Review
Cell- & Tissue-based Therapy
Transplantation of cultured
progenitor cells to the
mammalian retina
1. Introduction
Henry Klassen
2. Retinal engraftment of CNS
Singapore Eye Research Institute, 11 Third Hospital Ave, #05-00 SNEC Building, 168751 Singapore
progenitor cells
3. Retinal progenitor
Multipotent progenitor cells have now been isolated from the brain and
retina, expanded in culture, and transplanted to the central nervous system
(CNS). Work in rodent models has shown that progenitor cells derived from
the CNS readily engraft in the diseased retina of mature recipients, where
they develop morphologies appropriate to the local microenvironment and
express mature markers, including the photoreceptor protein rhodopsin.
There is also evidence for graft-associated rescue of host photoreceptors
and preservation of light sensitivity in the degenerating retina. Graft
survival does not necessarily require immune suppression, as CNS
progenitors can behave as an immunoprivileged cell type. The use of
biodegradable polymers results in an organised implant and further
improves graft survival. Efforts are underway at present to extend this work
to the pig, with initial results showing engraftment in both the neural
retina and retinal pigment epithelium (RPE).
cells: characterisation
and engraftment
4. Immunological properties of
CNS progenitor cells
5. Biodegradable polymers as a
scaffold for progenitor
cell transplantation
6. The pig model:
xenotransplantation of
murine retinal progenitor cells
to the pig retina
7. Expert opinion and conclusion
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Keywords: biodegradable polymers, immune privilege, neural stem cells, regenerative medicine,
retinal degeneration, tissue engineering
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Expert Opin. Biol. Ther. (2006) 6(5):xxx-xxx
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1.
Introduction
Numerous breakthroughs in the treatment of cataract and corneal disease have
greatly decreased the incidence of blindness from these causes. In contrast, advances
in the treatment of retinal and optic nerve disease have been more limited, with
these conditions now representing major causes of incurable visual loss in the
developed world. The barriers to effective treatment are many and include the
general lack of endogenous regeneration within the mammalian central nervous
system (CNS), the complex phenotypes of retinal cells, in particular photoreceptors,
and the requirement for an extremely precise retinal cytoarchitecture, particularly in
the macula. Although no restorative treatments for retinal cell loss exist at present,
stem cell transplantation and tissue engineering have emerged as especially
promising strategies. In addition, the concept of delaying retinal cell death through
the use of neuroprotective agents, although not strictly regenerative in the literal
sense, certainly has considerable merit. Interestingly, stem or progenitor cells may
represent useful platforms for delivery of neuroprotective cytokines.
The application of regenerative medicine to the retina therefore encompasses two
rather different strategies. On the one hand there is the preservation of vision in the
setting of progressive retinal deterioration. This would be achieved through the
rescue of host neurons, particularly photoreceptors or ganglion cells, depending on
the disease; examples include retinitis pigmentosa and glaucoma, respectively.
Clearly, preservation of neurons represents the simpler approach. The more
challenging goal is the restoration of vision in patients who have already suffered
extensive retinal cell loss. This will require the replacement of lost retinal cells, either
10.1517/14712598.6.5.xxx © 2006 Informa UK Ltd ISSN 1471-2598
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Transplantation of cultured progenitor cells to the mammalian retina
through the engraftment of new cells or implantation of a
bioelectronic prosthesis. Digital image technology is already
quite sophisticated; however, application to ‘chip’ implants is
frustrated by problems interfacing the electronics with the
diseased visual system. Success will probably require the
advent of bioengineering and biomaterials solutions not
available at present. Therefore, the author and colleagues have
directed attention to the use of cell transplantation for the
treatment of retinal cell loss, and it is this work that forms the
basis of this review.
Cells and tissues of many types survive transplantation to
the subretinal space, in part because this location exhibits
characteristics of an immunoprivileged site [1]. Both
photoreceptors [2] and retinal pigment epithelium (RPE)
cells [3,4] survive transplantation beneath the retina; however,
failure of donor photoreceptors to integrate with surviving
host circuitry and failure of donor RPE cells to adhere to
Bruch’s membrane have frustrated attempts to achieve
functional repair of the outer retina [5]. In the case of
photoreceptors, there is a fundamental problem that must be
overcome, namely the physical barrier to neurite outgrowth
posed by the outer limiting membrane (OLM), a structure
formed by retinal Mueller cells. In the setting of photoreceptor
degeneration, the OLM undergoes hypertrophy with
upregulation of CD44 and neurocan. Regenerating neurites
are largely unable to cross this barrier [6]. The phenomenon of
glial hypertrophy is certainly not unique to the retina. Glial
hypertrophy and scar formation are commonly seen in the
setting of CNS disease and have frequently been implicated in
the failure of endogenous regenerative mechanisms to bridge a
lesion [7], particularly after spinal cord injury [8].
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Retinal engraftment of CNS progenitor cells
In previous work, the author’s group have shown that
transplanted CNS progenitor cells are not impeded by the
hypertrophied OLM and cross this barrier in large
numbers [9,10]. The ability to migrate into the mature retina is
one remarkable characteristic of CNS progenitor cells that
recommends them as a subject for further investigation. In fact,
these cells do not simply migrate into the retina, but exhibit
widespread integration into the retinal cytoarchitecture, with
pronounced tropism for areas of injury or disease. This is
extremely important as undirected migration is a characteristic
of malignant processes and not of itself a desirable outcome. In
addition, progenitor cell migration is not a phenomenon limited
to the neural retina. Long-distance migration of brain progenitor
cells along degenerating fibre tracts has been seen in the injured
spinal cord of mice (Schnell et al., unpublished data).
CNS progenitor cells possess considerable phenotypic
plasticity, both in vitro and in vivo, as has been previously
demonstrated by a large number of laboratories. This is
exemplified in the retina by work showing that hippocampal
progenitors transplanted to the vitreous of neonatal rats
frequently integrate into the retina and develop morphologies
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appropriate to their layer of residence [11]. In the dystropic
Royal College of Surgeons (RCS) rat, the author’s group
showed that grafted hippocampal progenitors residing in the
outer nuclear layer developed rod photoreceptor-like
morphologies, and donor cells in the ganglion cell layer
extended neurites into the optic nerve [9]. Although
brain-derived progenitors can express neuronal markers,
expression of retina-specific markers by these cells was not seen
in the rat; however, recoverin was seen following transplantation to the very immature retina of the Brazilian opossum [12].
This apparent lineage restriction, however incomplete, may
relate to tissue of origin, as very different results have been
obtained working with progenitor cells from the retina.
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Retinal progenitor cells: characterisation
and engraftment
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Progenitor cells have been expanded from the immature
neural retina of mice using methods similar to those
developed for brain progenitors [10]. These cultured retinal
progenitor cells (RPCs) expressed a range of markers
consistent with their primitive neuroepithelial origin,
including the intermediate filament nestin, the transcription
factor Sox2, as well as other neurodevelopmental genes,
including Notch1, Hes1, Hes5, Prox1, Mash1, Numb and
NeuroD. Also expressed were the surface markers GD2
ganglioside, CD15, CD9 and CD81, while immunocytochemical analysis revealed distinct subpopulations positive for
the
neuronal
marker
β-III
tubulin
and
the
microtubule-associated protein doublecortin (DCX). DCX is
associated with migrating neuroblasts [13]. Cells staining for
this marker were morphologically distinct with small, round
cell bodies and long, thin processes.
In the same study it was shown that mouse RPCs differentiate into cells of photoreceptor morphology and express both
rhodopsin and recoverin following transplantation to the
subretinal space [10]. In addition, evidence of in vitro differentiation into bipolar cells following treatment with ciliary
neurotrophic factor has been obtained, as evidenced by
morphology and expression of protein kinase C-α [14].
Interestingly, no evidence of expression by RPCs of
oligodendrocyte markers has been seen. Whereas production
of oligodendrocytes is a cardinal feature of neural stem cells,
these opaque cells are not generated in the retina; hence, this
result is compatible with normal lineage restriction in this
location. Although various stem or progenitor cell types could
potentially generate photoreceptors, only progenitor cells
derived from the neural retina do this as part of normal
development. RPCs, therefore, should exhibit the greatest
propensity in this regard, thus providing the rationale for the
author’s interest in these particular cells.
In terms of integration into the retina, the author’s group
have seen grafted RPCs express photoreceptor-associated
genes and photoreceptor-like morphology in both the retinal
detachment and rd1 dystropic models [10]. Widespread
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Expert Opin. Biol. Ther. (2006) 6(5)
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integration of grafted RPCs in the retina of the rhodopsin
double knockout (rho-/-) mouse, a transgenic model of
photoreceptor dystrophy, has also been reported. In this
model, donor cells migrated extensively, took up residence
within the three cellular layers of the neural retina, and
assumed morphological configurations consistent with cell
types found in these layers, including horizontal cells, bipolar
cells, amacrine cells and retinal ganglion cells. In addition, a
few RPC-derived donor cells exhibited immunoreactivity for
the photoreceptor marker cone opsin, suggestive of cone
differentiation, although the morphology of these cells was
anomalous. In addition to cell replacement, there was evidence
of substantial preservation of host rod photoreceptors.
Behavioural testing of rho-/- mice suggested that animals
with RPC grafts were more light-sensitive than age-matched
dystropic littermates, the latter being either sham-operated or
treated with subretinal fibroblasts as controls. As the rods of
rho-/- animals are non-functional, rescue of these cells is by
itself insufficient to explain the behavioural results. In
addition, as there was little evidence of RPC-derived
photoreceptor differentiation in rho-/- hosts, behavioural
improvements were unlikely to owe their origin to a cell
replacement-based mechanism. A likely albeit more
convoluted possibility is that non-functional host rods,
rescued by the grafted RPCs, in turn rescued functional host
cones. Still another possibility is the preservation or facilitation of endogenous melanopsin-based signalling. Given the
difficulties associated with investigating the various alternative
mechanisms, it would be of interest to know whether RPC
transplantation leads to functional improvements in large
animals, where the results of electrophysiological and visual
testing are generally more accurate and meaningful.
The author’s group have begun development of a porcine
model of progenitor cell transplantation to the retina, as
discussed below. First, the important issues of stem cell
immunology and tissue engineering are considered,
specifically, the application of synthetic biomaterials to
stem cell transplantation.
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Transplantation in mammals has a long history as an experimental strategy, but a comparatively short history of success.
The key to achieving graft survival was an understanding of the
immune system and elucidation of the mechanisms underlying
immunological rejection. Rejection is the norm for grafts
between individuals of disparate genetic background. However,
this tendency towards rejection of allografts can be overcome in
a number of situations. One possibility is systemic immune
suppression. Alternatively, grafts can be placed in an immunoprivileged site. Another equally interesting possibility is that the
grafts themselves can exhibit immune privilege. These two
latter possibilities are of particular interest in the setting of
progenitor cell transplantation to the subretinal space.
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MHC expression by CNS progenitor cells
The importance of MHC molecules to transplant survival is
implied by their alternative designation as transplantation
antigens. In a series of studies, the author’s group have used
flow cytometry to evaluate the expression of MHC class I and
II molecules by CNS progenitor cells cultured from the brain
and retina of various mammalian species. Following is a
description of results in mice, rats and humans, and a
summary across species.
Progenitor cells from the newborn mouse brain did not
express detectible MHC class I or II antigens under baseline
culture conditions [17]. This was also the case for
retina-derived progenitor cells (Figure 1, top row). Rat
progenitors derived from the adult hippocampus consistently
expressed low levels of MHC class I (Figure 2), including both
heavy chain and β2 microglobulin, but did not express MHC
class II molecules [15]. Human forebrain-derived progenitors
expressed MHC class I at high levels (Figure 2), but did not
express detectible MHC class II [17,18]. Human RPCs
exhibited the same expression pattern as the brain-derived
cells, with strong expression of MHC class I, but no
expression of class II by flow cytometry [19].
From these data a number of trends emerge. The first is
that MHC class I expression is consistent for progenitors from
different individuals or strains within a given species. This was
the case for multiple examples of CNS progenitors from the
brain and retina of mice and humans. This trend is consistent
with the concept that class I expression levels are species-specific for CNS progenitor cells. Again, it would be very useful
to confirm this trend across additional mammalian species,
and a good place to start would be the rat, as brain progenitor
information has already been obtained [15] and rat RPCs have
been cultured by other laboratories [20,21].
A related trend is that levels of MHC class I expression differ
between species (Figure 2). Interestingly, the trend of increasing
expression of class I from mouse to rat to human parallels an
increase in phylogenetic complexity. In other words, mouse CNS
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Immunological properties of CNS
progenitor cells
4.
There is now compelling evidence that the subretinal space
of the rodent exhibits features of immune privilege [1]. This
does not mean that grafts to this site cannot be rejected;
however, it does mean that grafts to the subretinal space benefit
from a decreased likelihood of rejection. In addition to the
phenomenon of site-specific immune privilege, as it applies to
grafts beneath the retina, evidence that rodent CNS progenitor
cells themselves exhibit properties of cell-specific immune
privilege has been found. Rat hippocampal progenitor cells
were not recognised by human mononuclear cells in vitro [15]
and murine brain progenitor cells survived transplantation to
the allogeneic kidney capsule, a conventional, that is,
non-privileged, site [16]. The mechanisms underlying
cell-specific immune privilege as exhibited by murine CNS
progenitor cells are not entirely understood and may be quite
complex. One area that has been investigated is expression of
the major histocompatibility complex (MHC) antigens.
Expert Opin. Biol. Ther. (2006) 6(5)
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Transplantation of cultured progenitor cells to the mammalian retina
H-2Kb
β2 microglobulin
I-Ad
Fas
Untreated
4 days on
7 days off
17 days off
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Figure 1. Reversible induction of MHC expression in cultured retinal progenitor cells. Flow cytometry was used to measure
expression of MHC antigens, before and after transient treatment with IFN-γ. At baseline (top row), murine retinal progenitor cells do
not express MHC class I heavy chain (H-2Kb), β2 microglobulin, or class II (I-Ad), nor do they express CD95 (Fas). After 4 days of
treatment with the pro-inflammatory cytokine IFN-γ (second row), class I and II MHC antigens were induced, as was CD95. After
termination of IFN-γ treatment, these markers remained elevated for at least 7 days (third row), but eventually returned to baseline
(bottom row). Filled contours = marker; empty contours = isotype control.
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progenitor cells expressed the least class I, rat progenitor cells a
small amount, and human progenitors a considerable amount.
Confirmation of this trend will require data from additional
mammalian species. One such candidate is the pig. The author’s
group have recently cultured CNS progenitors from the pig
forebrain [22], and these cells provide a means of looking for
further evidence of a relationship between phylogenetic
complexity and MHC class I expression by CNS progenitor
cells. If confirmed as a widespread phenomenon, it would be of
great interest to know whether this trend relates directly to
increasing sophistication of the immune system or something
else entirely, such as a role for immunological molecules in neural
development, as has been recently proposed [23].
A final trend, of considerable importance to transplantation
studies, is an absence of detectible MHC class II expression.
This was the case for all CNS progenitor cells examined from
mice, rats and humans. The classical mechanism of graft
rejection involves the nonspecific recognition of foreign
MHC class II molecules by CD4+ host lymphocytes. Hence,
an absence of class II molecules would allow grafted progenitor
cells to evade immune rejection mediated by this important
mechanism. CNS progenitor cells, therefore, differ from solid
tissue grafts of either brain or retina, both of which contain
class II-expressing cell types, such as resident microglia,
passenger leukocytes and vascular endothelial cells.
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Although work to date has shown that murine CNS
progenitors do not express detectable MHC molecules at
baseline and are immunoprivileged as allografts, this does not
mean they cannot be rejected, as has been shown following
sensitisation of a previously grafted host [16]. Therefore, CNS
progenitor cells do express alloantigens that are detected by
the host immune system; however, these are not antigenic and
only lead to rejection under certain circumstances. In
addition, it has been shown that MHC antigens, both class I
and II, can be reversibly induced by stimulation of CNS
progenitors with IFN-γ [15,16]. This result has also been
replicated for murine RPCs (Figure 1) and implies that the
immune privilege of grafted progenitor cells is not absolute,
but instead subject to modulation by cytokines present in the
local microenvironment.
Progenitor cells produce cytokines as well as responding to
them, and these include members of the transforming growth
factor-β family [24], which are known to downregulate
immune activity. Active immune suppression, therefore,
represents another potential mechanism by which stem or
progenitor cells might influence their own survival following
transplantation. Additional agents that might potentially
contribute to active induction of immune tolerance include
Fas ligand and indolamine 2,3-dioxygenase, as implicated in
other systems [25,26]. Such agents could either be expressed by
Expert Opin. Biol. Ther. (2006) 6(5)
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Mouse
Rat
Human
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Figure 2. Differences in MHC I expression by species.
Comparative flow cytometric analysis of MHC class I expression by
CNS progenitor cells revealed species-related differences, ranging
from no detectible expression in mouse (top), to moderate
expression in rat (middle), and strong expression in human
(bottom), as indicated by the progressive shift-to-the-right of the
filled contours (marker) versus the empty contours (isotype control).
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the grafted cells or produced by cells of the host, either
constitutively (as may be the case in an immunoprivileged
site) or in response to the graft.
It is also important to note that the immunogenicity of
human, or large mammal, CNS progenitor cells has yet to be
systematically evaluated. Furthermore, there are additional
variables to consider when using progenitor cells in the setting
of xenotransplantation, as discussed in section 6.
The absence of immune rejection alone is insufficient to
assure the survival of grafted progenitor cells. Although
frequently difficult to quantify, massive death of donor cells
following transplantation is not an uncommon observation,
even in the absence of overt rejection. This has motivated the
search for methods of cell delivery capable of improving graft
survival. One such method is the use of polymer scaffolds.
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polymers poly(lactic-co-glycolic acid) (PLGA) and
poly(L-lactic acid) (PLLA) [27,28]. Following transplantation
to the mouse eye, cell survival and overall delivery were
strongly enhanced by seeding of RPCs onto scaffolds
composed of a PLGA/PLLA blend. Quantitative studies
showed an order of magnitude increase in cell survival at
4 weeks when compared with injections of cells alone [28]. In
addition, adherence of the cells to the polymer resulted in
improved delivered to the subretinal space, as cells were not
lost as a result of reflux through the retinotomy. Taken
together, there was 16-fold greater effective cell delivery for
polymer composite grafts.
Cellular differentiation also appears to be enhanced by the
use of PLGA/PLLA composite grafts, both in vitro [27] and
in vivo [28]. For instance, the rho-/- mouse, as a rhodopsin
double knockout model, does not express functional
rhodopsin molecules. The author’s previous work in the rho-/mouse showed no expression of rhodopsin by either host or
grafted RPCs [10]. In contrast, RPCs delivered as composite
grafts to the retina of rho-/- animals showed colocalisation of
rhodopsin staining with green fluorescent protein (GFP)+
progenitor cells, suggesting that the polymer facilitates
differentiation of the grafted cells [28]. This interesting
phenomenon has not been extensively explored, and the
underlying mechanism, as well as the extent to which it can be
generalised to other polymers, cell types and species, remains
to be determined.
An important benefit of using composite grafts – indeed,
the original impetus behind such studies – is the ability to
influence the organisation of donor cells post-transplantation.
Tissue engineering represents an important strategy for work
on retinal degeneration because of the multi-layered organisation of the retina, the tendency of grafted progenitor cells to
migrate, and the need for a tightly organised photoreceptor
matrix to provide useful spatial resolution of visual objects.
Working in the pig, initial evidence of the utility of
PLGA/PLLA blends in this setting has now been obtained.
Transplanted to the subretinal space as a polymer
composite graft, RPCs remained confined to the polymer
scaffold and showed little migration into the retina at the time
points chosen [29]. RPCs also maintained a high degree of
radial alignment in this location, propbably as a result of the
pore structure of the polymer sheet.
These findings present an alternative method of RPC
delivery as compared with bolus injections. The use of
polymer scaffolds has a promising role in strategies aimed at
the reconstruction of multiple cellular layers lost to
degeneration, such as the photoreceptor and RPE layers, and
may therefore be useful in the setting of retinitis pigmentosa,
choroideraemia, macular degeneration and other conditions.
One caveat to consider is the ever-present potential for
increased macrophage activity in response to the presence of
artificial material. Activated macrophages could increase the
likelihood of rejection of co-grafted foreign cells, although
this potential scenario may in turn be contained by the
Biodegradable polymers as a scaffold for
progenitor cell transplantation
5.
Work from the author’s group has shown that murine RPCs
survive well in culture when seeded onto the biodegradable
Expert Opin. Biol. Ther. (2006) 6(5)
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5
Transplantation of cultured progenitor cells to the mammalian retina
incorporation into the polymer of agents capable of
moderating an immune response.
In summary, the author’s group’s studies of murine RPCs
grown on biodegradable polymers and transplanted to the
subretinal space of the mouse and pig indicate that composite
grafts of this sort provide a number of advantages over bolus
injections. These include cell survival, cell delivery,
phenotypic differentiation and cytoarchitectural organisation.
6. The pig model: xenotransplantation of
murine retinal progenitor cells to the
pig retina
Considerable progress has been made transplanting RPCs in
rodent models; however, assessing the full potential of both
RPCs and RPC/polymer composite grafts is more readily
accomplished, not to mention more clinically meaningful, in
an animal with a large eye and retina of human dimensions.
As indicated in the previous section on polymers, the author’s
group have begun studies in the eye of the pig. From a
surgical standpoint, the pig eye clearly models the human eye
more closely than does that of the rodent. Like humans, the
pig also has an immune system considerably more
sophisticated than that of rodents.
The first progenitor cell grafts into the pig utilised RPCs
from GFP-transgenic mice. The possibility of xenograft
rejection was well known, but the graft proceeded for a
number of reasons. First of all, it was wished to develop a large
animal model of subretinal RPC transplantation, and only
murine RPCs were available at that time. In addition, as the
subretinal space of the rodent is an immunoprivileged site,
there was reason to believe that the same would apply to the
pig and, therefore, xenografts would at least survive long
enough to show some degree of integration into the retina.
Finally, as murine RPCs had shown evidence of being
immunoprivileged cells as allografts, the possibility remained
that they would not be rejected as xenografts either. Although
it is common knowledge that xenografts tend to do poorly,
previous studies have demonstrated extended survival of
mouse retinal transplants in the rat brain [30] and of mouse
brain progenitor cells in the eye of the Brazilian opossum [31],
despite a lack of exogenous immune suppression. There was
in fact no experimental precedence for predicting the
outcome of the work the author’s group were undertaking.
Following pars plana vitrectomy and application of
endolaser burns to the pig retina, murine RPCs were injected
into the subretinal space, either as a single cell suspension, as
spheres, or in association with a polymer sheet as described in
the previous section [29]. No immune suppression was used.
The results of the polymer composite grafts have been
discussed above and will not be revisited here. In terms of the
non-polymer data, this work showed that RPCs from the
mouse could migrate into the neural retina of the pig,
apparently homing to areas of laser-induced injury and
exhibiting signs of cellular integration (Figure 3). Interestingly,
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there was also evidence of integration into the adjacent RPE
layer, something these same murine RPCs had not done as
allografts. Cells survived well for 2 weeks, and a few cells
survived for over a month; however, a mononuclear reaction
began to appear in the inner choroid well before then
(Warfvinge et al., unpublished data). The cellular response
continued to build in intensity and by 5 weeks had formed
very prominent choroidal infiltrates, yet surprisingly few
immune cells were evident in the adjacent retina.
Nevertheless, the grafted mouse cells showed markedly
decreased survival at this time point.
Clearly the degree of immune privilege afforded murine
progenitor cells as allografts, and as xenografts in the Brazilian
opossum, is insufficient to protect them in the pig, even when
grafted to a site that is presumed to exhibit immune privilege.
Additional work is being performed to further evaluate the
mechanism of graft rejection in this model (Warfvinge et al.,
unpublished data); however, given the severity of the response
to xenogeneic cells, the next strategy was to derive progenitor
cells from the pig so that RPC transplantation could be
explored in a porcine allograft model where immune rejection
should be considerably less likely. To do this, it was first
necessary to derive CNS progenitor cells from the pig.
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6.1 The pig model: characterisation of progenitor cells
from the pig forebrain
Progenitor cells from both the brain [22] and retina
(Klassen et al., unpublished data) of the pig have now been
cultured. In the case of progenitors from the porcine
forebrain, derivation had been previously reported by other
groups [31-33]; however, the contribution of the author’s group
was to examine a broader range of markers and compare this
expression profile with analogous cells from the human
forebrain. The properties of these cells were investigated using
reverse transcriptase-polymerase chain reaction (PCR), gene
microarray, immunocytochemistry and flow cytometry.
Although the number of available pig-specific reagents is
extremely limited, it was found that probes developed for use
on human material can frequently be applied to porcine
specimens as well. This work revealed gene expression
patterns in porcine cells similar to those seen in the author’s
previous work with human neural progenitors. There was,
however, a preponderance of negative data in the studies of
porcine gene expression, particularly with respect to the
microarray data. This was not unexpected, as detection of
porcine gene products by human reagents is a hit and miss
proposition. For example, nestin, although faintly detected by
human PCR primers, is difficult to demonstrate using
available antibodies. Nevertheless, a broad range of markers
can be identified in porcine CNS progenitor cells.
Markers expressed by both porcine and human forebrain
progenitor cells include the neurodevelopmentally regulated
nuclear transcription factors Sox2, Hes1 and Pbx1, the cell
cycle genes nucleostemin and cyclin D2, the cytoskeletal
genes nestin (weak signal in pig), vimentin, glial fibrillary
Expert Opin. Biol. Ther. (2006) 6(5)
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Figure 3. Integration of GFP+ murine RPCs in the laser-injured porcine retina. RPCs from GFP-mice were transplanted to the
subretinal space of the laser-injured pig. RPCs (green) integrated into both the neural retina and RPE layer of porcine hosts at 2 weeks
post-transplantation. Donor cells showed tropism for areas of laser injury, evident here as a prominent gap in the well-defined stripe of
synaptophysin labelling (red) corresponding to the host OPL. More donor cells are seen in the inner plexiform layer, seen as the broader
and more diffuse band of synaptophysin staining below the OPL, as well as along the vitreal surface of the retina (bottom). The green
profiles along the top of the image are in the RPE layer. Photomicrograph courtesy of Dr K Warfvinge.
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GFP: Green fluorescent protein; OPL: Outer plexiform layer; RPC: Retinal progenitor cell; RPE; Retinal pigment epithelium.
acidic protein, DCX and β-III tubulin, as well as the surface
molecules neural cell adhesion molecule, aquaporin-4 and
nogoA. Although the author’s work to date has evaluated
brain progenitors more extensively than pig-derived RPCs,
there are indications that, in general, porcine CNS progenitor
cells share a close similarity to the expression profile of
analogous human cells from the brain [18] and retina [19], as
compared with the analogous murine CNS progenitor
cells [10]. This similarity between pig and human data
provides additional justification for investigation of
progenitor allografts to the pig retina, as it now appears that
both the donor cells and the recipient eye more closely
resemble their human counterparts than do those of rodents.
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Expert opinion and conclusion
The experiments described here were directed towards the
establishment of a novel strategy for retinal repair. This work
involved the isolation and characterisation of progenitor
cells from the brain and retina of different mammalian
species, followed by the transplantation of these cells to the
retina of rats, mice and pigs, with subsequent evaluation of
the degree of cytoarchitecture integration displayed by
donor cells and the immunological responses of the host.
These experiments included work in which progenitor cells
were seeded onto biodegradable polymers. The results to
date have been encouraging.
Notably, it has been shown that, unlike solid tissue
transplants of mature or immature neural retina, CNS
progenitor cells engraft in the dystropic retina of mature
mammalian recipients. Engraftment in this context includes
widespread migration in the host retina, evidence of
cytoarchitectural organisation, differentiation into cells
resembling local populations, elaboration of appropriately
oriented neurites and expression of appropriate genes.
Specifically, it has been shown that cultured retinal
progenitors can differentiate into photoreceptors, as
evidenced by morphology and expression of the markers
rhodopsin and recoverin. Evidence of graft-associated benefits
at the behavioural level has also been seen, specifically with
respect to light sensitivity. Additional work is needed to
determine whether this last finding reflects functional
replacement of photoreceptors or the equally important
phenomenon of host cell neuroprotection. In the author’s
opinion it is likely to be the latter in this instance, although it
is believed that functional photoreceptor replacement is
achievable, particularly in conjunction with the further
development of tissue engineering approaches. The functional
consequences of photoreceptor rescue and replacement, as
associated with progenitor cell transplantation, can be better
addressed in large animal models.
It is the opinion of the author that work with large animals
is a critical and frequently neglected aspect of the translational
process for novel therapies. This is particularly important in
Expert Opin. Biol. Ther. (2006) 6(5)
7
Transplantation of cultured progenitor cells to the mammalian retina
the arena of regenerative medicine, where relatively little
clinical experience exists at present. Whereas work in mice is
relatively inexpensive and rapid, success in a large animal
model provides a more realistic basis for the initiation of
clinical trials. Unfortunately, at present, many research
institutions lack the infrastructure for work of this kind. In
the case of retinal progenitor transplantation, an international
collaborative team has come together with the common goal
of developing a porcine allograft model. The overall aim of
Bibliography
1.
2.
WENKEL H, CHEN PW, KSANDER BR,
STREILEIN JW: Immune privilege is
extended, then withdrawn, from allogeneic
tumor cell grafts placed in the subretinal
space. Invest. Ophthalmol. Vis. Sci. (1999)
40:3202-3208.
SILVERMAN MS, HUGHES SE:
Transplantation of photoreceptors to
light-damaged retina. Invest. Ophthalmol.
Vis. Sci. (1989) 30:1684-1690.
11.
12.
LI LX, TURNER JE: Inherited retinal
dystrophy in the RCS rat: prevention of
photoreceptor degeneration by pigment
epithelial cell transplantation. Exp. Eye Res.
(1988) 47:911-917.
4.
LOPEZ R, GOURAS P, KJELDBYE H
et al.: Transplanted retinal pigment
epithelium modifies the retinal degeneration
in the RCS rat. Invest. Ophthalmol. Vis. Sci.
(1989) 30:586-588.
13.
5.
BERSON EL, JAKOBIEC FA: Neural
retinal cell transplantation: ideal versus
reality. Ophthalmology (1999) 106:445-446
14.
7.
8.
9.
10.
8
A
SILVER J, MILLER JH: Regeneration
beyond the glial scar. Nat. Rev. Neurosci.
(2004) 5:146-156.
DAVID S, LACROIX S: Molecular
approaches to spinal cord repair.
Annu. Rev. Neurosci. (2003) 26:411-440.
YOUNG MJ, RAY J, WHITELEY SJ,
KLASSEN H, GAGE FH: Neuronal
differentiation and morphological
integration of hippocampal progenitor cells
transplanted to the retina of immature and
mature dystropic rats. Mol. Cell. Neurosci.
(2000) 16:1997-2005.
KLASSEN HJ, NG TF, KURIMOTO Y
et al.: Multipotent progenitor cells from the
The author thanks all coauthors for their contributions to the
work cited here, in particular M Young, who has been an
inspiration to us all.
TAKAHASHI M, PALMER TD,
TAKAHASHI J, GAGE FH: Widespread
integration and survival of adult-derived
neural progenitor cells in the developing
optic retina. Mol. Cell. Neurosci. (1998)
12:340-348.
VAN HOFFELEN SJ, YOUNG MJ,
SHATOS MA, SAKAGUCHI, DS:
Incorporation of murine brain progenitor
cells into the developing mammalian retina.
Invest. Ophthalmol. Vis. Sci. (2003)
44:426-434.
15.
16.
17.
18.
o
h
P
r
GLEESON JG, LIN PT, FLANAGAN LA,
WALSH CA: Doublecortin is a
microtubule-associated protein and is
expressed widely by migrating neurons.
Neuron (1999) 23:257-271.
t
u
ZHANG Y, KARDASZEWSKA AK,
VAN VEEN T, RAUCH U, PEREZ MT:
Integration between abutting retinas: role of
glial structures and associated molecules at
the interface. Invest. Ophthalmol. Vis. Sci.
(2004) 45:4440-4449.
Acknowledgements
retina express developmental markers,
differentiate into neurons, and preserve
visually mediated behavior. Invest.
Ophthalmol. Vis. Sci. (2004) 45:4167-4173.
3.
6.
this ongoing effort is the development of a regenerative
therapy for blinding conditions, such as retinitis pigmentosa,
macular degeneration and retinal detachment.
ZAHIR T, KLASSEN H, YOUNG MJ:
Effects of ciliary neurotrophic factor on
differentiation of late retinal progenitor cells.
Stem Cells (2005) 23:424-432.
KLASSEN H, IMFELD K, RAY J,
YOUNG MJ, GAGE FH, BERMAN M:
The immunological properties of adult
hippocampal progenitor cells. Vision Res.
(2003) 43:947-956.
HORI J, NG TF, SHATOS M,
KLASSEN H, STREILEIN JW,
YOUNG MJ: Neural stem cells lack
immunogenicity and resist destruction as
allografts. Stem Cells (2003) 21:405-416.
KLASSEN H, SCHWARTZ M, BAILEY A,
YOUNG MJ: Surface markers expressed by
multipotent human and mouse neural
progenitor cells include tetraspanins and
non-protein epitopes. Neurosci. Lett. (2001)
312:180-182.
SCHWARTZ PH, BRYANT PJ, FUJA TJ,
SU H, O’DOWD DK, KLASSEN H:
Isolation and characterization of neural
progenitor cells from post-mortem human
cortex. J. Neurosci. Res. (2003) 74:838-851.
Expert Opin. Biol. Ther. (2006) 6(5)
19.
KLASSEN H, ZIAEIAN B, KIROV II,
YOUNG MJ, SCHWARTZ PH: Isolation
of retinal progenitor cells from postmortem
human tissue with comparison to autologous
brain progenitors. J. Neurosci. Res. (2004)
77:334-343.
o
r
f
o
20.
AHMAD I, DOOLEY CM,
THORESON WB, ROGERS JA, AFIAT S:
In vitro analysis of a mammalian retinal
progenitor that gives rise to neurons and
glia. Brain Res. (1999) 831:1-10.
21.
YANG P, SEILER MJ, ARAMANT RB,
WHITTEMORE SR: Differential lineage
restriction of rat retinal progenitor cells
in vitro and in vivo. J. Neurosci. Res. (2002)
69:466-476.
22.
SCHWARTZ PH, NETHERCOTT H,
KIROV II, ZIAEIAN B, YOUNG MJ,
KLASSEN H: Expression of
neurodevelopmental markers by cultured
porcine neural precursor cells. Stem Cells
(2005) 23:1286-1294.
23.
HUH GS, BOULANGER LM, DU H,
RIQUELME PA, BROTZ TM, SHATZ CJ:
Functional requirement for class I MHC in
CNS development and plasticity. Science
(2000) 290:2155-2159.
24.
KLASSEN HJ, IMFELD KL, KIROV IL
et al.: Expression of cytokines by
multipotent neural progenitor cells.
Cytokine (2003) 22:101-106.
25.
OSAWA H, MARUYAMA K,
STREILEIN JW: CD95 ligand expression
on corneal epithelium and endothelium
influences the fates of orthotopic and
heterotopic corneal allografts in mice.
Invest. Ophthalmol. Vis. Sci. (2004)
45:1908-1915.
26.
KWIDZINSKI E, BUNSE J, KOVAC AD
et al.: Indolamine 2,3-dioxygenase is
expressed in the CNS and down-regulates
autoimmune inflammation. FASEB J.
(2005) 19:1347-1349.
27.
LAVIK EB, KLASSEN H,
WARFVINGE K, LANGER R,
YOUNG MJ: Fabrication of degradable
Klassen
polymer scaffolds to direct the integration
and differentiation of retinal progenitors.
Biomaterials (2005) 26:3187-3196.
28.
29.
TOMITA M, LAVIK E, KLASSEN H,
ZAHIR T, LANGER R, YOUNG MJ:
Biodegradable polymer composite grafts
promote the survival and differentiation of
retinal progenitor cells. Stem Cells (2005)
23:1579-1588.
WARFVINGE K, KIILGAARD JF,
LAVIK EB et al.: Retinal progenitor cell
xenografts to the pig retina: morphological
integration and cytochemical
differentiation. Arch. Ophthalmol. (2005)
123:1385-1393.
A
t
u
30.
31.
32.
YOUNG MJ, RAO K, LUND RD:
Integrity of the blood-brain barrier in
retinal xenografts is correlated with the
immunological status of the host.
J. Comp. Neurol. (1989) 283:107-117.
SMITH PM, BLAKEMORE WF: Porcine
neural progenitors require commitment to
the oligodendrocyte lineage prior to
transplantation in order to achieve
significant remyelination of demyelinated
lesions in the adult CNS. Eur. J. Neurosci.
(2000) 12:2414-2424.
ARMSTRONG RJ, HARROWER TP,
HURELBRINK CB et al.: Porcine neural
xenografts in the immunocompetent rat:
o
h
P
r
Expert Opin. Biol. Ther. (2006) 6(5)
immune response following grafting of
expanded neural precursor cells.
Neuroscience (2001) 106:201-216.
33.
UCHIDA K, OKANO H, HAYASHI T
et al.: Grafted swine neuroepithelial stem
cells can form myelinated axons and both
efferent and afferent synapses with
xenogeneic rat neurons. J. Neurosci. Res.
(2003) 72:61-69.
Affiliation
Henry Klassen MD, PhD
Senior Clinician-Scientist, Singapore Eye
Research Institute, 11 Third Hospital Ave,
#05-00 SNEC Building, 168751 Singapore
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