TISSUE-SPECIFIC STEM CELLS
Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem
Cells with Potential for Retinal Repair, Fail to Differentiate into New
Rod Photoreceptors
SARA GUALDONI,a MICHAEL BARON,a JÖRN LAKOWSKI,a SARAH DECEMBRINI,a ALEXANDER J. SMITH,b
RACHAEL A. PEARSON,b ROBIN R. ALI,b JANE C. SOWDENa
Developmental Biology Unit, Institute of Child Health; bDepartment of Genetics, Institute of Ophthalmology,
University College London, London, UK
a
Key Words. Adult stem cells • Transgene expression
cells • Retina • Tissue-specific stem cells
•
Green fluorescent protein
•
Lentiviral vector
•
Neural differentiation
•
Progenitor
ABSTRACT
The ciliary margin in lower vertebrates is a site of continual retinal neurogenesis and a stem cell niche. By contrast,
the human eye ceases retinal neuron production before
birth and loss of photoreceptors during life is permanent
and a major cause of blindness. The discovery of a proliferative cell population in the ciliary epithelium (CE) of
the adult mammalian eye, designated retinal stem cells,
raised the possibility that these cells could help to restore
sight by replacing lost photoreceptors. We previously demonstrated the feasibility of photoreceptor transplantation
using cells from the developing retina. CE cells could provide a renewable source of photoreceptors for transplantation. Several laboratories reported that these cells
generate new photoreceptors, whereas a recent report
questioned the existence of retinal stem cells. We used
Nrl.gfp transgenic mice that express green fluorescent pro-
tein in rod photoreceptors to assess definitively the ability
of CE cells to generate new photoreceptors. We report
that CE cells expanded in monolayer cultures, lose pigmentation, and express a subset of eye field and retinal
progenitor cell markers. Simultaneously, they continue to
express some markers characteristic of differentiated CE
and typically lack a neuronal morphology. Previously
reported photoreceptor differentiation conditions used for
CE cells, as well as conditions used to differentiate embryonic retinal progenitor cells (RPCs) and embryonic stem
cell-derived RPCs, do not effectively activate the Nrl-regulated photoreceptor differentiation program. Therefore,
we conclude that CE cells lack potential for photoreceptor
differentiation and would require reprogramming to be
useful as a source of new photoreceptors. STEM CELLS
2010;28:1048–1059
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Degeneration of photoreceptor cells in the neural retina (NR)
is a major cause of untreatable blindness. Multiple retinal diseases such as retinitis pigmentosa, cone dystrophy and agerelated macular degeneration are all characterized by photoreceptor degeneration. Recent studies have demonstrated that
photoreceptor transplantation is feasible in mice [1–3]. To establish photoreceptor transplantation therapy in humans, a
source of postmitotic photoreceptor precursors is required [1].
Human fetal and embryonic material, not withstanding ethical
implications, is very limited and the access is strictly regulated. Stem cells, with properties of self-renewal and with the
potential to produce large numbers of neurons in vitro, offer
an ideal donor source of cells to generate photoreceptors.
Recently, independent groups reported the differentiation of
human and mouse embryonic stem (ES) cells to a retinal progenitor-like fate, which were further committed to generate
cells expressing photoreceptor markers in vitro [4–7].
Although ES-derived retinal cells may be useful for transplantation, the problem of host rejection remains.
Ideally, stem cells would be acquired from the patient to
avoid the problem of graft rejection. Many investigators have
recently focused their research interest on the reprogramming
of somatic cells by virus gene transfer to generate induced
pluripotent stem (iPS) cells [8–10]. However, the mutagenesis-risk associated with random lentivirus integration and the
Author contributions: S.G.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript
writing; M.B.: collection and/or assembly of data, data analysis and interpretation; J. L.: collection and/or assembly of data, data
analysis and interpretation; S. D.: collection and/or assembly of data, data analysis and interpretation, financial support; A.S.: provision
of study material or patients, collection, and/or assembly of data; R.A.P.: conception and design, manuscript writing, financial, support;
R.R.A.: conception and design, manuscript writing, financial support; J.C.S.: conception and design, data analysis and interpretation,
manuscript writing, final approval of manuscript, financial support.
Correspondence: Jane C. Sowden, PhD, Developmental Biology Unit, UCL Institute of Child Health, 30 Guilford Street, London,
WC1N 1EH, UK. Telephone: þ44(0)20 79052121; Fax: þ44(0)20 78314366; e-mail: j.sowden@ich.ucl.ac.uk Received January 5,
C AlphaMed Press
2010; accepted for publication March 11, 2010; first published online in STEM CELLS EXPRESS March 31, 2010. V
1066-5099/2009/$30.00/0 doi: 10.1002/stem.423
STEM CELLS 2010;28:1048–1059 www.StemCells.com
Gualdoni, Baron, Lakowski et al.
1049
teratoma-forming potential of such cells requires further attention to make iPS cells practicable.
Adult tissue-specific stem cells theoretically do not bear
these risks, but could be harvested from the patient and
expanded for auto-transplantation. It has long been known that
lower vertebrates, such as teleosts or amphibians, display the
ability to generate new retinal neurons throughout life from a
region called the ciliary marginal zone (CMZ), at the anterior
rim of the retina [11–13]. A less potent CMZ was also discovered in the chicken [14] which displays a limited capacity for
regeneration. Although the adult mammalian eye does not display these regenerative features, in 2000, Tropepe et al. reported
that the ciliary epithelium (CE) of the murine eye contains a
population of multipotential retinal stem cells [15]. Single pigmented ciliary epithelial cells clonally proliferate in vitro in the
presence of mitogens to form sphere colonies of cells (neurospheres) [15]. When cultured under differentiating conditions
(high levels of fetal bovine serum (FBS) and without mitogens),
they were reported to differentiate into retinal-specific cell
types, including rod photoreceptors, bipolar neurons, and Müller
glia [15]. Multipotential neural progenitors in the adult mammalian CE were independently reported in the same year [16]. Follow-up research with human and pig CE cells introduced techniques to expand these cells in monolayer cultures [17, 18].
These cells may be ontogenetically closer to multipotential retinal progenitor cells (RPCs), which during development generate
all of the NR cell-types, than ES or neural stem cells and thus
easier to differentiate into photoreceptors. Indeed, Coles et al.
reported that when these cells were transplanted into adult mice
they generated new photoreceptors, albeit in small numbers.
These studies indicated that if the numbers of CE-derived cells
could be increased and their differentiation optimized, then they
offered the prospect of providing a cell source for photoreceptor
cell replacement. Most importantly, they could raise the possibility of autologous transplantation when derived from the
patient’s own eye. A number of laboratories have pursued these
objectives and have reported the expansion of cell numbers
and/or generation of photoreceptors from CE-derived cells [17–
24]. However, a recent report showed evidence that the clonogenic spheres derived from the mouse and the human CE are
made up of differentiated pigmented CE cells rather than a distinct retinal stem cell population [25] Cicero et al. conducted a
rigorous analysis of the phenotype of cells in CE-derived
spheres and concluded that differentiated pigmented CE cells
proliferate in culture, and express low levels of pan-neuronal
markers such as b-3-tubulin without forming retinal neurons.
Both theories are based on studies relying solely on the analysis
of the cellular morphology and expression profiles (reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry) of the differentiated photoreceptor cells.
In this study, we use instead an Nrl.gfp transgenic reporter
line that expresses green fluorescent protein (GFP) in developing photoreceptors to provide a definitive assessment of the
ability of murine CE cells, previously designated retinal stem
cells [15], to generate new photoreceptors in vitro. The Nrl
gene is essential for rod differentiation and is expressed in
developing and mature rods [26–28].
January 1995. The Nrl.gfp line was a kind gift of A. Swaroop
and has been described previously [26]. Mice defined as ‘‘adult’’
were at least 6 weeks old.
CE Dissociation, Spheres, and Monolayer Cultures
Dissection and cultures of adult murine CE to generate spheres
were performed as previously described [29] and monolayer cultures were established as detailed in the Supporting Information
Methods. CE monolayer cultures were grown for 3 (early passage) to 5 (late passage) weeks in proliferation medium before
transfer to standard differentiation medium or retinal differentiation medium. Cells were examined daily by epifluorescence and
transmitted light.
Differentiation of Embryonic RPC Spheres,
CE Spheres, and CE Monolayers Cultures
CE monolayer cultures or embryonic RPCs (prepared as
described in Supporting Information Methods) were grown at
high densities on poly-L-ornithine (100 lg/ml)/laminin (5 lg/ml)coated 24-well dishes in either of the following media.
Standard Differentiation Medium. Dulbecco’s modified
Eagle’s medium (DMEM)-F12 plus Glutamax (Invitrogen, U.K.)
containing N2 supplement (1:100; Invitrogen, U.K.), Penicillin–
Streptomycin solution (1:100; Invitrogen, U.K.), and 10% FBS.
Medium was changed every second day and the cells were
allowed to differentiate over the course of 14–21 days.
Retinal Differentiation Medium (as Described in Osakada
et al. [6]). DMEM-F12 plus Glutamax (Invitrogen, U.K.) containing N2 supplement (1:100; Invitrogen, U.K.), Penicillin–
Streptomycin solution (1:100; Invitrogen, U.K.), and 10 lM N[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester
(DAPT; Calbiochem, U.K.) for 3 days. Medium was then
replaced with complete medium DMEM-F12 plus Glutamax
(Invitrogen, U.K.) containing N2 supplement (1:100; Invitrogen,
U.K.), Penicillin–Streptomycin solution (1:100; Invitrogen, U.K.),
10 lM DAPT (Calbiochem, U.K.), FGF2 (10 ng/ml; Peprotech
EC, U.K.), heparin (5 ng/ml; Peprotech EC, U.K.), Sonic hedgehog (Shh, 3 nM; R&D Systems Inc., Minneapolis, http://
www.rndsystems.com), Taurine (100 lM; Sigma, U.K.), all-trans
Retinoic acid (500 nM; Sigma, U.K.), and 1% FBS. Medium was
changed every second day and the cells were allowed to differentiate over the course of 14 days.
Immunofluorescence on CE Cultures and
Immunohistochemistry on Retinal Sections
As described in Supporting Information Methods.
RNA Isolation and RT-PCR
As described in Supporting Information Methods.
Lentivirus Production and CE Monolayer Infection
MATERIALS
AND
METHODS
Animals
Mice were maintained in the animal facility at University College
London. All experiments have been conducted in accordance
with the Policies of the Use of Animals and Humans in Neuroscience Research, approved by the Society of Neuroscience in
www.StemCells.com
The coding sequences of Dsred, Gfp, mouse NeuroD, Chx10, Nrl,
and human CRX genes were cloned into lentiviral vectors.
Viruses were produced and harvested as previously described
[30] and detailed in Supporting Information Methods. CE monolayer cells grown in proliferation medium for 2 weeks were
replated in differentiation medium, at 90% confluence. After 24
hours, each lentivirus was applied to the cells at a multiplicity of
infection (MOI) of 15. Medium was exchanged after 1 day and
every other day for a total time of 14 days.
CE Cells Fail to Differentiate into Photoreceptors
1050
RESULTS
Nrl.gfp Labels Rod Photoreceptors in Adult Retina
Whereas It Is Not Detected in the CE
In rodents, rod photoreceptor cells are predominantly born in
the late embryonic and early postnatal period [31] and make
up 70% of the adult neural retinal cell population [32]. The
Nrl.gfp transgene is specifically expressed in rod photoreceptors in the adult retina as previously described [26] and GFP
is localized throughout the cell (Fig. 1A, 1B). Nrl.gfp expression is detected in newly born photoreceptors (Supporting
Information Fig. 1), but it is not detected in the mature CE,
or the retinal pigmented epithelium (RPE) (Fig. 1A–1D).
Notably, the adult CE expresses Pax6 and Chx10 (Fig. 1E–
1L), both markers of RPCs in the embryonic NR (Supporting
Information Fig. 2), and of bipolar (Fig. 1I, 1J), amacrine and
ganglion cells (Fig. 1E, 1F), respectively, in the adult retina.
In addition, we found that Connexin 43, a component of Gap
junctions, is highly expressed in the adult CE (Fig. 1M–1P).
As only a few adult CE markers have been identified, Connexin 43 may be used to better define this cell population.
Cytokeratin expression was recently described as a CE marker
[25]. Here, weak cytokeratin expression was detected in the
CE (arrowhead; Fig. 1Q–1T), although it was difficult to distinguish from nonspecific staining of the vasculature (open
arrowhead; Fig. 1T, 1X), also seen with the control secondary
antibody A594 (open arrowhead; Fig. 1Y–1b). As expected,
the RPE marker RPE65 is restricted to the RPE and does not
extend into the CE (Fig. 1U–1X). Based on the Nrl.gfp
expression pattern, the adult CE shows no evidence of photoreceptor genesis or differentiation in vivo. These findings are
in contrast to a recent article which described new photoreceptor generation within the CE in adult mice with inherited
retinal degeneration [33].
Expansion and Passaging of Ciliary
Epithelium-Derived Neurosphere-Forming
Cells in Monolayer Culture
Whereas brain neural stem cells can be efficiently expanded
as neurospheres, the neurospheres derived from adult mouse
CE tissue do not expand well in vitro [29]. To improve the
expansion of CE-derived cells and test their differentiation
potential using the Nrl.gfp reporter, we first developed a
monolayer culture system based on previous protocols used in
pig and human CE-derived cultures [17, 18]. Addition of low
levels (1–4% [v/v]) of FBS allowed neurospheres to adhere to
a poly-L-ornithine/fibronectin-substratum and expand into
high-density monolayer cultures (Fig. 2B). The morphology
of monolayer cultured cells was heterogeneous; the majority
of cells possessed several processes and were of variable size,
depending on cell density. Optimal survival and proliferation
was achieved by plating at high density, whereas cultures
with a low seeding density proliferated poorly. CE-derived
monolayers were readily passaged using Accutase, whereas
culture growth rapidly retarded with high amounts of cell
death observed after passaging using trypsin. Typically, cell
counts of a few million cells were reached after 2–3 weeks
and 8–10 million after 6–8 weeks in culture (>5 independent
experiments). An important concern regarding the use of stem
cells maintained for long periods in culture is their potential
to undergo transformation and immortalization [24]. Immortalized cells continue to divide even after reaching confluence.
By contrast, CE monolayers require passaging to continue
dividing and stop dividing when they are cultured in differentiation medium (removal of mitogens and addition of 10%
FBS). Beyond 10–12 weeks in culture the number of dividing
cells was dramatically reduced compared with early passage
numbers. Immunostaining for the mitotic marker phosphoHistone3 (pH3) showed 21% 6 8% (SD) of CE cells, cultured at low density at early stages in proliferating conditions,
to be in M-phase (1–2 weeks, n ¼ 3). On the contrary, only
2.5% 6 3.5% (SD) of cells cultured either at high density, or
in long-term cultures (10–12 weeks, n ¼ 3) in proliferating
conditions, or in differentiating conditions are pH3 positive
(data not shown). These features are typical of primary progenitor cells, rather than stem cells, maintained in culture as
monolayers [34, 35].
Although CE-derived neurospheres are always pigmented,
either partially or heavily, the cells in monolayer cultures lose
this dense pigmentation over time in culture (Fig. 2B–2E). To
better define the pigmentation state of monolayer cells, we analyzed the melanosome morphology of monolayer cells at
early and later stages (2 weeks and 8 weeks) by electron microscopy (EM). Although the function of melanosomes in the
CE and RPE is not well-understood, it is thought that they
have a protective function against light and oxidation [36].
The mammalian adult CE and RPE do not normally synthesize melanin. The majority of melanin synthesis occurs during
embryonic development, in which immature unpigmented
melanosomes (stages I, II) undergo modifications that lead to
complete maturation into pigmented functional melanosomes
(stage IV). The pigmented CE monolayer cells analyzed at
early stages retain stage IV melanosomes, in which melanin
deposition is complete and the organelle striations are
obscured (Fig. 2D). In late stage CE cultures, we saw no evidence of mature stage IV melanosomes or any sign of immature elongated unpigmented stage II melanosomes. Instead,
we detected round-shaped organelles with varying amounts of
membrane accumulation and scattered melanin deposits
(Fig. 2E). These latter organelles were never present in the
early stage cultures. We therefore assume that pigmentation
in CE monolayers is lost by melanosome degeneration over
time. These data suggest that a process of dedifferentiation is
occurring with loss of the mature differentiated pigment epithelium cell phenotype.
Differentiated epithelial cells, including CE cells in vivo,
display epithelial-specific morphological features, such as basal and lateral membrane interdigitations and epithelial cell–
cell junctions. EM analysis of CE-derived cells in monolayer
cultures did not reveal cell–cell junctions (tight junctions,
adherens junctions). After time in culture (late passage stage),
cells developed membrane interdigitations typical of epithelial
cells in vivo (Fig. 2F, 2G), although they remained
depigmented.
Expression Profile of CE-Derived Proliferating
Cells in Monolayer Culture
To characterize the extent to which the CE-derived monolayer
cells display a retinal progenitor expression profile, we performed RT-PCR and immunocytochemical analyses. After
expansion as CE monolayers (late and early passages),
mRNA was isolated and analyzed for expression of pluripotency markers (Fig. 3A), forebrain and eye field markers (Fig.
3B), and for genes involved in retinal histogenesis (Fig. 3C)
and pigmentation pathways (Fig. 3D). This CE expression
profile was compared with that of ES cells and embryonic
RPCs from E11.5 eyes.
Recent work has identified Klf4, Oct3/4, c-Myc, Nanog,
and Sox2 as pluripotency genes, essential in ES cell maintenance [37, 38]. Transcripts of c-Myc and Klf4 were expressed
in CE cells and immunostaining revealed that 98% of cells
Figure 1. Expression of epithelial and retinal progenitor markers in mouse adult retina and CE. (A–D): Sections of adult retina from an Nrl.gfp
transgenic mouse show green fluorescent protein expression specifically in the ONL (A, B). The transgene is not expressed in the CE (C, D). (E–
X): Immunostaining on sections of adult retina stained with the RPC, amacrine, ganglion cell marker, Pax6 (E–H), the RPC and bipolar cells
marker Chx10 (I–L), the epithelial and RPE markers, Connexin 43 (M–P), Cytokeratin (Q–T), and RPE65 (U–X). The inset in (L) shows Chx10
single channel immunostaining. Cytokeratin specific signal is shown by a white arrowhead (T) and nonspecific staining of the vasculature is indicated by open arrowhead (T, X, B). Control using the secondary antibody anti-mouse A594 is shown (Y–b). Higher magnification pictures of the
central neural retina and CE (white arrows) are shown for each panel. Sections were counter-stained with the nuclear dye Hoechst 33342 (blue).
Abbreviations: RPE, retina pigmented epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; CE, ciliary epithelium. Scale bars: 25 lm.
1052
CE Cells Fail to Differentiate into Photoreceptors
Figure 2. Development of CE monolayer. (A): Diagram of the mouse adult eye showing the anatomical position of the CE and associated
structures. (B): CE cells were grown as neurospheres for 7 days in vitro (D), before expansion as monolayers in proliferating medium. Scale bar
50 lm. (C–G): Electron microscopy images of primary RPE cultures (C), early (2 weeks; D, F) and late (5 weeks; E, G) passage CE monolayers
showing stage IV mature melanosomes (C, D), and multivesicular organelles with scattered melanin deposition (E), white arrows. Inset in (E)
shows a higher magnification image of typical melanosome at late stages in culture. (F, G) Plasma membrane at early passages (F) does not
show membrane interdigitations, whereas at late passages (G) they are clearly visible (black arrows). Scale bars: 0.5 lm (C–E), 200 nm (inset in
E), 1 lm. Abbreviation: CE, ciliary epithelium.
express Sox2 (Supporting Information Fig. 3). The presumptive eye field is defined by expression of a group of transcription factor genes, including, Rx, Pax6, Six3, Lhx2, Tll1, Tbx3,
and Six6 [39, 40], all of which were detected by RT-PCR in
embryonic RPCs (Fig. 3). In Xenopus embryos, simultaneous
overexpression of these transcription factors and Otx2 is sufficient to promote eye development including retinal cell differentiation [40]. We found that Tbx3, Tll1, Lhx2, Six6, and
Pax6 were expressed in CE monolayers (Fig. 3B), although Rx
was absent (Fig. 3C). Chx10 is expressed in RPCs throughout
retinal development from the earliest stage of retinal specification in the embryonic optic vesicle, whereas Mitf becomes restricted to the presumptive RPE and Pax6 and Otx2 are
expressed in both RPE and NR (Supporting Information Fig. 2).
The CE monolayers expressed Pax6, Otx2, and Mitf, but Chx10
mRNA was barely detectable, and a number of other markers of
retinal differentiation, including Fgf15, NeuroD and the photoreceptor marker Crx were not expressed (Fig. 3C). Early and late
passage cultures showed similar profiles indicating that the phenotype is maintained in monolayer cultures. These data suggest
that proliferative CE monolayer cells display aspects of the gene
expression profile of optic cup progenitors.
As EM analysis indicates loss of a pigmented phenotype,
we next sought to establish whether any mature pigment and
epithelial markers were present. Dissected uncultured adult
CE, RPE, and NR were used as controls. Palmdelphin was
recently described as a differentiated CE marker [25], but was
found to be expressed in all control tissues (RPE, CE, and
Gualdoni, Baron, Lakowski et al.
1053
a defining characteristic of RPCs throughout development
(Supporting Information Fig. 2). Although Chx10 protein was
found in neurospheres [15, 29], it was not detected by immunocytochemistry in monolayer cultures (Supporting Information Fig. 3). Neither RPE65 nor the other epithelial markers
including ZO-1 and E-cadherin were detected (data not
shown). CE monolayers consistently expressed cytokeratin
and Cx43 (Fig. 4), which although not CE-specific markers,
are both expressed in the adult CE in vivo. Taken together,
these data show that although CE-derived monolayers proliferate and exhibit aspects of a retinal gene expression profile,
a number of critical markers of RPCs, such as Rx and Chx10
are largely absent, whereas some characteristics of differentiated CE persist. These results indicate a lack of evidence that
proliferating CE cells exhibit a bona fide RPC state.
Differentiation of Nrl.gfp Ciliary
Epithelium-Derived Cells
Figure 3. mRNA expression profile of CE monolayer cells at early
and late passages in vitro. RT-PCR of CE monolayers cultures, maintained in proliferating conditions, shows consistent expression of proliferation markers (Klf4, c-Myc) (A). They express only a subset of
forebrain and retinal progenitor cell markers (Tbx3, Tll1,Lhx2, Pax6,
Chx10, Otx2, Mitf) (B, C). In addition, they lose the pigmentation
markers Tyrosinase, Rpe65, and the Mitf isoformD (D). E11.5 eye,
embryonic stem cells, dissected CE, RPE, and CNR are used as positive controls. Abbreviations: CE, ciliary epithelium; CNR, central
neural retina; early, early passages; ES, embryonic stem cells; late,
late passages; RPE, retinal pigmented epithelium.
NR). Tyrosinase and RPE65 mRNA were detected in CE and
RPE, whereas the D isoform of Mitf was only detected in CE.
RPE65 protein was only detected in dissected RPE and not in
CE (Fig. 1U–1X), suggesting post-transcriptional regulation.
In CE monolayer cells, we only detected palmdelphin,
whereas all pigmentation and other RPE marker were absent,
consistent with the nonpigmented appearance of these cells
(Fig. 3D).
To assess the significance of these mRNA profiles for protein localization at single cell resolution, early (2 weeks) and
late (>5 weeks) stage cultures were analyzed by immunocytochemistry. Significant and consistent expression of markers
characteristic of neural progenitors (Pax6, Sox2, Nestin, pH3)
and of neurogenesis (b3-tubulin) were detected (Fig. 4 and
Supporting Information Fig. 3), although levels decreased
over time in culture (data not shown). Despite presence of the
mRNA, Otx2 protein was never detected. Chx10 expression is
www.StemCells.com
Previously we showed that Nrl.gfp-positive cells isolated from
the developing retina are able to mature into functional rod
photoreceptors after transplantation into the adult retina [1].
Hence, Nrl.gfp transgene activation provides a reliable and robust assay for cells with the potential to differentiate into rod
photoreceptors. As the expression profile of CE-derived cells
suggests some potential for activation of a retinal differentiation program, we next utilized CE cells from the Nrl.gfp
transgenic line to provide a simple assay for commitment to a
photoreceptor differentiation program.
We first identified and validated conditions for culture of
embryonic RPCs in vitro that were able to activate the Nrl.gfp
transgene and initiate photoreceptor differentiation, before
testing the same conditions on CE cells. RPCs dissected from
the E11.5–12.5 retina were expanded and then transferred to
two independent differentiation-promoting conditions. The
first utilized conditions described in the original paper reporting the discovery of retinal stem cells (standard differentiation
medium; addition of serum and removal of mitogens [15]).
The second employed a combination of factors recently
shown to effectively induce photoreceptor differentiation from
ES cell-derived RPCs (retinal differentiation medium [6]).
RPCs in both conditions activated the Nrl.gfp transgene after
4–6 days in culture indicating that these conditions support in
vitro photoreceptor commitment. High-level GFP activation
was observed after DAPT treatment, the first step of the retinal differentiation medium indicating that these conditions
were highly effective at activating rod photoreceptor development in vitro (Fig. 5A).
CE-derived neurospheres were cultured for 7 days in vitro
before exposure to the retinal differentiation medium. The
spheres expressed Chx10, Rx, and other progenitor markers,
as previously described ([29], Supporting Information Fig. 3
and data not shown). However, activation of the Nrl.gfp transgene was not observed in CE neurospheres either with DAPT
alone or the full retinal differentiation condition (Fig. 5B), or
the standard differentiation conditions, previously reported to
activate photoreceptor differentiation from CE cells [15] (data
not shown). Similarly, these same conditions failed to promote transgene activation when tested on CE monolayer cultures (n ¼ 3 separate experiments with multiple independent
cultures monitored daily; Fig. 5C). Other culture conditions,
including the addition of Noggin, insulin-like growth factor
(IGF), and Dkk1, previously shown to generate RPCs from
ES cells were also tested [5]. These did not generate Nrl.gfp
expressing cells, as assessed by GFP fluorescence (data not
shown). qPCR was also performed for the early photoreceptor
1054
CE Cells Fail to Differentiate into Photoreceptors
Figure 4. Protein expression profile of
CE monolayer cells at early passages in
vitro. Immunostaining of CE monolayers
cultures, maintained in proliferating conditions, shows expression of neural progenitor (Nestin), retinal progenitor (Pax6),
epithelial (Cytokeratin), and the gap junction marker (Connexin 43). By contrast,
cells are negative for RPE65. RPE cultures
are used as positive control for RPE65
staining. Scale bar: 25 lm (Connexin43
panel) and 50 lm (Nestin, Pax6, Cytokeratin, and RPE65). Abbreviations: CE, Ciliary epithelium; RPE, retinal pigmented
epithelium.
marker Crx, and other retinal markers, but no evidence for
photoreceptor differentiation was detected (data not shown).
In addition, we performed immunostaining on the differentiated cultures to assess expression of neuronal and photoreceptor-specific markers. The cultures failed to show positive
immunoreactivity for photoreceptor markers including rhodopsin, recoverin, and blue opsin. All antibodies effectively labeled photoreceptors in both retinal sections and dissociated
retinal cells (Supporting Information Fig. 6C and data not
shown). Furthermore, CE monolayers cultured for 2 weeks in
either differentiation media lost expression of b-3 tubulin and
displayed a dramatic reduction in the number of Pax6-positive
cells (Supporting Information Fig. 4). When these cells were
cultured in standard differentiation medium, they showed partially reduced nestin and dramatically increased vimentin
expression (Supporting Information Fig. 5), whereas in retinal
differentiation medium they develop distinct epithelial fea-
tures, including formation of cortical actin and accumulation
of ZO-1, a marker of the tight junctions at cell–cell contacts
(Fig. 6). The apparent reversion of CE cultures toward an epithelial phenotype, when cultured in a medium that supports
and promotes photoreceptor differentiation from RPCs, provides additional evidence that the CE-derived cell population
does not contain bona fide RPCs.
Taken together, these data indicate that CE-derived cells
do not undergo photoreceptor differentiation.
CE Progenitors Rarely Activate the Nrl.gfp
Transgene Under Photoreceptor Differentiation
Conditions Even with Exogenous Expression of
Retinal Transcription Factors
Finally, we asked whether CE monolayer cells could differentiate into photoreceptors, upon exogenous expression of the
Gualdoni, Baron, Lakowski et al.
1055
Figure 5. Retinal differentiation protocols do
not activate Nrl.gfp transgene in CE-derived
neurospheres and monolayer cultures. (A):
Cultures of embryonic retinal progenitor cells
(RPCs) activate the Nrl.gfp trangene and initiate photoreceptor differentiation. RPCs dissected from the E12.5 retina were expanded in
proliferation medium followed by differentiation in standard or retinal differentiation
media. The first step of the retinal differentiation medium alone (DAPT) was sufficient to
activate Gfp expression. Gfp activation is significantly higher in retinal differentiation medium. (B): CE-derived neurospheres expanded
for 7 days in vitro and transferred to proliferation medium or retinal differentiation medium
for an additional 2 weeks. No Gfp activation is
detected. (C): CE-derived monolayers in proliferation medium, retinal differentiation medium, or in standard differentiation medium,
cultured for 2 weeks. No Gfp activation is
detected. Scale bars: 75 lm and 50 lm in
poliferation medium in B. Abbreviations: prolif. medium, proliferation medium; ret. diff.
medium, retinal differentiation medium; st.
diff. medium, standard differentiation medium.
retinal-specific transcription factor genes that they lacked. We
used lentiviral vectors that encode for NeuroD, Chx10, CRX,
and Nrl together with a Dsred viral vector, to transduce CE
monolayer cells in an attempt to reprogram them along a photoreceptor differentiation pathway. We imaged live cells after
10–14 days, to evaluate the levels of photoreceptor differentiation, using the Nrl.gfp transgene activation assay. Neither
CE cells transduced with a single transcription factor gene
nor transduction with multiple transcription factor genes or
the control Dsred marker alone led to a significant number of
Nrl.gfp positive cells in culture. Less than 0.003% of the cells
activated the Nrl promoter and presented GFP fluorescence
(Fig. 7B) and there were no differences between controls and
cells transduced with retinal transcription factor genes. Moreover, RT-PCR analysis showed no expression of the endogenous photoreceptor-specific transcription factor gene Crx, an
essential player in photoreceptor development [41, 42]
(Fig. 7A), whereas the virally expressed human CRX mRNA
was detectable. Immunostaining analysis for rhodopsin and
blue opsin did not show any positive cells (data not shown).
No obvious change in cell morphology was observed after
transduction. We also performed RT-PCR analysis for photoreceptor-specific gene expression (Supporting Information
Fig. 6A, 6B). Rhodopsin and blue opsin mRNA were detected
(18/24 samples; three independent experiments) using two different primer sets for each gene (Supporting Information
Fig. 6A and data not shown). Additional photoreceptor
markers Irbp and S-arrestin were not detected, whereas the
CE markers Palmdelphin and Connexin43 were expressed
(Supporting Information Fig. 6A). Real-time quantitative RTPCR was used to assess the level of rhodopsin and blue opsin
www.StemCells.com
expression in the transduced CE cultures cultured in differentiating conditions. Rhodopsin and blue opsin mRNA were
found to be present at around 30,000- and 300-fold lower levels, respectively, compared with adult NR (Supporting Information Fig. 6B). Palmdelphin expression was 20-fold higher
in transduced CE cells compared with CE cells maintained in
proliferating conditions. These data, together with a consistent
lack of Nrl.gfp activation, strongly indicate the absence of a
coherent rod photoreceptor differentiation program.
DISCUSSION
As Tropepe et al. [15] proposed that the adult CE contains a
population of retinal stem cells able to differentiate into retinal neurons, different groups have invested effort into repeating these experiments and investigating the potential of CEderived retinal stem cells for retinal repair. Success in this
area has been limited, with reports of 1% of opsin-positive
cells generated from rat CE-derived cells [20], 30% of
Rho4D2-positive cells generated from human CE-derived
cells [17], no fully differentiated photoreceptors generated
from porcine CE-derived cells [18] and threefold increase of
photoreceptor markers such as rhodopsin and blue opsin in
Crx-electroporated CE cells [22]. Recently, Cicero et al. [25]
argued against the validity of the existing theory. The earlier
studies relied only on analysis of the cellular morphology and
the expression profile of the differentiated cells as assessed by
immunocytochemistry and PCR [15-17, 20, 23]; such techniques can lead to false positives and mis-interpretation of the
1056
CE Cells Fail to Differentiate into Photoreceptors
Figure 6. Ciliary epithelium
(CE) monolayer cells cultured in
retinal differentiation medium
show an increase of cortical
actin and ZO-1 at the cell–cell
contacts. Immunostaining of CE
monolayers cells cultured in
proliferation medium, retinal differentiation medium or standard
differentaiation medium using
ZO-1 (green) antibody and phalloidin for F-actin staining (red).
Cells were counter-stained with
the nuclear dye Hoechst 33342
(blue). Insets show higher magnification images. Scale bar:
75 lm and 20 lm in the insets.
Abbreviations: prolif. medium,
proliferation medium; ret. diff.
medium, retinal differentiation
medium; st. diff. medium, standard differentiation medium.
results due to nonspecific labeling or the presence of mRNA,
but not the corresponding proteins. In this study, we used a
genetic tool, the Nrl.gfp transgenic line that expresses GFP in
developing and mature photoreceptors under the control of
the Nrl promoter [26]. This novel approach based on the activation of a rod-specific promoter has allowed us to assess
more definitively the potential for generating photoreceptors
from CE-derived cells.
It has been previously reported that CE-derived neurospheres have a poor expansion potential [17, 18]. To use cells
for autologous cell therapy, a high number of cells are
required. In this study, we established the conditions for
expanding, as monolayer cultures, the mouse CE-derived
cells. CE-derived monolayer cultures are highly proliferative;
a robust expansion was obtained when compared with floating
neurospheres, reaching 10 million cells in 3 weeks. CE monolayer cells express a number of markers known to be associated with neural stem cells, such as Nestin and Sox2. They
express the forebrain and eye field marker Pax6, although Rx
and Chx10 were difficult to detect. Occasionally, the pan-neuronal marker b3-tubulin was detected in proliferating monolayer cultures, without specific differentiation conditions.
Moreover, mRNA transcripts of additional early eye field-specific genes such as Otx2, Tbx3, Tll1, Lhx2, and Six6 were also
identified. We found the presence of Otx2 transcript in CE
cells, but we did not detect any Otx2-positive cells by immunostaining indicating post-transcriptional regulation of specific
genes might be occurring. The expression profile of the mouse
CE monolayer cells we have identified here is consistent with
the previous findings from human and porcine CE-derived
cells [17, 18].
Although CE-derived neurospheres are always pigmented,
either partially or heavily, as also recently described [25], we
found that the cells in monolayer cultures lose their dense
pigmentation with time in culture suggesting that a dedifferentiation to a proliferating progenitor-like status occurs with
consequent loss of the pigmented status. This process may be
analogous to the transdifferentiation of RPE to NR, which in
lower vertebrates can lead to the regeneration of the retina
[43–45]. Notably, Pax6 and Chx10 are both detected in the
adult differentiated CE in vivo and may be important for their
apparent progenitor characteristics. We found that although
the CE monolayer cultures resemble progenitor cells of the
developing embryonic eye (by virtue of their depigmentation,
proliferation characteristics and expression of eye field and
retinal-specific genes), they also simultaneously display CE
features including Cx43 and cytokeratin expression and formation of membrane interdigitations. Furthermore, the lack of
Gualdoni, Baron, Lakowski et al.
1057
Figure 7. Ciliary epithelium (CE)
cells rarely activate the Nrl-gfp transgene under retinal differentiation conditions even with exogenous expression
of retinal transcription factors. (A)
Scheme of the experimental procedure
for the transduction of CE monolayers
cells using NeuroD, Chx10, CRX, and
Nrl lentiviral vectors. (B) RT-PCR
shows no differences between controls
and retina transcription factor-transduced cells. The dsred lentiviral transduced (red) and the untransduced cells
were used as controls. Live cells were
imaged 10 days after transduction and
GFP expression (green) was assessed.
Less than 0.003% of the cells activate
the Nrl promoter (C, white arrows).
Scale bar: 100 lm. Abbreviation: GFP,
green fluorescent protein.
Chx10 protein as well as other retinal markers in the proliferating cultures argues against the existence of a bona fida RPC
state.
Photoreceptor differentiation experiments were performed
primarily on monolayers at the stage at which they had started
to dedifferentiate and showed the highest resemblance to
RPCs. We tested differentiation conditions that support the
commitment of embryonic RPCs and ES cell-derived RPCs to
photoreceptors in vitro, on CE neurospheres, as well as CE
monolayer cultures, using the expression of the Nrl.gfp transgene as a marker of rod photoreceptor commitment. The removal of mitogens and the addition of 10% FBS was previously reported to generate photoreceptors from mouse and
human CE cells [15, 17]. Different reports have recently shown
that by using a combination of different factors, ES cells could
be directed toward an RPC state. In particular, the use of
Dkk1, a Wnt/b-catenin signaling pathway antagonist, and
LEFTY-A, a nodal antagonist and noggin, a potent inhibitor of
the bone morphogenetic protein (BMP) pathway, were shown
to effectively increase the Rxþ/Pax6þ cell population in ES
cells [5]. Furthermore, inhibition of Notch signaling induces
www.StemCells.com
photoreceptor differentiation in vivo [46, 47], and the use of
the c-sectretase inhibitor DAPT on ES-derived Rxþ cells,
increases the number of Crx-positive photoreceptor precursors
[6]. Here, unequivocally we did not observe Nrl.gfp transgene
activation after exposing both CE-derived neurospheres and
monolayers to any of these previously reported differentiation
conditions. Moreover, we did not detect immunoreactivity for
any photoreceptor markers, despite testing a wide array. By
contrast, these protocols effectively induced Nrl.gfp transgene
activation in embryonic RPCs in control experiments. These
data confirm that CE cells show limited potential to generate
photoreceptor cells; on the contrary, CE cells seem to revert
their phenotype toward the original differentiated epithelial status, as confirmed by the cortical actin formation and ZO-1
accumulation at the cell–cell contacts.
Viral vector-mediated transduction of retinal transcription
factors in different cell types has been reported to increase
the number of cells expressing photoreceptor markers [19, 22,
48]. We also evaluated this approach, however, we found that
a negligible proportion of cells (less that 0.003%) transduced
with retinal transcription factors activate the Nrl.gfp
CE Cells Fail to Differentiate into Photoreceptors
1058
transgene, a finding that was independent of the vector combination used. RT-PCR data indicated a low level of ectopic
transcription of some photoreceptor genes, but not the photoreceptor transcription factor gene Crx, and may explain previous reports that concluded photoreceptors could be successfully generated from adult retinal stem cells.
CONCLUSION
In summary, we conclude that CE-derived cells do proliferate
in vitro, do express some of the eye field and neural/retinal
progenitor markers and undergo a process of dedifferentiation
including the loss of pigmentation. However, they do not
effectively activate the Nrl-regulated rod differentiation program and are unable to generate new rod photoreceptors.
These data do not support the conclusions drawn from earlier
immunostaining experiments [15, 17] which indicated high
levels of new rod photoreceptor generation by retinal stem
cells derived from the adult CE. The range of strategies we
have used to test for activation of rod photoreceptor differentiation in CE cultures, including the Tropepe protocol [15],
combined with the reliability of Nrl.gfp transgene activation
in vivo, in transplanted rod photoreceptors [1], and in embryonic RPC differentiation control experiments in vitro
strengthen this conclusion. Recently, Cicero et al. reported an
extensive analysis designed to identify the putative retinal
stem cell located within the CE and were unable to find such
a cell [25]. Instead, they reported that the mature pigmented
CE cells exhibit the properties of neurophere formation and
clonal expansion as reported by Tropepe et al. [15]. This lat-
REFERENCES
1
2
3
4
5
6
7
8
9
10
11
12
13
14
MacLaren RE, Pearson RA, MacNeil A et al. Retinal repair by transplantation of photoreceptor precursors. Nature 2006;444:203–207.
Lamba DA, Gust J, Reh TA. Transplantation of human embryonic
stem cell-derived photoreceptors restores some visual function in Crxdeficient mice. Cell Stem Cell 2009;4:73–79.
Bartsch U, Oriyakhel W, Kenna PF et al. Retinal cells integrate into
the outer nuclear layer and differentiate into mature photoreceptors after subretinal transplantation into adult mice. Exp Eye Res 2008;86:
691–700.
Ikeda H, Osakada F, Watanabe K et al. Generation of Rxþ/Pax6þ
neural retinal precursors from embryonic stem cells. Proc Natl Acad
Sci USA 2005;102:11331–11336.
Lamba DA, Karl MO, Ware CB et al. Efficient generation of retinal
progenitor cells from human embryonic stem cells. Proc Natl Acad
Sci USA 2006;103:12769–12774.
Osakada F, Ikeda H, Mandai M et al. Toward the generation of rod
and cone photoreceptors from mouse, monkey and human embryonic
stem cells. Nat Biotechnol 2008;26:215–224.
Meyer JS, Shearer RL, Capowski EE et al. Modeling early retinal development with human embryonic and induced pluripotent stem cells.
Proc Natl Acad Sci USA 2009;106:16698–16703.
Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent
stem cells from adult human fibroblasts by defined factors. Cell 2007;
131:861–872.
Park IH, Zhao R, West JA et al. Reprogramming of human somatic
cells to pluripotency with defined factors. Nature 2008;451:141–146.
Lowry WE, Richter L, Yachechko R et al. Generation of human
induced pluripotent stem cells from dermal fibroblasts. Proc Natl
Acad Sci USA 2008;105:2883–2888.
Hollyfield JG. Differential addition of cells to the retina in Rana
pipiens tadpoles. Dev Biol 1968;18:163–179.
Straznicky K, Gaze RM. The growth of the retina in Xenopus laevis:
An autoradiographic study. J Embryol Exp Morphol 1971;26:67–79.
Moshiri A, Close J, Reh TA. Retinal stem cells and regeneration. Int J
Dev Biol 2004;48:1003–1014.
Fischer AJ, Reh TA. Identification of a proliferating marginal zone of
retinal progenitors in postnatal chickens. Dev Biol 2000;220:197–210.
ter study did not pursue the differentiation potential of these
cells and left open the possibility that they could undergo retinal neurogenesis. Our study has pursued this question and
concludes that the photoreceptor potential of these cells is
negligible. if these cells are to be useful for cell-based therapies in the treatment of retinal disease, it is likely that they
will require reprogramming and/or trans-differentiation. Other
more promising alternatives are the use of fetal tissue-derived
RPCs, pluripotent ES, or iPS cells [6, 7]. Recently, significant
progress toward retinal differentiation was achieved by using
low-molecular-mass compounds on both ES cells and iPS,
indicating the possibility of avoiding animal-derived factors to
induce differentiation [49].
ACKNOWLEDGMENTS
We gratefully thank Anand Swaroop for the Nrl.gfp mice, Elena
Sokolskaja for assistance with lentivirus production, and Peter
Munro for electron microscopy analysis. This work was supported by the Medical Research Council UK (G03000341), the
Macula Vision Research Foundation, Fight for Sight, EMBO,
the Ulverscroft Foundation, the Royal Society, and the NIHR
Biomedical Research Centre for Ophthalmology.
DISCLOSURE
OF
OF
POTENTIAL CONFLICTS
INTEREST
The authors indicate no potential conflicts of interest.
15 Tropepe V, Coles BL, Chiasson BJ et al. Retinal stem cells in the
adult mammalian eye. Science 2000;287:2032–2036.
16 Ahmad I, Tang L, Pham H. Identification of neural progenitors in the
adult mammalian eye. Biochem Biophys Res Commun 2000;270:
517–521.
17 Coles BL, Angenieux B, Inoue T et al. Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci USA
2004;101:15772–15777.
18 MacNeil A, Pearson RA, MacLaren RE et al. Comparative analysis of
progenitor cells isolated from the iris, pars plana, and ciliary body of
the adult porcine eye. Stem Cells 2007;25:2430–2438.
19 Akagi T, Mandai M, Ooto S et al. Otx2 homeobox gene induces photoreceptor-specific phenotypes in cells derived from adult iris and ciliary tissue. Invest Ophthalmol Vis Sci 2004;45:4570–4575.
20 Das AV, James J, Rahnenfuhrer J et al. Retinal properties and potential of the adult mammalian ciliary epithelium stem cells. Vision Res
2005;45:1653–1666.
21 Jomary C, Jones SE, Lotery A. Generation of light-sensitive
photoreceptor phenotypes by genetic modification of human adult ocular stem cells using Crx. Invest Ophthalmol Vis Sci 2010;51:
1181–1189.
22 Jomary C, Jones SE. Induction of functional photoreceptor phenotype
by exogenous Crx expression in mouse retinal stem cells. Invest Ophthalmol Vis Sci 2008;49:429–437.
23 Giordano F, De Marzo A, Vetrini F et al. Fibroblast growth factor and
epidermal growth factor differently affect differentiation of murine retinal stem cells in vitro. Mol Vis 2007;13:1842–1850.
24 Djojosubroto M, Bollotte F, Wirapati P et al. Chromosomal number
aberrations and transformation in adult mouse retinal stem cells in
vitro. Invest Ophthalmol Vis Sci 2009;50:5975–5987.
25 Cicero SA, Johnson D, Reyntjens S et al. Cells previously identified
as retinal stem cells are pigmented ciliary epithelial cells. Proc Natl
Acad Sci USA 2009;106:6685–6690.
26 Akimoto M, Cheng H, Zhu D et al. Targeting of GFP to newborn
rods by Nrl promoter and temporal expression profiling of flow-sorted
photoreceptors. Proc Natl Acad Sci USA 2006;103:3890–3895.
27 Liu Q, Ji X, Breitman ML et al. Expression of the bZIP transcription
factor gene Nrl in the developing nervous system. Oncogene 1996;12:
207–211.
28 Mears AJ, Kondo M, Swain PK et al. Nrl is required for rod photoreceptor development. Nat Genet 2001;29:447–452.
Gualdoni, Baron, Lakowski et al.
29 Kokkinopoulos I, Pearson RA, Macneil A et al. Isolation and characterisation of neural progenitor cells from the adult Chx10(orJ/orJ) central neural retina. Mol Cell Neurosci 2008;38:359–373.
30 Demaison C, Brouns G, Blundell MP et al. A defined window for efficient gene marking of severe combined immunodeficient-repopulating
cells using a gibbon ape leukemia virus-pseudotyped retroviral vector.
Hum Gene Ther 2000;11:91–100.
31 Cepko CL, Austin CP, Yang X et al. Cell fate determination in the
vertebrate retina. Proc Natl Acad Sci USA 1996;93:589–595.
32 Young RW. Cell differentiation in the retina of the mouse. Anat Rec
1985;212:199–205.
33 Nishiguchi KM, Kaneko H, Nakamura M et al. Generation of immature
retinal neurons from proliferating cells in the pars plana after retinal
histogenesis in mice with retinal degeneration. Mol Vis 2009;15:
187–199.
34 Zhang H, Zhao Y, Zhao C et al. Long-term expansion of human neural progenitor cells by epigenetic stimulation in vitro. Neurosci Res
2005;51:157–165.
35 Sherr CJ, DePinho RA. Cellular senescence: Mitotic clock or culture
shock? Cell 2000;102:407–410.
36 Schraermeyer U, Heimann K. Current understanding on the role of
retinal pigment epithelium and its pigmentation. Pigment Cell Res
1999;12:219–236.
37 Knoepfler PS. Why myc? An unexpected ingredient in the stem cell
cocktail. Cell Stem Cell 2008;2:18–21.
38 Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 2008;132:567–582.
39 Hatakeyama J, Tomita K, Inoue T et al. Roles of homeobox and
bHLH genes in specification of a retinal cell type. Development 2001;
128:1313–1322.
1059
40 Zuber ME, Gestri G, Viczian AS et al. Specification of the vertebrate
eye by a network of eye field transcription factors. Development
2003;130:5155–5167.
41 Furukawa T, Morrow EM, Cepko CL. Crx, a novel otx-like homeobox
gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. Cell 1997;91:531–541.
42 Chen S, Wang QL, Nie Z et al. Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific
genes. Neuron 1997;19:1017–1030.
43 Zhao S, Rizzolo LJ, Barnstable CJ. Differentiation and transdifferentiation of the retinal pigment epithelium. Int Rev Cytol 1997;171:
225–266.
44 Fischer AJ, Reh TA. Transdifferentiation of pigmented epithelial cells:
A source of retinal stem cells? Dev Neurosci 2001;23:268–276.
45 Liang L, Yan RT, Li X et al. Reprogramming progeny cells of embryonic RPE to produce photoreceptors: Development of advanced photoreceptor traits under the induction of neuroD. Invest Ophthalmol Vis
Sci 2008;49:4145–4153.
46 Jadhav AP, Mason HA, Cepko CL. Notch 1 inhibits photoreceptor
production in the developing mammalian retina. Development 2006;
133:913–923.
47 Yaron O, Farhy C, Marquardt T et al. Notch1 functions to suppress
cone-photoreceptor fate specification in the developing mouse retina.
Development 2006;133:1367–1378.
48 Akagi T, Akita J, Haruta M et al. Iris-derived cells from adult rodents
and primates adopt photoreceptor-specific phenotypes. Invest Ophthalmol Vis Sci 2005;46:3411–3419.
49 Osakada F, Jin ZB, Hirami Y et al. In vitro differentiation of retinal
cells from human pluripotent stem cells by small-molecule induction.
J Cell Sci 2009;122:3169–3179.
See www.StemCells.com for supporting information available online.