I
August 2010 vol. 5 no. 4
Expert Review of
Ophthalmology
Eorronrnl
Primary optic nerve sheath meningioma
lnrrnvrew
lnternational eye health: a 20-year perspective
DEvrce Pnonlrs
Aspheric toric intraocular lenses for cataractous eyes
iStento: trabecular micro-bypass stent for open-angle glaucoma
Revrews
Does structural damage precede functional loss in glaucoma?
the best method for diagnosing glaucoma?
Developments in diagnostic tools for corneal ectasia
What
is
Advances in pediatric orbital magnetic resonance imaging
Eye
growth transformation: uveal melanoma
Genetics of retinoblastoma
New concepts for reconstruction of retinal and pigment epithelial tissues
Mechanisms of corneal allergic reaction: new options for treatment
Comprehensive review of the effects of diabetes on ocular health
XPERT
REvrewd
Expenr
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NeW,COnCeptSfuir',.,.,,
I Rpvrpwsreitnit,ruction',o-f.'fetinal,and
pr'g nt'e-
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ExBert&eu.Apfithalmol:5(41,58:5a1t2910),, ',,,,.'..
Paul G Layerlt,
Masasuke Araki2 and
Astrid Vogel-Hdpkert
Tech n isch e U n iversifit Da rmstadt,
Entwicklungsbiologie und
Neurogenetik, Schnittspahnstrasse 13,
D-64287 Darmstadt, Germany
Developmenta I Neu robiol ogy
'z
Laboratory, Department of Biological
Sci ences, N a ra Women's U n iversity,
Nara 630-8506, Japan
tAuthor for correspondence :
1
Tel.: +49 615 116 3800
61 5 1 16 6548
Fax: +49
I aye r@ b i o.tu
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d a rm sta dt. d e
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The rise of stem cell-based regenerative medicine has created great hopes for novel therapies
for major blinding diseases. lntensive relevant research is grounded on a deep cellular and
molecular knowledge of the complex embryonic development of the neural retina and retinal
pigmented epithelium (RPE) from the eye vesicle. This research similarly relies on a long history
of transdifferentiation studies, having revealed an innate capacity to regenerate a more or less
complete retinal tissue from RPE. To analyze principles of self-organization that govern retinal
tissue (re-)construction under normal or regenerative conditions on a 'cell-by-cell' basis, the
reaggregate approach of dispersed embryonic progenitor cells into retinotypic cellular spheres
has been instrumental. Based on this knowledge, a multitude of fascinating studies using
embryonic. induced pluripotent, adult stem cells, or permanent cell lines from various species
have been carried out over the past two decades, and directed production of human retinal
and RPE cell types has become possible. Moreover, reconstruction of complete retinal tissue, of
functioning RPE monolayers, or even eye-like structures has become feasible. After their
implantation into appropriate animal models for blinding diseases, some functional recovery
has been observed. Here, we review some historical, cellular and molecular perspectives of this
vast research program.
Ktvwonm: BMPs r cell reaggregation
In Europe, every 5 s one
o cell specification
person
will
become
blind. Nearly everybody at some point during
their lifetime suffers from decay or loss of eyesight. The list of ophthalmic defects, endangering lesions ofthe eye or blinding diseases is
nearly endless Forl; for many of them there is no
therapy or even a rational treatment. Therefore,
with the rise of stem cell-based regenerative
medicine, great hopes have been raised from
both
researchers and patients.
!7hile recruit-
ment of uncommitted cells from embryonic
stem cells (ESC$ and primordial germ cells are
still interesting research topics, adult stem cells
(ASCs), and in particular induced pluripotent
stem cells (iPSCs), now offer the most exciting new therapeutic avenues tt,zl. To further
promote stem cell-based regenerative medicine, technologies that lead from stem cells to
implantable tissues could become instrumental, since it is likely that only in rare cases will
injected stem cells directly and autonomously
restore a lesioned tissue part in situ lt,+1. More
typically, stem cells have to be amplified to large
www.expen- reviews,com
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t0.t586lEOP.t0.42
.
eye diseases
.
ocular development
.
regenerative
quantities in suspension cultures, in the presence ofappropriate growth factors, in order to
establish 3D histotypical cell spheres that can
then be implanted into a lesioned organ. Both
the transdifferentiation ofASCs into diverse cell
types [5], as well as their transformation into
functioning tissues and implantation, will need
much further elaboration.
Blinding diseases
Retinal degenerative diseases, including agerelated macular degeneration (AMD) and
retinitis pigmentosa (RP), are the predominant
causes of human blindness in the world; however, these diseases are
difficult to treat. During
the last two decades, a tremendous amount of
research has been invested into developing cellbased therapies for some of the most devastating
blinding
diseases.
Age-related macular degeneration is a complex disorder of the eye and the third leading
cause of blindness worldwide. AMD results in
progressive loss of central vision affecting the
@ 2010 Expert Reviews
Ltd
ISSN 1246-9899
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Loyer, Arqki & Vogel-H6pker
macular region of the eye in the elderly. An inrraocular lens
implanted into eyes ofAMD patienrs can improve their vision tel.
lVhile the prevalence is higher in Caucasian populations, AMD
has gradually become a major public health issue worldwide due
to changing demographics and lifestyle factors 121. AMD has
a multifactorial etiology. For insrance, in the prevention and
treatment of neovascular AMD, inhibition of angiogenesis is
critical. Pathologic states such as hypoxia, ischemia or infammation may favor the formation of new blood vessels by driving
the synthesis of VEGF tsl.
Retinitis pigmentosa is a heterogeneous group of retinal
initially affecting the rod photoreceptor
(PR). Inherited retinal degeneration, which includes conditions such as RP and Leber congenital amaurosis (LCA), affects
degenerative diseases
approximately I in 3000 of the population in the'Western world.
Patients suffer from night blindness, loss of peripheral vision
and, finally, the loss ofcentral vision as a consequence ofdeath
of cone PRs. There are currenrly no effective treatments. RP
is a genetic disease, showing inheritance of autosomal domi
nant, autosomal recessive or Xlinked recessive traits, although
some patients have no family hisrory of RP (simplex RP). RP
can result from mutations in any one of more than 100 different genes, many of which have now been identified and their
functions elucidated, providing a major impetus to develop
gene-based treatments.
The genetic defects that can lead ro PR degeneration are innu-
merable and multifactorial. Over 500 mutations in the gene
encodingABCA4 are associated with a specrum of related autosomal recessive retinal degenerative diseases, including Stargardtt
macular degeneration, cone-rod dystrophy and a subset ofRP.
ABCA4 is a member of the ABCA subfamily of ATP-binding
cassette transporters that is expressed in rod and cone PRs ofthe
vertebrate retina. ABCA4 (a retinylidene-phosphatidyl-ethanolamine transporter) facilitates the removal of potentially reactive retinal derivatives from PRs following photoexcitation lrl.
Similarly relevant, peripherin/RDS is an integral membrane
glycoprotein located in the rod and cone outer segments. Thus
far, over 90 human peripherinlRDS gene mutations have been
identified and are associated with a variety of retinal dystrophies t101. Furthermore, RPE65 is an isomerohydrolase expressed
in retinal pigment epithelium (RPE). It is critical for the regeneration of the visual pigments necessary for both rod- and conemediated vision. Mutations in human RPE65 cause LCA and
other forms of autosomal recessive RP that are associated with
early-onset blindness. LCA, although its incidence is very rare,
is the most severe retinal dystrophy causing blindness or severe
visual impairment before the age of I year. Linkage analysis,
homozygosity mapping and candidate gene analysis facilitated
the identification of 14 genes mutated in LCA and juvenile retinal degeneration. As a first gene therapy for a blinding disease,
adeno-associated viral vectors encoding RPE65 were subretinally
administered in patients affected with LCA2 and were shown to
be safe and long lastingwithout displaying negative immunologic
responses tlll. Many blind people hope for an early introduction
of cell-based therapies.
524
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Scope of this article
Both retinal and RPE tissues are most relevant in treating particular eye diseases. This article is driven by looking ar basic
knowledge of the interactions between the outer and inner layer
of the early optic cup, eventually forming into the RPE and the
retina. How can cell biological and molecular knowledge of the
ontogenetic formation of both tissues be instrumental to develop
methodologies to become capable of regenerating human retinal
tissue zz uitro (or, ifpossible, directly in uiuo), and how can ir be
used for transplantation into a diseased eye? The field ofcell-based
repair of eye tissues has become so huge that it is impossible ro
cover it wholly in one article. Therefore, we restrict the scope of
this article to the following topics:
. Molecular and cellular biology of eye developmenr;
. Capacities and constraints of retinal and RPE regeneration;
. Advances in engineering ofeye rissues;
. Further aspects of ophthalmic regenerarive medicine.
Since major topics in this 6eld have been covered by excellent
recent reviews, here we will not (or only marginally) deal with
the following topics:
,
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Other highly relevant approaches to achieve retinal tissue repair,
particularly from ASCs, as derived from the aduh ciliary
margin, adult iris epithelium or Miiller glial cells (MCs) trz-rzl;
.
.
t
I
Gene therapy approaches to repair PRs;
The fascinating bioelectronic approach of constructing rerinal
chip implants (bioelectronic technologies; see Usl).
t
)
Molecular & cellular biology of eye development
The neural retina & the RPE
The neural retina (NR) is the light-sensitive layer of the eye.
The laminar organization of the vertebrate NR is stereotypic
across species (Frcunr lA a. c and FrcuRE 2, right), whereas thick-
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ness ofeach cell layer, relative numbers ofeach cell type, and so
on, can vary between species. The retina consists of seven cell
types in all vertebrates: PRs (rods and cones), MCs, bipolar,
horizontal (HCs), amacrine (ACs) and ganglion cells (GCs).
The cell bodies of retinal neurons are arranged in three layers,
while neuropil and synaptic connections are resrricted to two
major laminae, the outer plexiform layer (OPL) and the inner
plexiform layer (IPL). Cell bodies of MCs are found in the inner
nuclear layer; their processes span the entire width ofthe rerina
and provide structural and functional support for retinal neurons.
The RPE is a monolayer of pigmented cuboidal cells and fulfills
multiple functions that are necessary for proper visual function
(reviewed in trgl). Thus, a functional unit is formed by both NR
and RPE, and abnormalities in one tissue can lead to secondary
degeneration of the other.
The mechanisms involved in eye developmenr have been
studied in a variety of model organisms, including Drosophila,
Xenopus, zebrafish, chicks and mice. This basic knowledge is useful for engineering stem cells into either RPE or NR rissues, since
Expert Reu. Ophthalnol. 5(4), (2010)
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New concepls for reconstruclion of retinol & pigmenl epithelioltissues
they
will
pass through the same states
of
specification and differentiation as observed
during normal vertebrate eye development
(Frcunr tA, B & c). Hence, the identification
ofintrinsic and extrinsic signals involved in
the processes of NR and RPE specification,
differentiation and transdifferentiation are
guiding researchers in directing the differentiation of human ESCs (hESCs) or iPSCs
into NR- and RPElike cells (see later).
Moreover, the signals involved in patterning the vertebrate eye are important markers required to distinguish between progenitor and mature states. Excellent reviews
about the molecular mechanisms that
direct growth and patterning of the vertebrate optic vesicle and cup have recently
been published tzo,zrl. Here, we will briefy
summarize the main mechanisms involved
in NR and RPE specification, mainly in
the chick, focusing on extrinsic signals
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that may similarly direct the differentiation of hESCs/iPSCs into RPE and NR.
This knowledge could be used as a basis
for the development of novel therapeutic
approaches for major eye disease, such as
RP,
AMD and diabetic retinopathy.
Patterning of the optic vesicle into
domains for RPE & NR
Eye development is initiated through the
evagination of the optic vesicle from the
anterior neuroepthelium of the forebrain.
OS
ONL
ONL
OPL
OPL
INL
IPL
GCL
GCL
Figure 1. Structure of an adult and postnatal rodent retina, and its nearly
complete tissue-engineered reconstruction from retinal progenitor cells.
(A) DAPI staining (light blue) of a section of an adult gerbil retina reveals a highly
ordered arrangement of neuronal cell bodies in three nuclear layers; an ONL, an INL and
a GCL. Unstained areas contain an OPL and an lPL, and the OSs of photoreceptors,
which are neighbored bythe RPE. (B) Partial section of a retinal spheroid, as
reaggregated after '10 days in vitro from dispersed cells of gerbil postnatal day 1 retinae,
cultured in the presence of RPE supernatant and Wnt3a; section was stained for DAPI
(blue), calretinin (red, for amacrine cells [including displaced amacrine cells] and ganglion
cells), and for Cern9o1 (green, for rod photoreceptors). ln comparing the laminar
structure of the reconstructed spheroid with a postnatal retina in (C) (homologous areas
and/or cell types in (B) and (C) are connected by white lines), its far advanced
differentiation of the inner retina (red), and onset of sublaminae formation in IPL is
evident. ln the outer retina, OPL is indicated by a DAPI-negative open ring, while rod
differentiation has only started (green) llevnnpG, uNpuor-rsnro Drrel Us21. (C) Section of
developing gerbil retina at postnatal day 10. Note that OS have not yet differentiated
(cf. with (A)).
GCL: Ganglion cell layer; INL: lnner nuclear layer; IPL: lnner plexiform layer; ONL: Outer
nuclear layer; OPL: Outer plexiform layer; OS: Outer segment; RPE: Retinal
pigment epithelium.
Reprinted with permission from A Bytyqi and M Rieke.
The optic vesicle then enlarges distally and
expands dorsally, dividing into three territories [22]: the proximal optic stalk, the presumptive NR and the
presumptive RPE. Formation of the lens placode occurs in the
surface ectoderm overlying the distal region of the optic vesicle.
The lens placode then invaginates to form the lens vesicle, and
this process results in the formation of a bilayered optic cup. The
inner layer develops into the multilayered NR, while the outer
layer develops into the single-layered, pigmented RPE. Initially,
a number of transcription factors are coexpressed throughout
the optic vesicle, such as Rxl, Pax6, Six3 and Otx2 (reviewed
in lzo.zr,nl). Extrinsic signals released from the prechordal plate,
surface ectoderm and the surrounding mesenchyme then pattern
the optic vesicle into the optic stalk, RPE and NR by inducing or
repressing specific transcription factors lFrcunr 3) [20,21]. These key
signals belong to a small number of protein families, including
In uiuo and in uino studies showed that Activin A, bone-morphogeneticproteins (BMPs) ormembersof theTGFsuperfamily, can all induce RPE-specific gene expressions in the optic
vesicle [2s,29]. Transcription factors that are required for correcr
formation of the RPE are the microphthalmia-associated transcription factor (Mitf), the orthodenticle homologue-l and -2
(Orxll2) and Rx3 in zebrafish. In zebrafish, Rx3 is involved
in RPE specification by regulating the expression of Mitf and
Otx2 1:01. In the optic vesicles of chicks and mice, Mitf expression is detected before NR development is initiated, and expression is localized to both the presumptive RPE and NR. At the
time when the neuroepithelium of the opric vesicle is specified
into RPE and NR, the surface ectoderm overlying the distal portion of the optic vesicle expresses BMP4 and BMPT (reviewed
thehedgehogs,TGF-B,FGFsand\Tnts,whichplaymultipleroles in;2t1, Frcunrl). At this stage, mesenchymal cells are absent in
during closely spaced periods of eye development. For example, the distal region of the chick optic vesicle (Frcunr 3 A &Bl lzz,zg,3tl,
sonic hedgehog signaling initially involved in separation of the eye suggesting that BMPs released from overlying ectoderm induce
field is also involved in specifying the proximal/ventral region of Mitf expression and hence RPE development. This hypothesis
the optic vesicle, and subsequently also plays a role during RPE is further supported by the finding that in mutant embryos,
development 1z<-221.
where the ocular mesenchyme is affected, RPE development is
www-exDert-revlews.com
525
Loyer, Aroki & Vogel-H6pker
(reviewed in t20,231). Removal of the surface
ectoderm at the time when Chxl0 expression is initiated (stage 10/11) prevents
the separation of the optic vesicle into a
NR and RPE domain, and a pigmented
vesicle containing a few neuronal cells will
develop t4u4. FGF can substitute for the
surface ectoderm to allow normal patterning of the optic vesicle t<2,+:1. Thus, the
separation of the optic vesicle into a ventral NR and a dorsal RPE domain seems
to be initiated through a FGF-mediated
induction of Chxl0. FGF signaling inhib-
its RPE development and induces NR
development l4z-491. However, recent
Figure 2. Tissue engineering of avian retina: complete tissue reconstruction
including all three layers of a chick retina, as derived from retinal progenitor
cells of the embryonic day 5 chick embryo. ln contrast to Frcunr 4, a correct
arrangement of layers is induced by supplementation with factors from retinal pigment
epithelium, ciliary margin or M0ller glial cells (tr/rzlfor review). Compare with E18 normal
chick embryonic retina (riqht). DAPI-stained cells are blue (in false green color for GC
layer) and rod photoreceptors are red (CERN901 antibody). For markers used to identify
all retinal cell types, IPL neuropil and synapses including transmission electron
microscopy, see U13,142,160l.
GC: Ganglion cell; INL: lnner nuclear layer; IPL: lnner plexiform layer; ONL: Outer nuclear
layer; OPL: Outer plexiform layer; OS: Outer segment.
studies suggest that BMP signaling might
also be involved in NR induction. First,
in double-BmpRla/b muant embryos,
Chx10 expression is not detected, suggesting that low levels of BMP signaling might
be involved in inducing Chxl0 expression
within the optic vesicle [so]. Second, BMPs
can induce retinal regeneration from the
ciliary marginal zone. Here, the BMP
pathway directs proliferation and regeneration through the activation of SMAD
and the upregulation of FGF signaling
by the MAPK pathway tsrt. Third, BMP
signaling can transdifferentiate proximal
RPE cells into NR, and this effect is also
observed in the absence of the NR 1vo..rHopKER A, UNpunrrsuro
initially unaffected. Pitx2 is expressed in the mesenchyme that
surrounds the optic vesicle during the initial stages ofvertebrate
In the neural crest-specific knockout of Pitx2,
the RPE initially develops normally, but defects in the central
RPE are observed at later stages of development 1:21. Similarly, in
homozygotes of the quail silver mutation, the proximal, but not
the peripheral, RPE transdifferentiates into NR at later stages
of development [33-35].
In the chick, the optic vesicle is subdivided into a ventral NR
and dorsal RPE domain at stage 10 (rrcunr3c). At this point, the
dorsal half of the optic vesicle is destined to develop into RPE,
while the ventral part of the optic vesicle is destined to develop
into NR 129.3r.36.37i.In agreement with this, Mitf expression initially detected in the entire distal region ofthe chick optic vesicle
(Frcuns 38) becomes ventrally downregulated at stage 10, possieye development.
bly through induction of ChxlO expression
(Frcurr
3c). Chx10,
a paired-like homeobox gene, is the earliest specific marker of
retinal progenitor cells, known to negatively regulate Mitf expression. Mitf andOull2 then cooperate to regulate expression of
melanogenic enzymes in the dorsal optic vesicle [38-41].
Members of the FGF family are expressed in the surface ectoderm at the right time to be involved in NR induction, and are
currently the prime candidates to be involved in NR specification
526
Drrel, Consistent with
this, BMPs are expressed in the surface ectoderm at the time of
NR induction, while phosphorylated SMADI appears to colocalize with the domain of Chx10 expression in the presumptive NR
underneath the BMP-expressing surface ectoderm t:z,trl. In this
respect, it has been suggested that high BMP concentrations are
required for RPE specification tzq,s<1, while low concentrations
specify the NR ptol.
The RPE and NR fate remains reversible for several days following the initial activation of differentiation, as shown by the
transdifferentiation of the RPE into NR and vice versa [55-57].
During optic cup stages, BMP family members are expressed in
the presumptive RPE, while FGFs are expressed in the adjacent
NR, and yet no respecification or transdifferentiation occurs.
At this stage, an antagonistic interaction between BMPs and
FGFs provides a useful mechanism to prevent transdifferentiation of these tissues, ensuring proper development of the chick
eye lze.4;l. In the absence of BMP signaling, FGFS expression
in the NR is upregulated 1521, and NR-specific gene expression
is observed in the outer layer of the optic cup Lzsl. By contrast,
raising the FGFS concentration by applying FGF8-soaked beads
adjacent to the presumptive RPE results in downregulation of
BMP expression in the presumptive RPE and surrounding
mesenchyme, and a mirror image duplicated NR develops [ae].
Expert Reu. Ophthalnol. 5(4), (2010)
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New concepls for reconslruclion of retinol & pigment epilheliol lissues
However, this antagonistic interaction is only required during
the initial stages of optic cup formation, as BMP expression
becomes downregulated in the proximal region of the optic cup
at stage 20 Vel.lnterestingly, it is at this time when BMPs are
able to induce transdifferentiation of the RPE into NR lsre r*r'n1,
UNrunrrsHlo Derel,
Members of the tVnt family are also important signaling
molecules during vertebrate eye development, being involved
in both NR and RPE development l5sl. Several 1Vnts, Frizzleds
and secreted Frizzled-related proteins are expressed in the eye at
'Wnt/B-catenin
sigoptic vesicle and cup stages. In the mouse,
naling is highly active in the developing RPE at the stage of optic
cup formation, and activity subsequently becomes restricted to
the ciliary margin tie-6:1. The Wnt/p-catenin pathway might
control dorso-ventral patterning in the NR by regulating Tbx5
expression. Analysis of T-cell factor/lymphoid enhancer factor reporters in chick and mouse show activation in the dorsal
optic vesicle, which represents the presumptive RPE. Indeed,
Wnt/B-catenin signaling is required for RPE differentiation by
directly regulating Mitf and Otx2 expression 164,651. Deletion
ofthe p-catenin gene results in transdifferentiation ofthe RPE
into NR. However, activation of \(nt/B-catenin is not sufficient
to induce RPE-specific gene expression
165-671, strggesting that additional signals
are required to allow nnr a.u.iopri.",.
Moreover, ectopic activation of \Vntlpcatenin signaling results in the disruption of RPE patterning, indicating that
precise spatial and temporal regulation of
'Wnt/B-catenin
signaling is also necessary
for normal RPE development [64].
Taken together, the elucidation of the
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crucial for the development of cellular therapies in the eye. Recent studies in animal
models give hope that in the future, stem
cell-based cellular therapies could become
a reality (see later).
retin
176,771.
Amphibian retinal regeneration & its molecular analysis
Urodelian retinal regeneration has been a classic subject of
experimental embryology. Retinal regeneration from RPE in
the newt is considered a representative case of true metaplasia, or transdifferentiation [rs]. Cell-cell interactions of RPE
cells are largely affected during retina regeneration [78,79], based
on regulation of certain genes in RPE cells taking place right
after retinectomy. In the newt, RPE cells become BrdU-labelled
lnitiation
RPE specification
NR specification
Stage 9
Stage 10
A
signaling networks involved in NR and
RPE specification and differentiation is
t
transdifferentiation from RPE 155.68.7i1, another cellular source
for growth and regeneration in fish and amphibians are multipotent neural precursor cells of the eye margin, located near
the Ora serrata, peripherally of the functional retina 174-76l.In
fish and amphibians, this distinct region represents a marginal
growth zone throughout life lz6,zzl. Cells sequestered within
the retinal tissue, including rod precursors or MCs, can also
be sources ofa regenerating retina, in addition to retinal stem
cells or precursor cells in the ciiiary marginal zore lrTl.In fish,
lost cellular elements of the retina can be replaced by yet a
third mechanism, based upon mitosis of neuroblasts located
in the entire outer nuciear layer (ONL) of the differentiated
Stage
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BMP expression
Mesenchyme
Neuroepithelium
Chxl Olr'sx2 expression
Mitf expression
Weak BMP signaling
Ventral
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Capacities & constraints of retinal
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regeneration
Retinal regeneration can take place to
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various extents in vertebrates, depending
on species. Classical studies have shown
(note that due to space limitations eminent original literature is not cited) that
under specific experimental conditions,
cells of the RPE have the capacity to
transdifferentiate into NR tesl. After retinectomy, in some urodeles retinal regeneration and restoration ofvisual function
occurs throughout life, while in anurans
it
has been considered
to be restricted
up to metamorphosis
l6e-72). Besides
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Figure 3. Model for the initiation of retinal pigment epithelium and neural
retina development during optic vesicle stages in the chick, (A) At stage 8, eye
development is initiated through evagination of the optic vesicle, hence the overlying
surface ectoderm (red) expresses BMP4 and BMP7. (B) At stage 9, the RPE is specified in
the distal portion of the optic vesicle. BMP expression (red) in the surface ectoderm
induces Mitf expression (orange) in underlying neuroepithelium of the optic vesicle,
while being (nearly) absent in its proximal portions. (C) At stage 10, neural retina
specification is initiated in the ventral region of the optic vesicle. Here, weak BMP
signaling perhaps induces FGF expression (e.g., FGF1, FGF2 and FGF19) within the
surface ectoderm, resulting in the induction of Chxl0/Vsx2 expression in the ventral
neuroepithelium. ln its dorsal region, Mitf expression is marntained by high BMP
concentrations in ectoderm. At this stage, Chx10/Vsx2-mediated inhibition of Mitf in the
ventral optic vesicle restricts RPE development to the dorsal optic vesicle. At later stages,
Activin A and/or BMPs in the presumptive RPE and surrounding mesenchyme maintain
RPE-specific gene expression in the dorsal optic vesicle (not shown).
BMP: Bone morphogenetic protein; NR: Neural retina; RPE: Retinal pigment epithelium.
527
Loyer, Aroki & Vogel-Hopker
Retina regeneration rn Xenopus.' a new model
nsd iffe renti ati o n
It has long been assumed that anurans can regenerate their
retina oniy up to metamorphosis [70,71,8t]. Recently, however,
we found that Xenopus laeuis can still regenerate the whole
retina after metamorphosis [72,s6]. A novel culture technology
was established for Xenopus by which studies on th€ regenerative histogenesis of retinal tissue became feasible. \(hole RPE
sheets were removed from the choroid, put on a filter membrane
and covered by matrigel. After culturingfur 3-4 weeks, retinal
tissues emerged consisting of all retinal layers, including PRs
with outer segments, and were eventually covered by RPE 1e21.
As with newts, detachment of RPE ceils from the choroid and
reattachment to the basement membrane is the most significant
step for initiation oftransdifferentiation lzrl. Thereby, drastic
alterations in cell adhesion states possibly trigger essential genes
necessary for the fate determination of retinal stem cells and to
downregulate RPE-specific genes, including Mitf. Instead, soon
after isolation of the RPE, Pax6 is upregulated but is sustained
only if FGF2 is administered lszl . Hence, a two-step mechanism
of RPE transdifferentiation was proposed (Frcunr 4): a first and
almost simultaneously approximately 4-5 days after rerinectomy
1801, and then produce retinal stem cells. This initial period is
supposedly the most critical for transdifferentiation. Expression
patterns of several genes have been described, including Pax6,
for tra
Msi-I and Notch 1ar s1.
In order to clarify the molecular events involved in transdifferentiation during the initial phase, tissue culture studies
are indispensable, since in situ manipulations are particularly
'\7e
tough.
have developed a new culture method by which the
newt RPE tissue is cultured on a filter membrane cup to then
differentiate into neuronal cells tsol . Thus, FGF2 plays a distinct
role. 'W'ithout FGF2, RPE cells of the newt neither proliferate
nor transdifferentiate into neural cells tsal. Other factors, such as
IGFl, are also involved. If FGF2 is not supplemented, RPE cells
transdifferentiate into neural ceils only when cocultured with
the choroid. The fact that the choroid is a rich source of FGF2
suggests that it could supply FGF2 to RPE cells, an assumption supported by these culture studies. Interestingly, RPE cells
responded to FGF2 administration only after the initial 4-5 days
and developed into neuronal cells lsal (similarly for Xenopus, see
later and
Frcunr
4).
tlg;*is:lijsffi
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FGF2-independentstep
{'nt-,;.---.
.-)1\*a?-::.-.,,
d"-L
,\
FGFZ "
u,
FGF2
FGF-2
RVM
::
$
FGF2-dependentstep
'tt!ii*g t&yr 1;!:riili1r;tst!*$i$*€,5t*:a:s{t'
la:. ,!;:'
l
$
f
Retinal regeneration
Cell
?
:!:
.
migration
..,
r -
.r
'- ..:i
'i:i:fi-.i;id*t*9i*l,9ji*i:e13tfjr,i;:\il
BM
FGF2
Figure 4. A two-step model of retinal regeneration from Xenopus retinal pigment epithelium. First, changes in cell-matrix and
cell-cell interactions trigger Pax6 expression in retinal pigment epithelium cells (yellow), which do not yet respond to FGF (curved arrows
in upper left and middle). ln a second step, FGF2 drives Pax6-positive retinal pigment epithelium cells into transdifferentiation. The first
step is reversible, the second is not.
BM: Basement membrane; NR: Neural retina; RVM: Retinal vascular membrane.
Adapted with permission from 1roe1.
525
Expert Reu. Ophthalmol. 5(4), (2010)
New concepls for reconslruclion ot retinol & pigmenl epilheliol lissues
reversible step is initiated by alteratious of interactions of cells
with the extracellular matrix or neighboring cells, which is followed by Pax6 upregulation. Only during a second step does
FGF2 come into action by driving RPE cells to dedifferentiate
into rerinal stem cells 1871.
Transdifferentiation of
RPE cells
in developing
avian embryos
In the avian embryo,
such a process ofRPE transdifferentiation
into the retina is limited to early stages ofembryonic development.
That this capacity can be extended iz zllra has been discovered
by reaggregate studies of dispersed or explanted retina and pigmented cells from the eye periphery ts8-er1. Further lz az'lro studies
revealed that FGF2 causes RPE cells to proliferate and differentiate into retinal neural cells 1ll,,ls], again showing regeneration
from RPE cells, which requires growth factors such as FGF for
the onset ofrransdifferentiation [34,,16,1771,r21 . All ofthe RPE cells
differentiated into neural cells in uitro as seen in uiuo, differing
from cultures ofamphibian RPE, where RPE cells adhering to
the choroid remain pigmented.
The molecular mechanism of avian RPE transdifferentiation
has been studied intensely. As outlined earlier, Mit{ a basic helixloop-helix leucine zipper protein, plays a major role in the fate
determination of RPE cells. Retrovirus-mediated overexpression
of Mit'inhibited FGF2-induced transdifferentiation of cultured
chick RPE cells, indicating that downregulation ofMitfexpression is essential for RPE transdifferentiation ts:1. A link between
rhe intracellular FGF signaling pathway and Mitf has been
revealed in the developing eye 141,4e,e41. FGF2 stimulates Pax6
expression during induction of transdifferentiation of the RPE
through the FGFR/MEK/Erk signaling c scadele4.When Pax6
is experimentally overexpressed in cultured RPE cells, or in normally developing eyes in the chick embryo, transdifferentiation
of NR from RPE cells was induced without addition of FGFs.
Pax6-mediated transdifferentiation can be induced even at later
stages
embryonic retina lz zlza, suggesting that iris pigmented epithelial
cells could provide a potential source for therapeutic application
for retinaldiseases lq9l. In facr, retinal stem cells have been isolated
from the ciliary marginal zone of rodent, porcine and human
eyes Ilor-lo4j, and transplantation experiments have confirmed
their potential for cell replacement in retinal degenerative diseases U021. However, it remains unclear whether they were from
pigmented or nonpigmented ciliary epithelial cells.
of development
1561.
A similar process was detected after
ectopic expression of neurogenin-2 in cultured RPE cells of the
chick embryo tetl.
Transdiffercntiation of pigmented cells from mammalian
iris to retinal cells
Retinal pigment epithelium transdifferentiation was also reported
in mammalian embryonic cultures, occurring only during very
early stages ofdevelopment under the influence ofFGF2 (mouse
E13 to El4, the optic cup stage t96l). It remains questionable
whether mammalian RPE cells at more advanced developmental
stages retain an ability to transdifferentiate into retinal cells [97].
Pigmented iris epithelial cells ofthe adult rat, however, hav€ been
shown to possess the ability to generate cells expressing neural/retinal antigens under culture conditions [981. Crx gene transfer into
cultured iris cells induced differentiation ofrod PR cells lssl. Even
without gene transfer, purely isolated pigmented iris cells could
form spheres in nonadherent cultures that contained cells expressing retinal progenitor markers [99,loo]. These sphere-forming cells
can also display phenotypes ofPRs and of MCs when grafted into
Advances in retinal &
RPE
tissue engineering
General approaches to tissue engineering
In attempts to develop cell-based therapies for blinding diseases,
two different approaches have to be distinguished. The first is a
more direct approach of implanting appropriate retinal or RPE
precursor cells. wirh rhe hope rhat rhey may integrate autonomously into the remaining (and diseased) target tissue. The second
strategy relies on a lesser degree of cell autonomy within the diseased environment. In this case, the bioengineer will first recon-
struct a piece ofretina or RPE tissue zz zr'lra, which can then be
implanted into the lesioned or diseased location. Only this latter
approach, which is technically much more challenging than the
first one, could be regarded as true tissue engineering (TE; see
FrcuRE rB for a tissue engineered rodent retina). The field ofTE has
become a huge research and industrial endeavor. It deals with the
artificial production ofalmost all tissues, and even whole organs
of the human body t:1. Besides a tremendous spectrum of biological issues, various technological challenges await solutions, for
example, to develop appropriate biomaterials as matrices for tissue attachment, develop preformed biodegradable rissue scaffolds,
improve handling and implantation oftissues, and so on (see larer).
Certainly, onewould only steer towards the rugged and strenuous
route of TE if the simple way did not provide satisfying results.
Only a case-by-case experimental assessment can help decide which
way will be the most rewarding for a given tissue or health problem.
The second strategy, true TE, can be divided up into two
separate challenges:
.
To produce (in most cases) a diverse population ofdifferentiated cells from an appropriate stem cell population in sufficient
amounrs wir hin a reasonable time frame;
.
To bring these cells into their natural tissue context (including
formation of ECM, cell-cell junctions, basal laminae, correct
neighborhood constellations, neural connections, and so on),
so
that
a
functional tissue will eventually emerge in uitro.
Often, material supports and technical tricks may be helpful
these endeavors; for example, shaping the forming tissue by
preformed scaffolds (e.g., cartilage replacements), or cell printing
into a prepatterned matrix (see later). Much recent experience
in the 6eld ofTE has clearly demonstrated that the best way of
in
achieving a complex tissue is to let nature take its course. That is
to say, one should take advantage of the highest possible degree
of self-organizing capacity of a given precursor cell population
without much external disturbance (which is, in fact, the basis
of directly injecting cells, e.g., approach 1), and keep technical
or molecular interference as marginal as possible. Once a certain
[oy€], Ardki & Vog€l-H6pker
degree oftissue formation has been achieved, one can then
further
focus on how to implant and integrate the tissue into the given
surrounds ofthe diseased tissue or organ.
Cellular hases ol rctinal tissue fomation
There is a huge difference between a fla! layer ofcells kept on a
plastic surface and a complex 3D tissue 11051. Th€ pric€ for going
into the third dimension is high, since producing and handling
3D cell spheres is time consuming and imaging is diffrcult 1roe1,
but this approach certainly has a brighr future. Organotypic
cultures have been widely used to develop engineered tissues
for mosr organs thar can eventually be employed in clinical
therapy; typically, they are based on reaggregated spheres (for
review see tll).
Self-organization is certainly one of the most stunning basic
features ofliving organisms at all scales oforganization, from the
molecular to population level. This becomes particularly evident
with developing or regenerative cell systems. The capacity ofcertain cells to reassemble and reform tissues or even whole animals
refers historically to the introduction of the so-called reaggregation approach, which nowadays repres€nts the basis of all TE.
This technique has a history more than a cenrury old, beginning
with the reaggregation of dissociated sponges into whole living
sponges Ir07]. Subsequently, this was followed by the reconstruc-
tion ofseveral tissues and more complex whole animals 007-llol.
Such organotypic cultures present a major advantage over conven-
tional monolayer cultures, in that their growth ofcells develops
into histotypic 3D tissues.
Reaggregated cell spheres can form functional tissues
The reaggregate approach amempts to achieve - under controlled
culture conditions - a complete reconsiruction oftissues from dispersed cells ofa particular origin (typically embryonic progenitor
cells, embryonic or ASCs; see later). Thereby, cell proliferation,
spatial sorting and self-organization of multipotent stem cells
play crucial roles. Technically, a tissue ofinrerest is enzymatically
and/or mechanically dissociated. Then, the dispersed cells are
reaggregated by constant rotation into cellular spheres. Spheres
are raised either as gyratory cultures in regular culture dishes,
in spinner flasks, in conical tubes, in a roller drum ot technically more advanced, in microcompartme nts (chips) on bioreac-
Il
l. In contrast to conventional monolayer cellcultures where
cells grow only into two dimensions on a flat plastic surface, in
suspension culture tissue growth will occur into all rhree dimensions (reviews [lr2-lr4]). Generally, in 3D spheres proliferation
rates are higher, and differentiation resembles tn ria conditions
more closely. The geometry of the conrainers and the speed of
lotation are all critical, The free-floaring roller tube culture syscem is parricularly suited for culturing tissue explants or slices
in suspension Illi], while reaggregate cultures aae more advantageous for studying the primary mixing ofcells, and their pattern
of differentiation and growth within growing spheres. Hanging
drop cultures, a special type ofsuspension culture requiring only
a small volume of culture medium, are parricularly suired for
raising mammalian blastocysts or embryoid bodies from ESCs
tors
530
under in uitro conditions [116l. In addition, the development of
high-throughput systems and manipulation ofindividual spheres
can be achieved tllll.
Before describing retinal tissue reconstruction by the reaggregate approach, some cell biological aspects ofcellular reaggregates
and their applications in basic rcsearch need a brief discussion.
Technically, short-term reaggregation experiments, lasting from
minutes to a few hours, should be distinguished from long-term
studies, ofwhich only rhe latter are ofinterest here. On a timescale of I day to severalweeks, the reaggregate approach allows the
strdy of the de nouoformation oftissue-like cell alrangements. As
an advantage, there is no cellular prepattern from which the tissue
originates. In a sense, the reaggregation approach is a distinctly
arrificial one: its primary goal is not ro mimic as closely as possible normal tissue formation (although eventually this might be
achieved and be applied in TE; see section on retina later), but
rarher ro unravel basic principles of tissue formation. Similar to
monolayer cultures where the individual 'naked cell' is studied
under controlled conditions, with reaggregates we follow tissue
formation 'cell-by-cell', from dispersed cells to organized spheres,
under a controlled environment. Thus, tissue-inherent formative
laws are becer revealed. This feature has been much exploited
in cancer research. Growth regulation in multicellular tumor
spheroids much resembles that in a solid tumor. The constraints
of tumor growth (supplementation, oxygen consumption, and
so on), its sensitivity to temperarure, drugs or radiation, infiltration into noncancerous tissues, and its dependency on capillary
supply are all issues rhat are analyzed by tumor sphere technologies tllT-llel. Using either primary or rransformed cells, sphere
technologies promote the development of gene therapies, since
dispersed cells can be efficiently transfected by genes ofinrerest.
A major technical advantage of using spheroid technologies is
that it is easy to perfolm loss-of-function experiments, including siRNA knockdown of relevant genes Uz0,l2ll. Thereafter,
rhe histological restoration or malformations in transfected
spheres can be analyzed. As anorher huge 6eld of application
of cellular spheres, they are increasingly employed as assay systems in toxicology, pharmacologl nutrition and environmental
biomonitoring lrzz rzll.
The retina reconstructed in vitro
In order to advance these various applicarions for TE, it is essential to undersrand how a tissue can be constructed step-by-step
from dispersed cells. The vertebrate rerina represenrs rhe mosr
powerful reaggregarion model developed to date for the analysis
of tissue regeneration. Fully dissociared cells of the embryonic
avian retina have the capacity to reconstiture different types of
with a more-o css complere arrangemenr ofretinal layers, allowing the analysis of basic principles of neural layer forspheres
mation
[108,110,112,125-128].
Histot]'pic self-organization in rerinal
not only due to the ortanization ofcells, but during a
secondary phase reaggregated cells proliferate and differentiate
to eventually establish a structured tissue. Spheres derived from
the avian embryonic retina reach a diameter of approximately
400 pm (Frcuix 5). Leading structures are intetnal rosettes, which
spheres is
E pct,
k,.
opr'haltuol. 514), 120t0)
at the onset of reaggregation hold mitotic
cells and later PRs, thus corresponding to
an ONL. Both rods and cones are found
at distinct ratios within rosettes t1291. All
major cell types are integrated in appropriate laminar positions. Processes of MCs
span through layered sections homologous
to ONLs and inner nuclear layers, separated by an OPL, to reach into a matrix
homologous to a circular inner plexiform
layer (IPL), where some displaced ACs
are loosely distributed. An inner limiting membrane is missing, GCs are rare,
and nonorganized areas are randomly
interspersed ln9,t3ol.
In these so-called rosetted retinal spheroids (F,cu*" ;), the differentiation of ACs,
HCs, GCs and MCs was detected by multiple markers. Differentiation of the various types of PRs could be particularly well
detected. Their expression depends on the
presence of MCs 1l3rl and growth factors
1t29,132,r331, but also on the type of spheroid, for example the conditions of a 3D
tissue environment [132]. The development
of synapses, calcium uptake and physioIogical functioning has been demonstrated
1t34,t35). Retinal spheroids were instrumental
to analyze the effects ofvarious growth factors on differentiation of retinal cell types
within a 3D cell context. 'W'hen rosetted
spheroids were cultured in the presence of
50 ng/ml glial derived neurotrophic factor
(GDNF), de nouo production ofrod PRs was
Figure 5. Organotypic reaggregation of retinal spheroids from dispersed retinal
progenitor cells from embryonic day 5 chick embryo. Note histotypic formation of
areas homologous to all specific layers of a normal retina; however, orientation of layers
is inverted compared with normal retina (FrcuRE2). 'lnner plexiform layer'-like areas
correspond to inner plexiform layer. which is surrounded by cells of the inner retina; 'ROS'
- rosette - holding an immature 'Outer nuclear layer' corresponds to the outer retina.
Pax6 (red) and DAPI (blue). Using other cell-specific markers, all cell types at appropriate
places were detected in these spheres (ganglion cells are rare; for other markers used to
identify all retinal cell types, IPL neuropil and synapses including transmission electron
microscopy. see [113,142,160]). Cells were cultured for 8 days in vitro. Scale bar: 200 pm.
AC; Amacrine cell; HC: Horizontal cell; IPL: lnner plexiform layer; ONL: Outer
nuclear layer.
Reprinted with permission from G Bachmann, Technische UniversitSt Darmstadt
(Darmstadt. Germany).
affected precursor cells, PRs and other cells l14l-l4s]. Recently, we have
supported
by managed to further advance the in uino reconstruction of the
ACs
were
strongly
ACs,
Among
dopaminergic
f1321.
GDNF. In the absence of GDNF, rod, but not cone PRs, under- gerbtl (Meriones unguiculatus) retina, which is comparable to
went apoprosis tt36l. In contrast to GDNF, pigment epithelium- stratified chicken spheres [111,14s].
Some disadvantages ofthe aforementioned reaggregate models
derived factor supported cones and inhibired rods, such that after
observed, whereas cone PRs, HCs, GCs and MCs were not
!
6,
\
1
t
\
l0 days i.c. the ratio between cones to rods was approximately tenfold. Moreover, while GDNF supported growth ofMCs in spheres,
pigment epithelium-derived factor inhibited it p:21. Remarkably,
FGF2 restricted the pool of PRs in favor of cells of the inner retina;
it increased and maintained rheir precursor pool, delayed their
differentiation, and protected them from apoptosis U301.
In mammals, similar reaggregation experiments have been
performed wirh retinal cells from the rat. In neonatal rat retinal
{
I
I
e
I
4
(
sis
and/or life imaging. Howevet this will be of utmost impor-
could
tance for future bioengineering; for example, replacements for
animal experimentation. Therefore , we comPared conventionally
be detected, including all major cell types except GCs. In a
study using pellet cultures of embryonic rat cells, GCs could be
found U:q,r+ol. Their histotypical arrangement was similar to that
in spheroids from chick rerinae . In the wake of stem cell biology,
the reaggregate approach has gained much impetus, and the use
of mammalian rerinal reaggregates or related approaches have
revealed significant new insights into the regulation of retinal
reaggregatedspheroidsderivedfromdissociatedretinalcellsfrom
neonatal gerbils with spheroids cultured on a novel microscaffold
cell chip (cf-chip) in a motion-free bioreactor. Further development of this approach should allow high-throughput systems, not
only for retinal but also for other tyPes of histotypic spheroids,
to become suitable for environmental monitoring, biomedical
diagnostics and reducing animal experimentation [11i,149,150].
reaggregares grlt1, significant histological differe ntiation
1
that the growing specimens have to be cultivated under motion,
the number of growing spheres is highly variable, and analysis of
individual spheres during growth is almost impossible. These
obstacles become particularly hindering if one wishes to develop
miniaturized, inexpensive, large-scale systems for high throughput and high reproducibility, possibly with multiparameter analyare
www-exDert-revlews.com
531
loyer, A]oki & Vogel-H6pker
Reversal of retinal tissue polarity in vilro
Albeit representing highly organized histotypical structures, it is
evident that rosetted retinal spheres resemble more an inverted
than a regular retina; for example, PRs are oriented internally
instead of forming the external surface of the dssue. Are there
means to restore a regular afiangement of layers within retinal
spheres? What are the constraints for avoiding rosette formation,
which - as a corollary - is a complication in some ophthalmic
? Indeed, procedures could be established frrst for the avian
system (for rodents, see tl48l) that allowed the complete reconstfuc-
diseases
tion ofa correct arrangement oflayers lz zlrra; so-called suatified
retinalspheres (Frcurt2). We were first ro note that fullylaminated
retinal spheres develop from retinal dispersed cells, provided that
RPE cells are included 1n,rs4, Remarkably, they were produced in
particularly high ratios ifcells were derived from the ciliary margin
t901, pointing to the fact that retinal stem cells are located in this
peripheral neuroepithelial zone F521. In subsequent srudies, it was
found that in these stratified retinospheres a complete arrangement oflayers is achieyed (FrcuRD2), provided that the population of
dispersed retinal progenitor cells is young and supplemented with
soluble factors from the RPE or from radial glial cells tl53l. Afrer
reaggregation and formation of rosettes,
a
conspicuous involurion
ofrosettes plus their adhering laminar tissue leads to a complete
inside-out rearrangement ofall cellular layers [1i4]. Thus, ov€r a
period of approximately l0 days, we watch the development ofa
'mini-eye' from fully dispersed cells to a completely laminated
sphere, merely missing a vitreous body inside and an RPE layer
outside. Recently, reaggregates ofretinal cells from the postnatal
gerbil have been brought to a similarly high degree oftissue organization (FrcuRx rB) [118] IRTEKE M, uNru!'rsEEo oerel, whereby not only
RPE supernatant but also supplementation with Wnt3a was essential. Furthermore, MCs, presenr in high numbers in reaggregates,
play a leading role in this process [112,r31,153].
Depending on the particular topics ofinterest, other cell culture
techniques ofretinal tissues may present distinct advantages over
using reaggregated spheroids as described earlier. For instance,
under roller culture conditions, floating retinal sections from
7-|2-day-oId rats form ball-shaped retinal bodies. Histological
studies of retinal bodies showed that their outer surface was
formed by the retina and completely retained the organotypic
cytoarchitectonics Il5tl; this method was similarly applied to
culturing the posterior sector ofthe eye tr56l. Organotypic retinal wholemount cultures provided an environment close to the
physiological in aiuo situation, whereby neuronal connecrions
and interactions were still preserved 0t71. Morcover, an explant
organotypic tissue culture system ofthe adult retina was suitable
a.s a.n in uitlo intr^ocular stem cell transplantation model U:sl.
Retina & eyeJike structures from stem cells ot cell lines
Much early work on eye differentiation and regeneration has indi-
regenerative medicine ofthe visual system, Indeed, under appropriate conditions stem cells ofvarious origins could become sources
for retinal TE and help repairvision defects; occasionally, they can
even form complete eye structures, Thereby, the environment
of
the eye cup provides important clues for proper growth,
Amphibians, whose eyes possess a relatively high regenerative
capacity, present a 6rst example. With glound-breaking work, the
Asashima group has managed the in uitro formation of complete
eyes from early gastrulae of Xenopus, whicb still consisted ofpluripotent stem cells. Using classical sandwich culture techniques, cells
from rhe dorsal lip and Iateral marginal zone ofthe embryo were dissected and then cultured for 4 days (equivalent to stage 42) between
two sheets of animal caps from late blastulae (stage 9). Under these
conditions, eye development was induced frequendy (867o) in the
cultured explants. The eyes could be transplanted into a stage-33
tadpole host, where they integrated and then established nerve connections to the aeclum ofthe host brain. The grafted eye remained
intact after meramorphosis and behavioral effects indicated that
the eyes were functioning in the adult frog tr5el. The capacity of
amphibian stem cells to produce retinalike tissues has also been
investigated under rz zzzo conditions. Pluripotent stem cells isolated
from the animal pole of Xenopus blasrulae were managed to differentiate into multipotent retinal progenitors and eventually into
complete eyes il60l. If the untreated pluripotent cells were transplanted into the fank or the eye field ofdeveloping embryos, they
formed epidermal cells. However, they could be directed to form
rednal cell q'pes after misexpression ofseven transcription factors
(i.e., Pax6, Tbxl, Rx1, Nr2e1, Six3, Six6 and Otx2). Funhermore,
in both places the cells now formed functional eyes, exhibiting a
complerc structure and electrical physiology. This supports the hope
that multipotent retinal progenitor cells can be used as a source ro
produce retinal cell types and form functional neural circuitries.
Embryonic stem cells from mouse, human and other origins
can be repeatedly differentiated into retinal cells, including PRs
and GCs n6l 1661. Such derivatives from ESCs were transplanted
into the vitreous ofa slow retinal regenerative mouse model 1167l.
Furthermore, PRs derived from ESCs were transplanted into Crxdeficient mice, where they partially restored visual function prrl,
In probing the capacity ofhESCs for the generation ofretinal dssues, aJapanese group has implanted undifferentiated hESCs into
the empty vitreous cavity ofmouse embryos, with some striking
results. This direct approach was intended to overcome the long
procedure ofconstructing a complicated organ, instead inducing
retinal organlike structures more directly within a narural environment. Hence, the authors tested the cultivation ofhESCs in
the adult mouse eye, expectingrhe ifi !;!o environmental cues of
the mouse eye to be favorable for eye developmenr. Approximarcly
1 month after transplantation, two-thirds ofthe mouse eyes had
teratomas that wele derived from the hESCs. Strikingly, in more
than 900/o of these teratoma-like masses, 3D-organized opric
cated that under certain circumstances (experimental or lesioned),
cup-like structures were detected, similar to rhe embryonic eye,
both layers ofthe optic cup, for example the retina and the RPE,
are intimately integrated with each other and can present tendencies to replace for the other. This earlier work (see previously)
has provided an indispensable basis for present day research into
The structures were reminiscent of the invaginating optic vesicle, including an inner retina-like and outer pigmented tissue
layer prll. !0hile this procedure certainlydoes not open the'direct
532
TE way to heaven', it does clearly show that the microenvironment
Eqat
Rer. Ophthalnol. 5/i4), (10t0)
tNew conc€pls tor reconslruclion ot letinol & pigmenl epitheliol tissues
ofa mouse eye devoid ofretina and RPE can provide major directives for inducing ESCs to form eyeJike structures. Clearly, the
3D microenvironment in which cells develop, as well as the inherent 3D environment thar rhe cells themselves will fom, both have
a
tremendous impact on proper tissue construction. Even progeni-
ror cells from the hippocampus transplanted into the recina of
mature dystrophic rats were correctly integrared and differentiated
into retinal cells within the retina
1rzo1.
The differentiation of
retinal progenitor cells derived from embryonic day 14 (El4\ or
E18 rat retina was assessed in retioal spheres cultured in conditioned medium from E3 chick retina. !0hen this medium, which
supposedly simulated the microenvironmenr of early retinal histogenesis, was separated inro rhree differenr acrive fracrions, rhey
could be associated with the differentiation ofdistinct retinal cell
types tr7ll. These studies show that particular constituents derived
from the eye microenvironment are responsible for direcring the
differentiation of speci6c retinal cell groups.
With detailed studies on the regularion of retinal progenitor
cells, the Gamm group is also taking advantage of the 3D in uitro
reaggregate apptoach lrzz-rz;1. Their neurospheres are derived
from human fetal retina, which are sectioned such that the original microenvironment may be preserved. Ifthe tissue was derived
from later stages, more glial cells and fewer neurons were produced 1ru1. Furthermore, ir was found that conditioned medium
secreted from human prenatal RPE prolonged and enhanced the
growth of prenaral rerinal neurospheres. The growth-promoting
activiry ofRPE-conditioned medium was associated wirh an acute
increase in transcription factor phosphorylation, and was dependent on mitogens. As rhe cuftures were expanded, they lost their
capacity ro produce neuronal cell types, an effect that could be
counteracted by misexpression of Mash-1. This indicates that
both extrinsic factors from the RPE but also intrinsic ones are
required to keep these cells on a retinogenic pathway lr7al. \X/ith
an excellent recent study, the same group has produced almost
complete retinal spheres from both human ESCs and iPSCs. In
particular, they could show rhat these cells are capable of following a normal developmental schedule to undergo a targeted,
stepwise differentiation process in uitro, as necessary for human
.etinogenesis {r7tl. More strikingly, all cells that formed within
a spherical 3D environment were capable of selForganizing and
establishing fully laminated retinal spheres. Thus, a complete
retinal tissue could be derived from various stem cells, representing a tissue construct that was very similar to what has been
described for avian and murine retinal spheroids derived from
dispersed embryonic retinal progenitors (see previously). This
study demonstrates thar as appropriate stem cells may become
available for rcutine use, particularly iPSCs, they could readily
form into highly structured retinal spheres, which may become
implantable. Moreover, this approach shows its potency to analyze
lineage-speci6c gene expression during neural and retinalcellfate
determinarion, and can help to undersrand the molecular events
governing rctinal specification from human plulipotentstem cclls.
Using a spontaneously immortalized human retinal progenitor
cell line, a similar study again points to rhe importance of rhe
3D environmenr as provided under rorary culture conditions to
achieve retinalike tissue conslrucrs 0761. \X/hen cultured alone
or in combination with RPE cells in a rotary system, agtregates
were formed within 10 days, with cells retaining a remarkable
spectrum of differenciation capabilities. The differentiation of
rod and cone PRs and other retinal cell types was escablished by
the expression of multiple markers. This technology could lead ro
the engineering ofretinal constructs, thus holding great promise
for the use of tissue-speci6c ASCs for therapy.
RPE tissue from stem cells or from RPE
The RPE ful6ls outstanding roles for the well-being ofthe retina
and particularly for the maintenance of PRs (see srart of this
article). Recently, this has been elegantly demonstrated by using
a 'foxed mice method', whereby 60-800/o ofall RPE cells were
knocked out. Although the remaining RPE cells in the resulting
RPE-(CreER)/DTA double-knockout mouse had reorganized
into a RPE monolayer, significant deficits in retinal histology
and electroretinography were revealed, whereby regions of PR
.osetting and degeneration became evident ttzzl. This showed
once again that healthy RPE cells are an absolute requiremenr
for proper visual function. As a monolayer ofcells so critical to
PR function and survival, the RPE is an ideally accessible target
for cellular therapy. Some fascinating research focuses on the
production of RPE cells from different types of stem cells to
be used in replacement therapies for damaged RPE. Important
potential applications ofhESCs would be the use ofRPE for the
treatment ofAMD, and also for Stargardt's disease, an untreatable form ofmacular dystrophy that leads to early-onset blindness
(see
previously).
Experiments using whole sheets of fetal or adulr RPE from
donors, or dissociated cells from such donor tissues, have been
performed and were partially successful. For instance, retinal
sheets including RPE from human fetuses were rransplanted into
blind patients, after which some remarkable sight improvements
were reported 1tzs1. Thc molecular nature ofthese results was not
fully clarified; however, in their extensive animal experiments the
authors point out that the RPE was essential 1tzr1. Besides unacceptable ethical issues, very limited amounts of available tissue
and poor cell integration would make treatments with primary
human tissues ineffrcient 0sol. Given these obstacles with primary
donor RPE tissues, human ESCs could possiblyserve as an unlimited source of RPE cells, which - after efGcienr multiplication
in uitro - codd be used for transplantation in a number ofblinding conditions. For instance, a culture system has been eseblished
to generate eyeJike structures consisting of lens, NR and RPE
cells from undifferentiated ESCs. RPE precursors differentiaring
in the cultures were rcsponsive to Vnt2b signaling. They were
shown to form secondary colonies within the eyeJike structures
consisting of RPE-Iike cells only. These transplanted eyeJike
structures were found to populate the developing chickeye as neuronal retina and RPE cells I169l. In another study, highly confuenr
hESCs or {loating embryoid bodies have presented pigmented
areas after 6-8 weeks Il8l.ls2). During generation of pigmented
foci Otxl'or Otx2'cell types were characterized as potenrial
RPE precursors. \Vhen excised and further c:uhlured, in uitro, rhese
loyer, A]okl & Vogel-H6pker
cells could - without signals from a developing retinal environment - produce polarized monolayers with distinctive apical and
basal features, thereby expressing markers ofboth developing
and mature RPE cells, including Otxl, Otx2, Pax6 and Pmel-17.
Their transplantation ino the subretinal space ofRCS rats, which
present a degenerating environment, allowed the cells ro survive
and maintain low levels of RPE65 expression, without relapse
into cell proliferation lls3l. This group has recently established a
novel coculture system ofhESC-derived RPE cells wirh porcine
outer segmen$ or, alternatively, with fresh human neural retinal dssue, which allowed molecular analysis of phagocytosis of
outer segments. Such hESC-derived RPE cells expressed relevanr
molecules required for phagocytosis ofouter segments, including
MeiTK psr;. Similarly differentiated from mouse ESCs, RPE cells
were transplanred into the Rpe65/Rpe65 C57BL6 mouse model
for RP in order to test restoration ofretinal funcrion 1ls5l. Again,
the findings were two-sided: one-quafier of the mice showed
iocteased electroretinogram responses in the transplanted eyes
(and nor in control eyes), but more than half of the mice wele
inflicted with retinal detachments or tumor developmenr,
Further aspects of ophthalmic regenerative medicine
Takahashi et al. haye foctsed on understanding and improving
the directed differentiation of mouse and human ESCs, and
also iPSCs into retinal and/or pigmented cells, with emphasis
on the production of PRs tls6,l87l. A newly designed serum-free
and floating aggregate culrure is supplemenred with the Alk4
I receptor acrivating
SMAD2/3 phosphor)'lation), the casein kinase I inhibircr Cki-7
and the Rho-associated kinase inhibitor Y-27632 trss). \Vith this
treatment, both types ofstem cells were induced into retinal
protenitors positive for Rx, MitC Pax6 and Chx10, and RPE
cells, defined by RPE65, CRALBP and ZO-l expressions. After
further treatment with retinoic acid and taurine, cells differentiated into PRs, which were characterized by the expression of
recoueri n, rh odops in and phototransduction-relevant genes. This
inhibitor SB-431542 (ALK4 is a type
novel procedure maymark a significant step towards introducing
iPSCs for retinal repair; for example, autologous cells derived
from the patient could be used to produce iPSCs, avoiding any
antigenic problems.
Although functional RPE cells can be generated from hESCs
via spontaneous differentiariol, the efficiency remained low and
the temporal rate of their ploducrion slow. Recently, the yield
of differentiated RPE cells from hESCs could be improved dramarically by novel defrned culture conditiols 1lsg]. The major
chemical player was nicotinamide, which was found to promote
the differentiation of hESCs first to a neural and subsequently
to the RPE fate. At the same time, apoptosis was inhibited, possibly leading ro an increased survival ofcells, supposedly in the
state of presumptive anterior neural plare cells. The presence of
nicotinamide and factors from the TGF-p superfamily (see previously) further directed the differentiation towards RPE. However,
replacing Activin A with FGF2 inhibired rather rhan promored
their passage towards RPE. The polygonal morphology ofcells,
their expression ofmarkers, the formation ofright junctions and
534
their capability to phagocytose PR oucer segments clearly established their RPE identity. To challenge their functiona.lity in uiuo,
RPE cells from a GFP-labeled hESC cell line were transplanted
into the subretinal space of RCS rats. Occasionally they were
integrated into the host RPE with some albeit rather weak functional recovery (e.g., phagocytosis, ERG light responses).
Luckily, tumor growth was not observed.
Could direct transfer of hESCs into patients' €yes become a
therapyi Indeed, the findings to date are quire promising. ESCs
were transplanted into the subretinal space ofRCS rats and were
shown to rescue degeneraring PR cells tryol. By simply transplanting stem cells, degeneration of PR cells could be delayed.
Moreover, a recent safety study suggests that transplantation of
RPE cells derived from ESCs in both the RCS rat and Elovl4
mouse, which are animal models of retinal degeneration and
Stargardq respectivel)', appear to be safe for the whole lifetime
ofthe tested mice. The RPE cells susrained visual function and
PR integrity without teratoma formation or other pathological
reactions. Near normal functional measurements were recorded
afrer 2 months survival in RCS rars tl9ll. Although thcsc safety
outcomes with ESCs are better rhan expected, Lund's group
is also using mesenchymal stem cells for RPE production and
transplantation tlst. This could be even more appropriate for
therapies, since the risks oftumor growth or antigenic reacrions
cannot be ignored. As a further cell source for RPE cells in therapy, hematopoietic stem cells (HSCs), were rhe easiesr to come
by. After chemical destruction of the RPE, which also induced
PR degeneration in mice, a structural and functional resroration
could be achieved by injection of HSCs that had been infected
ex uitto wirh a.lenriviral vecror expressing the RPE-specific gene
RPE65. A RPE layer was re-established, with typical phenotype including the coexpression of CRALBP as a RPE-specific
marker, and PR outer segment phagocytosis. Moreover, retinal
degeneration was prevented and visual funcrion, as measured by
electrorerinography, was restorcd to levels similar to that found
in normal animals. Since adenylate cyclase was associared wirh
RPE65 expression and differentiation rowards RPE cells, the
authors applied an agonist of this enzyme. Thereby, rhey could
transdifferentiate HSCs into RPE cells ln uitro ar'd tse those for
implantation tr92l. Indeed, this procedure could become a method
of choice, if the efficiency and purity of RPE cell production
are sufficient.
In summary, the studies using ESCs show both the potential
ofhESCs as a possible cell source to replenish RPE in blinding
diseases, while at the same time reminding us of the problems
of using ESCs for therapies (possibility of tumor growth) and
the complexity of directing them cleanly into a well-differentiated 6nal cell type. Ifstem cell biology is to lead to real
applicable therapies, a brighter furure is promised by the field
of iPSCs tre3-re5l. Although this field is still in its beginnings,
there are first r€ports on the attempts of differenriating iPSCs
into functional RPE cells 0e61, both from mouse and human
iPSCs trszl. The iPSCs, generated by the expression of Oct4,
Sox2, Nanog and Lin28, were shown to sponraneously differenriate into RPE cells. After their isolation, they were cultured to
E,pat R.v. oftthalno|.
5(),
{2010)
New concepls tor reconslruclion ol relinol & pigmenl epilheliol tissues
form differentiared RPE monolayers. Remarkably, the expression
ofRPE markers and their phagocytotic capacity was similar to
that seen in RPE cells derived from fetal RPE or hESCs. In a
further study, RPE cells from iPSCs were transplanted into RCS
rats, where they facilitated the short'term maintenance of PRs
through phagocytosis of PR outer segments 0981. Strikingly, the
authors also report that long-term visual function was maintained in this model, even though the xenografted cells were
eventually lost. Certainly, more information will be needed to
support the author's suggestion that a secondary protective host
cellular response may explain this perplexing observation. Both
studies have identified an alternative source ofreplacement tissue
for use in human retinal cellular therapies, thus clearly showing the high potential that iPSCs also have in the treatment of
severe
vision defects.
Technological aspects of engineering of eye tissues
At this point, of course, rhe question remains whether histo-
typical cellular spheres as produced by reaggregation can be
implanted into a diseased organ; for example, into a lesioned
retina. Retinal spheres have been implanted into chick embryos
whose retinae had been extracted at an earlyeye vesicle stage. The
results indicated that the spheres'opened up' after implantation
to achieve a more Iaminar, planar appearance and then began to
integrate into an eye that was devoid ofretina and RPE [LAyERPG,
urrurusaro lerel. Similarly, ifone transfers spheres from rotation
into a standing cuhure, the spheres will eventually form a flat
piece oftissue. Howevet in this respect, much more research will
be required. In some recent attempts, spheroids from rat retina
were prepared as tissues for transplantation 1r+:1; the spheres were
labeled with a lipophilic dye and placed onto rat retinal explants.
This allowed the analysis of tissue interactions after 2-6 days
of culture. Retinal spheres, including differentiated PRs and
other retinal cell types that were grafted onto retinal explants
demonstrated integration into the host tissue with a consistent
penerration ofglial cell processes into the explanted tissue tl99l.
Vith this intelligent approach, spheroids were instrumental to
study tissue interactions of an implanted tissue sphere into the
host tissue.
The choice ofappropriate biomaterials as carriers or substrate
matrices for cells or tissues will be a major issue for their safe
handling and implantation; however, it is out of the scope of
this article to deal with this topic in any detail I3l. As just one
example, it was shown thar if PR cells were micropatterned on
biodegradable PLGA-PHBV8 blend frlms (poly-rJactic acid-co-
glycolic acid/polylhydroxybutyrate-co-hydroxyvaleric acidl),
they could be delivered in a more organized manner than after
injections without this carrier. Thus, well-chosen matrices have
the potential to deliver PRs to the subretinal space and ensure
laminar organization and maintenance of differentiation.
Furthermore, it was also demonstrated that incorporation of
intrinsic factors within the scaffold enhances the survival rate of
transplanted PRs [200]. To master the difficulty oftransplanting
the fragile and soft retinal tissue, fabricated biodegradable gelatin membranes were formed into sandwiches, in which retinal
www.€xpert-rev,ews.com
grafrs were encapsulated for transplantation , ln their in uioo
rabbit study, the gelatin membranes demonstrated satisfactory
biocompatibility without any inflammation. The transplanted
retinal sheets survived well and developed into laminar structures [2ol]. In terms ofpracticability ofsurgical procedures, the
handling and transfer ofRPE monolayer sheets that have been
successfully cultured in uitro could represent a major obstacle.
To facilitate this, ARPE-19 cells were labeled with magnetite
cationic liposomes. Using a magnet, the magnetically labeled
cells could first be organized into a flat sheet ofRPE cells. After
removal ofthe magnet, the cell sheet could be easily detached.
Again applying the magnet, they could be transferred into a
location ofinterest [zo2]. Generally in TE, appropriate artificial
scaffold architectures may become instrumental to support the
integration ofa preformed tissue. For instance, unidirectional
channels and microgrooves, oriented longitudinally within
cylindrical sponge scaffolds, supported astrocytic infiltration
into a rat brain regeneration paradigm t20ll. Obviously, much
more research needs to be invested imo these technical aspects;
however, it appears that these obstacles should not be of an
insurmountable nature.
Are there other technologies for the production of a laminar tissue, such as the vertebrate retina, if the appropriate cells
were available? Recently, the inkjet printing technique has been
introduced as a promising approach for the creation of cellular patterns on substrates. The printing, either of living cells
or of substrate prepatterns, allowed the patterned culturing of
cells pzoal. Various cellular patterns including figures, letters and
gradients could be fabricated by seeding mouse fibroblasts or
neuroblastoma cells onto a printed polyethyleneimine-patterned
substrate, which was crosslinked to an albumin substrare [zo;].
For the human eye, microcontact printing, a modern material
fabrication technique, has been used to pattern the spatial distribution ofinhibitory molecules and thus direct the growth ofRPE
or iris pigment epithelial cells on human lens capsules t2061. Since
the retina represents a highly organized neural network with a
simple threefold laminar arrangement of its cell bodies, recent
advances with regular inkjet printers used as cell printers (socalled 'bioprinters') are fascinating and rhey may become a useful
method for speci6c applications ;zoz1. However, the production of
fitting numbers ofall necessary cell types from a precursor cell
population (e.g., stem cells), their identification and handling,
to be finally applied in a bioprinter, seems an extremely difficult
problem. The self-organising power of a reaggregating system
(as discussed previously) presents superior advantages ofraising
cells
within growing
spheres.
Expert commentary
Are stem cell researchers fuelling too much hope? Are blind
people too optimistic when they await the coming of a new
age in ophthalmic therapiesl Vithout doubt, we are witnessing
exciting times in new cell-based therapies for ophthalmology.
Although the human retina does not normally show any sign
of regeneration, such capacities in other vertebrates indicate
that some residual hidden activity may still reside even in adult
Loyer, Aroki & Vogel-H6pket
human eyes. Vith this in mind, researchers have argued that
new cell-based technologies may allow us to somehow reactivate this potency or, alternatively, to frod ways to regenerate
rhe necessary cell types (e.g., PRs or RPE cells) and reconsri
tute a lesioned retina. Ifproperly handled, rhis may even apply
to ASCs.
Indeed, stem cell research has now brought us to the point
where more than a narrow brightstripe can be seen at the horizon.
'Where have we seen the greatest advance? Remarkable progress
in the production of
PRs and other retinal cell types has been
achieved from all possible cell sources, be it ESCs, embryonic
germ cells (not dealt with in this article), fetal progenitor cells
or various sources ofASCs (only briefy mentioned here, including transdifferentiation from mesenchymal cells). For instance,
hESCs were transformed into functional RPE cells, and retinalike spheroids were produced from a human progenitor cell line,
very similar to the retinalike spheroid structures produced from
avian and rodent retinal progenitor cells. Thus, a basic knowledge
of retinal tissue formation and regeneration as provided by the
reaggregate approach is stillvery instrumental for the whole field.
As the hottest topic presently in the 6eld, iPSCs were shown to
differentiate towards retinal cells along normal schedules and
arrange properly within growing spheroids. In addition, whole
RPE sheets were derived from iPSCs and led to some functional recovery after their implantation in dysttophic rats. It is
also promising rhat implanted fetal retinal sheets could be both
structurally and functionally inregrated into a lesioned retina.
Due to space limitations, some fascinating approaches could not
be discussed; for example, a genetic transfection ofinner retinal
cells to transform them inro functional PRs (not discussed in
detail in this article).
As remarkable as the progress may already be, significant quesif implantation of a whole piece of
tions still await an answer:
tissue may become feasible, how does it integrate into a lesioned
eye, and can
it establish functional connections with the brain?
How long does the therapeutic effect last? Therefore, a full
understanding of the molecular details of transdifferentiation
.
.
.
.
.
.
.
.
.
of retinal cells is essential, including genetic cascades, the roles
of growth factors and signaling cascades, and the influence of
artificial or natural environments for the production and maintenance ofsuch tissues. There is also a need to address the diverse
technological aspects; for example, how to improve handling
procedures, issues of technological supports and biomaterials, and finally all safety issues need further exploration and
firm documentation.
Five-year view
This 6eld of ophthalmology has now arrived at a most exciting
and promising point of basic research. Nevertheless, it is hard to
predict whether we can expect dramadc breakthroughs within the
next 5 years. Clinical trials for a first gene-based therapy for Leber
congenital amaurosis are ongoing [208,209], and more are certainly to
come. Much more basic research will be necessary to undersand the
reliable amplification and directed differentiation ofany cell source
into a complete functional tissue. The present state ofresearch makes
us confident that the production ofhuman retinal and RPE tissues is
about to become technically feasible. It may depend on rhe specific
eye disease, whether a direct injection of appropriate cells can be
applied, or - ifthis does not suffice implantation ofa reconstituted
piece oftissue into a lesioned eye will be required.
From all possible cell sourcesJ two appear as the most promising.
For sweral reasons, choice number one would be ASCs from the
eye periphery (ciliary margin, iris or RPE) or other sources 02-11.
Howwer, a major problem - as with many ASCs - is still their insufficient rate of amplifi cation in uitro, Ho,Never, it appears likely that
culturing procedures of their amplifrcation and directed differentiation will be much improved in the near future. At present, iPSCs
rWe expect their
as the alternative cell source seem very promising.
reliable differentiation into PRs, GCs, RPE cells and also whole
retinal and RPE tissues to be achieved soon. Major issues remaining
with iPSCs are the need to better understand the environmental
influences on their differentiation and handling, and safety issues.
With the invention of iPSCs, ESCs and human fetal tissues, which
harbor many safety and ethical problems, may fall out offavor, and
Stem cellbased regenerative medicine holds great potential for novel therapies for major blinding diseases.
Two approaches are envisioned: direct injection of appropriate stem cells or their derivatives into a lesioned eye, or the reconstruction
of a retinal or retinal pigmented epithelium (RPE) tissue n vltro (tissue engineering), which then is implanted.
The directed generation of cells or the reconslruction of tissues requires a deep knowledge of the embryonic development of the eye
vesicle into neural retina and the RPE.
transdifferentiation sludies in various vertebrates indicate an innate, albeit sometimes hidden capacity to regenerate a
more-or-less complete retinal tissue from RPE.
The classic reaggregate approach of dispersed embryonic progenitor cells forming retinotypic cellular spheres has allowed analysis of
the principles of self-organization governing retinal tissue (re-)construction (e.9., retinal tissue engineering).
Classic
Based on this knowledge, a multitude of fascinating studies from the last decade using embryonic stem cells (EScs), induced
pluripotent slem cells (iPSC5). adult stem cells (ASCs) or permanent cell lines from various species have demonstrated that the
production of human retinal and RPE cell types is feasible.
of complete retinal tissue, functioning RPE monolayers or eye{ike structures has become a possibility.
After their implantation into appropriate animal models for blinding dlseases, some functional recoveries could be observed.
Considering all possible cell sources, ASC5 and iPSC5 (but not ESCs) hold the greatest potential for future eye therapies.
Even reconstruction
536
E,pot R.". O?ttralnol. 5t4), l2o1o)
New concepls lol leconslruction ol retinol & plgmenl epilheliol llssues
-
as we see it - will play no maior therapeutic role. In concluding
this extensive but still incomplete chapter, it seems likely thar rhe
field ofcell-based vision therapies may soon emerte as a successful
one in regenerative medicine.
Financial & competing interests disclosure
Thtu worb was *pported bl the Deutsche Forcbungsgemeinscbajl (DFG La
379/12-1), European Space Organiation ESA and ECSST Japan. The
duthofi hate no other releuafit afrliations or fnancial innluement tuith
dn! olgnization ot ehtit! aith afnalldal ikterest in orfnancial confict
Acknowledgements
with the sabject matter or mdteriab d*crsted in the manuscript apartfi'om
G Bachmann, A Bltyqi, F Frohns, M Rielee, A Rotltermel,
L Spcrlin& J SteinfeLl and E \Villbold for doclmettation, tecfttaridl
*pport and belpful discassions.
IYe thanh
l1
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