2002. Generating neuronal diversity in the
retina: one for nearly all. Trends Neurosci. 25 (1), 32–38.
Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R.,
Guillemot, F., Gruss, P., 2001. Pax6 is required for the multipotent
state of retinal progenitor cells. Cell 105 (1), 43–55.
Martinez-Barbera, J.P., Beddington, R.S., 2001. Getting your head
around Hex and Hesx1: forebrain formation in mouse. Int. J. Dev.
Biol. 45(1 Spec. No.), 327–336.
Masland, R.H., 2001. The fundamental plan of the retina. Nat.
Neurosci. 4 (9), 877–886.
Mathers, P.H., Grinberg, A., Mahon, K.A., Jamrich, M., 1997. The
Rx homeobox gene is essential for vertebrate eye development.
Nature 387 (6633), 603–607.
McDonald, J.W., Liu, X.Z., Qu, Y., Liu, S., Mickey, S.K., Turetsky,
D., Gottlieb, D.I., Choi, D.W., 1999. Transplanted embryonic stem
cells survive, differentiate and promote recovery in injured rat
spinal cord. Nat. Med. 5 (12), 1410–1412.
McFarlane, S., McNeill, L., Holt, C.E., 1995. FGF signaling and
target recognition in the developing Xenopus visual system. Neuron
15 (5), 1017–1028.
McLoon, L.K., Lund, R.D., McLoon, S.C., 1982. Transplantation of
reaggregates of embryonic neural retinae to neonatal rat brain:
differentiation and formation of connections. J. Comp. Neurol. 205
(2), 179–189.
Medawar, P., 1948. Immunity to homologous grafted skin. III. The
fate of skin homografts transplanted to the brain, to subcutaneous
tissue, and to the anterior chamber of the eye. Br. J. Exp. Pathol.
29, 58–69.
Mellerick, D.M., Modica, V., 2002. Regulated vnd expression is
required for both neural and glial specification in Drosophila. J.
Neurobiol. 50 (2), 118–136.
Merzenich, M.M., Kaas, J.H., Wall, J., Nelson, R.J., Sur, M.,
Felleman, D., 1983. Topographic reorganization of somatosensory
cortical areas 3b and 1 in adult monkeys following restricted
deafferentation. Neuroscience 8 (1), 33–55.
Merzenich, M.M., Nelson, R.J., Kaas, J.H., Stryker, M.P., Jenkins,
W.M., Zook, J.M., Cynader, M.S., Schoppmann, A., 1987.
Variability in hand surface representations in areas 3b and 1 in
adult owl and squirrel monkeys. J. Comp. Neurol. 258 (2),
281–296.
Mey, J., Thanos, S., 2000. Development of the visual system of the
chick. I. Cell differentiation and histogenesis. Brain Res. Rev. 32
(2–3), 343–379.
Mizumoto, H., Mizumoto, K., Whiteley, S.J., Shatos, M., Klassen, H.,
Young, M.J., 2001. Transplantation of human neural progenitor
cells to the vitreous cavity of the Royal College of Surgeons rat.
Cell Transplant. 10 (2), 223–233.
179
Modolell, J., 1997. Patterning of the adult peripheral nervous system
of Drosophila. Perspect. Dev. Neurobiol. 4 (4), 285–296.
Morrow, E.M., Furukawa, T., Lee, J.E., Cepko, C.L., 1999. NeuroD
regulates multiple functions in the developing neural retina in
rodent. Development 126 (1), 23–36.
Niederkorn, J., 1990. Immune privilege and immune regulation in the
eye. Adv. Immunol. 48, 191–226.
Ohtsuka, T., Ishibashi, M., Gradwohl, G., Nakanishi, S., Guillemot,
F., Kageyama, R., 1999. Hes1 and Hes5 as notch effectors in
mammalian neuronal differentiation. EMBO J. 18 (8), 2196–2207.
Ohtsuka, T., Sakamoto, M., Guillemot, F., Kageyama, R., 2001. Roles
of the basic helix–loop–helix genes Hes1 and Hes5 in expansion of
neural stem cells of the developing brain. J. Biol. Chem. 276 (32),
30467–30474.
Okada, T.S., 1980. Cellular metaplasia or transdifferentiation as a model
for retinal cell differentiation. Curr. Topics Dev. Biol. 16, 349–380.
Okochi, M., Steiner, H., Fukumori, A., Tanii, H., Tomita, T., Tanaka,
T., Iwatsubo, T., Kudo, T., Takeda, M., Haass, C., 2002.
Presenilins mediate a dual intramembranous gamma-secretase
cleavage of Notch-1. EMBO J. 21 (20), 5408–5416.
Opas, M., Dziak, E., 1994. BFGF-induced transdifferentiation of RPE
to neuronal progenitors is regulated by the mechanical properties
of the substratum. Dev. Biol. 161 (2), 440–454.
Otani, A., Kinder, K., Ewalt, K., Otero, F.J., Schimmel, P.,
Friedlander, M., 2002. Bone marrow-derived stem cells target
retinal astrocytes and can promote or inhibit retinal angiogenesis.
Nat. Med. 8 (9), 1004–1010.
Otteson, D.C., Hitchcock, P.F., 2003. Stem cells in the teleost retina:
persistent neurogenesis and injury-induced regeneration. Vision
Res. 43 (8), 927–936.
Palmer, T.D., Schwartz, P.H., Taupin, P., Kaspar, B., Stein, S.A.,
Gage, F.H., 2001. Cell culture. Progenitor cells from human brain
after death. Nature 411 (6833), 42–43.
Pan, D., Rubin, G.M., 1998. Targeted expression of teashirt induces
ectopic eyes in Drosophila. Proc. Natl. Acad. Sci. USA 95 (26),
15508–15512.
Park, C.M., Hollenberg, M.J., 1989. Basic fibroblast growth factor
induces retinal regeneration in vivo. Dev. Biol. 134 (1), 201–205.
Park, C.M., Hollenberg, M.J., 1993. Growth factor-induced retinal
regeneration in vivo. Int. Rev. Cytol. 146, 49–74.
Park, K.I., 2000. Transplantation of neural stem cells: cellular & gene
therapy for hypoxic-ischemic brain injury. Yonsei Med. J. 41 (6),
825–835.
Park, K.I., Teng, Y.D., Snyder, E.Y., 2002. The injured brain interacts
reciprocally with neural stem cells supported by scaffolds to
reconstitute lost tissue. Nat. Biotechnol. 20 (11), 1111–1117.
Patel, N.H., Schafer, B., Goodman, C.S., Holmgren, R., 1989. The
role of segment polarity genes during Drosophila neurogenesis.
Genes Dev. 3 (6), 890–904.
Perron, M., Opdecamp, K., Butler, K., Harris, W.A., Bellefroid, E.J.,
1999. X-ngnr-1 and Xath3 promote ectopic expression of sensory
neuron markers in the neurula ectoderm and have distinct inducing
properties in the retina. Proc. Natl. Acad. Sci. USA 96 (26),
14996–15001.
Piccini, P., 2002. Dyskinesias after transplantation in Parkinson’s
disease. Lancet Neurol. 1 (8), 472.
Pignoni, F., Hu, B., Zavitz, K.H., Xiao, J., Garrity, P.A., Zipursky,
S.L., 1997. The eye-specification proteins So and Eya form a
complex and regulate multiple steps in Drosophila eye development. Cell 91 (7), 881–891.
Pittack, C., Jones, M., Reh, T.A., 1991. Basic fibroblast growth factor
induces retinal pigment epithelium to generate neural retina in vitro.
Development 113 (2), 577–588.
Prada, C., Puga, J., Perez-Mendez, L., Lopez, R., Ramirez, G., 1991.
Spatial and temporal patterns of neurogenesis in the chick retina.
Eur. J. Neurosci. 3 (6), 559–569.
ARTICLE IN PRESS
180
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Rager, G.H., 1980. Development of the retinotectal projection in the
chicken. Adv. Anat. Embryol. Cell Biol. 63, 1–90.
Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C.,
Melton, D.A., 2002. Stemness: transcriptional profiling of embryonic and adult stem cells. Science 298 (5593), 597–600.
Rapaport, D.H., Dorsky, R.I., 1998. Inductive competence, its
significance in retinal cell fate determination and a role for DeltaNotch signaling. Semin. Cell Dev. Biol. 9 (3), 241–247.
Raymond, P.A., 1991. Retinal regeneration in teleost fish. Ciba Found.
Symp. 160, 171–186 (discussion, pp. 186–191).
Reich, A., Shilo, B.Z., 2002. Keren, a new ligand of the Drosophila
epidermal growth factor receptor, undergoes two modes of
cleavage. EMBO J. 21 (16), 4287–4296.
Reyer, R.W., 1977. The amphibian eye: development and regeneration.
In: Criscitelli, F. (Ed.), Handbook of Sensory Physiology, Vol. VII/
5. The Visual System of Vertebrates. Springer, Berlin, Heidelberg,
pp. 309–390.
Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes
from isolated cells of the adult mammalian central nervous system.
Science 255 (5052), 1707–1710.
Richards, L.J., Kilpatrick, T.J., Bartlett, P.F., 1992. De novo
generation of neuronal cells from the adult mouse brain. Proc.
Natl. Acad. Sci. USA 89 (18), 8591–8595.
Rosser, A.E., Dunnett, S.B., 2003. Neural transplantation in patients
with Huntington’s disease. CNS Drugs 17 (12), 853–867.
Rothermel, A., Layer, P.G., 2003. GDNF regulates chicken rod
photoreceptor development and survival in reaggregated histotypic
retinal spheres. Invest. Ophthalmol. Vis. Sci. 44 (5), 2221–2228.
Ryter, J.M., Doe, C.Q., Matthews, B.W., 2002. Structure of the DNA
binding region of prospero reveals a novel homeo-prospero
domain. Structure (Camb) 10 (11), 1541–1549.
Sah, D.W., Ray, J., Gage, F.H., 1997. Bipotent progenitor cell lines
from the human CNS. Nat. Biotechnol. 15 (6), 574–580.
Sakaguchi, D.S., 1989. The development of retinal ganglion cells
deprived of their targets. Dev. Biol. 134 (1), 103–111.
Sakaguchi, D.S., Janick, L.M., Reh, T.A., 1997. Basic fibroblast
growth factor (FGF-2) induced transdifferentiation of retinal
pigment epithelium: generation of retinal neurons and glia. Dev.
Dyn. 209 (4), 387–398.
Sakaguchi, D.S., Van Hoffelen, S.J., Young, M.J., 2003. Differentiation and morphological integration of neural progenitor cells
transplanted into the developing mammalian eye. Ann. N. Y.
Acad. Sci. 995, 127–139.
Sasaki, H., Coffey, P., Villegas-Perez, M.P., Vidal-Sanz, M., Young,
M.J., Lund, R.D., Fukuda, Y., 1996. Light induced EEG
desynchronization and behavioral arousal in rats with restored
retinocollicular projection by peripheral nerve graft. Neurosci.
Lett. 218 (1), 45–48.
Scharenberg, C.W., Harkey, M.A., Torok-Storb, B., 2002. The
ABCG2 transporter is an efficient Hoechst 33342 efflux pump
and is preferentially expressed by immature human hematopoietic
progenitors. Blood 99 (2), 507–512.
Schroder, N., Gossler, A., 2002. Expression of Notch pathway
components in fetal and adult mouse small intestine. Gene Exp.
Patterns 2 (3–4), 247–250.
Schuurmans, C., Guillemot, F., 2002. Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr.
Opin. Neurobiol. 12 (1), 26–34.
Schwab, M.E., Caroni, P., 1988. Oligodendrocytes and CNS myelin
are nonpermissive substrates for neurite growth and fibroblast
spreading in vitro. J. Neurosci. 8 (7), 2381–2393.
Seo, H.C., Drivenes, O., Ellingsen, S., Fjose, A., 1998. Expression of
two zebrafish homologues of the murine Six3 gene demarcates the
initial eye primordia. Mech. Dev. 73 (1), 45–57.
Shatos, M.A., Mizumoto, K., Mizumoto, H., Kurimoto, Y., Klassen,
H., Young, M.J., 2001. Multipotent stem cells from the brain
and retina of green mice e-biomed. J. Regenerative Med. 2,
13–15.
Sheedlo, H.J., Li, L.X., Turner, J.E., 1989. Functional and structural
characteristics of photoreceptor cells rescued in RPE-cell grafted
retinas of RCS dystrophic rats. Exp. Eye Res. 48 (6), 841–854.
Shen, W., Mardon, G., 1997. Ectopic eye development in Drosophila
induced by directed dachshund expression. Development 124 (1),
45–52.
Sidman, R.L., 1961. Histogenesis of mouse retina studied with
thymidine-3H. In: Smelser, G. (Ed.), The Structure of the Eye.
Academic Press, New York.
Silverman, M.S., Hughes, S.E., 1989a. Photoreceptor transplantation
in inherited and environmentally induced retinal degeneration:
anatomy, immunohistochemistry and function. Prog. Clin. Biol.
Res. 314 (687), 687–704.
Silverman, M.S., Hughes, S.E., 1989b. Transplantation of photoreceptors to light-damaged retina. Invest. Ophthalmol. Vis. Sci. 30
(8), 1684–1690.
Skeath, J.B., 1998. The Drosophila EGF receptor controls the
formation and specification of neuroblasts along the dorsal-ventral
axis of the Drosophila embryo. Development 125 (17), 3301–3312.
Skeath, J.B., Carroll, S.B., 1994. The achaete–scute complex: generation of cellular pattern and fate within the Drosophila nervous
system. FASEB J. 8 (10), 714–721.
Spence, S.G., Robson, J.A., 1989. An autoradiographic analysis of
neurogenesis in the chick retina in vitro and in vivo. Neuroscience
32 (3), 801–812.
Stenkamp, D.L., Gregory, J.K., Adler, R., 1993. Retinoid effects in
purified cultures of chick embryo retina neurons and photoreceptors. Invest. Ophthalmol. Vis. Sci. 34 (8), 2425–2436.
Stone, L.S., 1950. Neural retina degeneration followed by regeneration
from surviving retinal pigment cells in grafted adult salamander
eyes. Anat. Rec. 106, 89–109.
Streilein, J.W., 1995. Immunological non-responsiveness and acquisition of tolerance in relation to immune privilege in the eye. Eye 9
(Pt 2), 236–240.
Streilein, J.W., 1996. Ocular immune privilege and the Faustian
dilemma. The Proctor lecture. Invest. Ophthalmol. Vis. Sci. 37 (10),
1940–1950.
Suhonen, J.O., Peterson, D.A., Ray, J., Gage, F.H., 1996. Differentiation of adult hippocampus-derived progenitors into olfactory
neurons in vivo. Nature 383 (6601), 624–627.
Suzuki, T., Ooto, S., Akagi, T., Amemiya, K., Igarashi, R.,
Mizushima, Y., Takahashi, M., 2003. Effects of prolonged delivery
of brain-derived neurotrophic factor on the fate of neural stem cells
transplanted into the developing rat retina. Biochem. Biophys. Res.
Commun. 309 (4), 843–847.
Svendsen, C.N., Caldwell, M.A., Shen, J., ter Borg, M.G., Rosser,
A.E., Tyers, P., Karmiol, S., Dunnett, S.B., 1997. Long-term
survival of human central nervous system progenitor cells
transplanted into a rat model of Parkinson’s disease. Exp. Neurol.
148 (1), 135–146.
Takahashi, M., Palmer, T.D., Takahashi, J., Gage, F.H., 1998.
Widespread integration and survival of adult-derived neural
progenitor cells in the developing optic retina. Mol. Cell Neurosci.
12, 340–348.
Toy, J., Yang, J.M., Leppert, G.S., Sundin, O.H., 1998. The optx2
homeobox gene is expressed in early precursors of the eye and
activates retina-specific genes. Proc. Natl. Acad. Sci. USA 95 (18),
10643–10648.
Tropepe, V., Coles, B.L., Chiasson, B.J., Horsford, D.J., Elia, A.J.,
McInnes, R.R., van der Kooy, D., 2000. Retinal stem cells in the
adult mammalian eye. Science 287 (5460), 2032–2036.
Tsai, R.Y., McKay, R.D., 2002. A nucleolar mechanism controlling
cell proliferation in stem cells and cancer cells. Genes Dev. 16 (23),
2991–3003.
ARTICLE IN PRESS
H. Klassen et al. / Progress in Retinal and Eye Research 23 (2004) 149–181
Tsunematsu, Y., Coulombre, A.J., 1981. Demonstration of transdifferentiation of neural retina from pigmented retina in cultures.
Dev. Growth Differ. 23, 297–311.
Turner, D.L., Cepko, C.L., 1987. A common progenitor for neurons
and glia persists in rat retina late in development. Nature 328
(6126), 131–136.
Uchida, N., Buck, D.W., He, D., Reitsma, M.J., Masek, M., Phan,
T.V., Tsukamoto, A.S., Gage, F.H., Weissman, I.L., 2000. Direct
isolation of human central nervous system stem cells. Proc. Natl.
Acad. Sci. USA 97 (26), 14720–14725.
Van Hoffelen, S.J., Young, M.J., Shatos, M.A., Sakaguchi, D.S., 2003.
Incorporation of murine brain progenitor cells into the developing
mammalian retina. Invest. Ophthalmol. Vis. Sci. 44 (1), 426–434.
VandeBerg, J.L., Robinson, E.S., 1997. The laboratory opossum
(Monodelphis domestica) in laboratory research. Ilar. J. 38 (1),
4–12.
Viczian, A.S., Vignali, R., Zuber, M.E., Barsacchi, G., Harris, W.A.,
2003. XOtx5b and XOtx2 regulate photoreceptor and bipolar fates
in the Xenopus retina. Development 130 (7), 1281–1294.
Vogel-Hopker, A., Momose, T., Rohrer, H., Yasuda, K., Ishihara, L.,
Rapaport, D.H., 2000. Multiple functions of fibroblast growth
factor-8 (FGF-8) in chick eye development. Mech. Dev. 94 (1–2),
25–36.
Wallis, D., Muenke, M., 2000. Mutations in holoprosencephaly. Hum.
Mutat. 16 (2), 99–108.
Wang, S.W., Kim, B.S., Ding, K., Wang, H., Sun, D., Johnson, R.L.,
Klein, W.H., Gan, L., 2001. Requirement for math5 in the
development of retinal ganglion cells. Genes Dev. 15 (1), 24–29.
Wassarman, K.M., Lewandoski, M., Campbell, K., Joyner, A.L.,
Rubenstein, J.L., Martinez, S., Martin, G.R., 1997. Specification of
the anterior hindbrain and establishment of a normal mid/
hindbrain organizer is dependent on Gbx2 gene function. Development 124 (15), 2923–2934.
Wehner, T., Bontert, M., Eyupoglu, I., Prass, K., Prinz, M., Klett,
F.F., Heinze, M., Bechmann, I., Nitsch, R., Kirchhoff, F.,
Kettenmann, H., Dirnagl, U., Priller, J., 2003. Bone marrowderived cells expressing green fluorescent protein under the control
of the glial fibrillary acidic protein promoter do not differentiate
into astrocytes in vitro and in vivo. J. Neurosci. 23 (12), 5004–5011.
Weimann, J.M., Charlton, C.A., Brazelton, T.R., Hackman, R.C.,
Blau, H.M., 2003a. Contribution of transplanted bone marrow
cells to Purkinje neurons in human adult brains. Proc. Natl. Acad.
Sci. USA 100 (4), 2088–2093.
Weimann, J.M., Johansson, C.B., Trejo, A., Blau, H.M., 2003b. Stable
reprogrammed heterokaryons form spontaneously in Purkinje
neurons after bone marrow transplant. Nat. Cell Biol. 5 (11),
959–966.
Weisz, J.M., Humayun, M.S., De Juan Jr., E., Del Cerro, M., Sunness,
J.S., Dagnelie, G., Soylu, M., Rizzo, L., Nussenblatt, R.B., 1999.
181
Allogenic fetal retinal pigment epithelial cell transplant in a patient
with geographic atrophy. Retina 19 (6), 540–545.
Wenkel, H., Streilein, J.W., Young, M.J., 2000. Systemic immune
deviation in the brain that does not depend on the integrity of the
blood–brain barrier. J. Immunol. 164 (10), 5125–5131.
West Greenlee, M.H., Swanson, J.J., Simon, J.J., Elmquist, J.K.,
Jacobson, C.D., Sakaguchi, D.S., 1996. Postnatal development and
the differential expression of presynaptic terminal-associated
proteins in the developing retina of the Brazilian opossum,
Monodelphis domestica. Dev. Brain Res. 96, 159–172.
Wetts, R., Fraser, S.E., 1989. Multipotent precursors can give rise to
all major cell types of the frog retina. Science 239, 1142–1145.
Wojciechowski, A.B., Englund, U., Lundberg, C., Wictorin, K.,
Warfvinge, K., 2002. Subretinal transplantation of brain-derived
precursor cells to young RCS rats promotes photoreceptor cell
survival. Exp. Eye Res. 75 (1), 23–37.
Wu, L., Aster, J.C., Blacklow, S.C., Lake, R., Artavanis-Tsakonas, S.,
Griffin, J.D., 2000. MAML1, a human homologue of Drosophila
mastermind, is a transcriptional co-activator for NOTCH receptors. Nat. Genet. 26 (4), 484–489.
Wu, L., Sun, T., Kobayashi, K., Gao, P., Griffin, J.D., 2002.
Identification of a family of mastermind-like transcriptional
coactivators for mammalian notch receptors. Mol. Cell Biol. 22
(21), 7688–7700.
Yandava, B.D., Billinghurst, L.L., Snyder, E.Y., 1999. Global cell
replacement is feasible via neural stem cell transplantation:
evidence from the dysmyelinated shiverer mouse brain. Proc. Natl.
Acad. Sci. USA 96 (12), 7029–7034.
Yasuda, K., Eguchi, G., Okada, T.S., 1981. Age-dependent changes in
the capacity of transdifferentiation of retinal pigment cells as
revealed in clonal culture. Cell Diff. 10, 3–11.
Young, M.J., Rao, K., Lund, R.D., 1989. Integrity of the blood–brain
barrier in retinal xenografts is correlated with the immunological
status of the host. J. Comp. Neurol. 283 (1), 107–117.
Young, M.J., Ray, J., Whiteley, S.J., Klassen, H., Gage, F.H., 2000.
Neuronal differentiation and morphological integration of hippocampal progenitor cells transplanted to the retina of immature and
mature dystrophic rats. Mol. Cell Neurosci. 16 (3), 197–205.
Young, R.W., 1985. Cell differentiation in the retina of the mouse.
Anat. Rec. 212 (2), 199–205.
Zhang, S.S., Fu, X.Y., Barnstable, C.J., 2002. Molecular aspects
of vertebrate retinal development. Mol. Neurobiol. 26 (2–3),
137–152.
Zhao, S., Thornquist, S.C., Barnstable, C.J., 1995. In vitro transdifferentiation of embryonic rat retinal pigment epithelium to neural
retina. Brain Res. 677 (2), 300–310.
Zuber, M.E., Gestri, G., Viczian, A.S., Barsacchi, G., Harris, W.A.,
2003. Specification of the vertebrate eye by a network of eye field
transcription factors. Development 130 (21), 5155–5167.