1. Loss of photoreceptor potential from retinal progenitor cell cultures,
despite improvements in survival
Fiona C. Mansergh a,*, Reaz Vawda a,b
, Sophia Millington-Ward a
, Paul F. Kenna a
, Jochen Haas c
,
Clair Gallagher d
, John H. Wilson e
, Peter Humphries a
, Marius Ader a,c
, G. Jane Farrar a
a
Ocular Genetics Unit, Smurfit Institute of Genetics, Trinity College Dublin, Lincoln Place Gate, Dublin 2, Ireland
b
Fighting Blindness Vision Research Institute, 1 Christchurch Hall, Dublin 2, Ireland
c
DFG-Center for Regenerative Therapies Dresden, Cluster of Excellence/TU Dresden, c/o MTZ, Fiedlerstr. 42, 01307 Dresden, Germany
d
National Institute of Cellular Biotechnology (NICB), Dublin City University, Glasnevin, Dublin 9, Ireland
e
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
a r t i c l e i n f o
Article history:
Received 6 May 2010
Accepted in revised form 7 July 2010
Available online xxx
Keywords:
retinal progenitor cells
cell culture
FACS
rhodopsin
photoreceptor
retina
a b s t r a c t
Retinal degeneration (RD) results from photoreceptor apoptosis. Cell transplantation, one potential
therapeutic approach, requires expandable stem cells that can form mature photoreceptors when
differentiated. Freshly dissociated primary retinal cells from postnatal day 2e6 (PN2e6) mouse retina
can give rise, post-transplantation, to photoreceptors in adult recipients. Unfortunately, incorporation
rates are low; moreover, photoreceptor potential is lost if the same PN2e6 cells are cultured prior to
transplantation. We investigated the identity of the cells forming photoreceptors post-transplantation,
using FACS sorted primary postnatal day (PN) 3e5 Rho-eGFP retinal cells. Higher integration rates were
achieved for cells that were expressing Rho-eGFP at PN3e5, indicating that post-mitotic photoreceptor
precursors already expressing rhodopsin form the majority of integrating rods. We then investigated
improvement of cell culture protocols for retinal progenitor cells (RPCs) derived from PN3e5 retinal cells
in vitro. We succeeded in improving RPC survival and growth rates 25-fold, by modifying retinal
dissociation, replacing N2 supplement with B27 supplement minus retinoic acid (B27 À RA) and coating
flasks with fibronectin. However, levels of rhodopsin and similar photoreceptor-specific markers still
diminished rapidly during growth in vitro, and did not re-appear after in vitro differentiation. Similarly,
transplanted RPCs, whether proliferating or differentiated, did not form photoreceptors in vivo. Cultured
RPCs upregulate genes such as Sox2 and nestin, markers of more primitive neural stem cells. Use of these
cells for RD treatment will require identification of triggers that favour terminal photoreceptor differ-
entiation and survival in vitro prior to transplantation.
Ó 2010 Elsevier Ltd. All rights reserved.
1. IntroductionQ1
Retinal degeneration (RD) involves the gradual loss of photo-
receptors by apoptosis, causing visual impairment and eventual
blindness. Inherited RD is genetically heterogeneous. To date, over
190 loci have been identified (RetNet; http://www.sph.uth.tmc.
edu/Retnet/). The disability associated with various forms of RD
carries a great social and economic cost. Retinitis pigmentosa (RP)
affects 1 in 3000 people, while age related macular degeneration
(AMD) affects as many as 1 in 10 over-65s. Gene therapy-based
approaches have shown therapeutic promise in clinical trials
(Maguire et al., 2008, 2009; Bainbridge et al., 2008; Cideciyan et al.,
2008), but require some surviving cells in order to work. Cell
therapy for advanced disease may provide a complimentary
approach.
A variety of stem cell sources have been identified, including
ciliary epithelial cells (CE), retinal progenitor cells (RPCs, derived
from embryonic or early postnatal neural retinas), embryonic stem
(ES) cells, and induced pluripotent stem (iPS) cells. Ciliary epithelial
cells (CE) are derived from the ciliary margin and can generate
spheres in culture which upregulate neuro-retinal genes (Tropepe
et al., 2000; Coles et al., 2004; Das et al., 2005), However, recent
reports note that these cells fail to form bona fide retinal neurons
and glia (Cicero et al., 2009; Gualdoni et al., 2010); this source of
stem cells has therefore not been investigated further here.
* Corresponding author. Tel.: þ353 1 8962484; fax: þ353 1 8963848.
E-mail addresses: mansergf@tcd.ie (F.C. Mansergh), reaz.vawda@fightingblindness.
ie (R. Vawda), sophia@maths.tcd.ie (S. Millington-Ward), pfkenna@tcd.ie (P.F. Kenna),
jochen.haas@crt-dresden.de (J. Haas), clair.gallagher@dcu.ie (J.H. Gallagher), jwilson@
bcm.tmc.edu (J.H. Wilson), pete.humphries@tcd.ie (P. Humphries), marius.ader@crt-
dresden.de (M. Ader), gjfarrar@tcd.ie (G.J. Farrar).
Contents lists available at ScienceDirect
Experimental Eye Research
journal homepage: www.elsevier.com/locate/yexer
0014-4835/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2010.07.003
Experimental Eye Research xxx (2010) 1e14
YEXER5575_proof ■ 21 July 2010 ■ 1/14
Please cite this article in press as: Mansergh, F.C., et al., Loss of photoreceptor potential from retinal progenitor cell cultures, despite
improvements in survival, Exp. Eye. Res. (2010), doi:10.1016/j.exer.2010.07.003
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2. Transplantation studies have shown that a sub-fraction of freshly
dissociated cells from PN2e6 mouse retinas can integrate into adult
host retina, show morphology characteristic of photoreceptors and
can ameliorate symptoms of RD (MacLaren et al., 2006; Bartsch
et al., 2008; West et al., 2008, 2009). Similar transplants using
cultured cells derived from embryonic to postnatal day 6 retinas
(RPCs) (or, indeed, freshly dissociated primary retinal cells from
embryonic retinas or retinas older than the first postnatal week) do
not result in photoreceptor morphology. Optimal integration
coincides with the birth of rod photoreceptors. Integrated cells are
thought to be post-mitotic (MacLaren et al., 2006; West et al.,
2009). Moreover the frequency of integration is low even at PN4
(0.6%), and transplantation is less successful in diseased retinas,
perhaps because of the gliosis that accompanies RD (West et al.,
2009). Disruption of the outer limiting membrane (OLM; a barrier
between the subretinal space and the outer nuclear layer) can
increase the proportion of integrating cells; however, percentages
are still small (West et al., 2008, 2009).
Primary retinal cells dissociated from late embryonic (E14.5 and
subsequent) and early postnatal mouse retinas can be expanded in
tissue culture. Adherent cultures are established within a few days,
can be passaged after 1 month, and can be grown indefinitely
(Klassen et al., 2004; Angénieux et al., 2006; Merhi-Soussi et al.,
2006). These cells are described here as retinal progenitor cells
(RPCs). RPCs are typically expanded in serum-free media optimized
for neural cultures with N2 supplement (or similar), epidermal
growth factor (EGF) and fibroblast growth factor 2 (FGF2), which
have both been shown to have a proliferative effect on these cells
(Kelley et al., 1995; Chacko et al., 2000; Das et al., 2005). Moreover,
there are indications that EGF, which biases towards glial cell fate in
neural stem cell cultures, can act as a potent neuralizingQ2 factor in
retinal cells (Angénieux et al., 2006). Plating on laminin, sometimes
with poly-L-ornithine, and sequential withdrawal of EGF, then FGF2
five days later, is used to differentiate RPCs in vitro, followed by
addition of B27 supplement. Adherent RPCs, classified variously as
retinal stem cells, retinal progenitor cells, retinal precursor cells,
radial glial cells and/or proliferating Mueller glia, show a capacity to
generate retinal neurons, including those expressing photoreceptor
markers (Klassen et al., 2004; Merhi-Soussi et al., 2006; Canola
et al., 2007). Cultured RPCs transplanted either subretinally or
intravitreally can integrate into the retina and have been shown to
generate some level of therapeutic effect (Klassen et al., 2004).
However, reports of photoreceptor morphology arising from
transplantation of postnatal rodent RPC cultures are discordant
(Klassen et al., 2004; Reh, 2006; Canola et al., 2007; Lamba et al.,
2008; West et al., 2009Q3 ), with the majority now suggesting that
such events are rare or non-existent (West et al., 2009).
Retinal differentiation protocols have been developed for RPCs,
embryonic neural stem cells, ES and iPS cells (Zhao et al., 2002;
Merhi-Soussi et al., 2006; Aoki et al., 2008; Meyer et al., 2006,
2009; Lamba et al., 2006, 2010; Ikeda et al., 2005; Osakada et al.,
2008; Jagatha et al., 2009; Hirami et al., 2009). ES or iPS-derived
cells also give relatively low integration rates of 0.1e0.5% of cells
initially transplanted, although some photoreceptor morphology is
achieved (Osakada et al., 2008; Lamba et al., 2009, 2010). The ability
to generate pure expandable cultures from which larger numbers of
photoreceptors can be obtained is a pre-requisite for RD cellular
therapies.
Firstly, we have investigated the identity of the primary retinal
cells that can integrate and give rise to photoreceptors. We have
found that PN3e5 Rho-eGFP primary retinal cells already expressing
rhodopsin at PN3e5 are more likely to integrate into the outer
nuclear layer (ONL) and form morphologically mature photorecep-
tors after transplantation than those Rho-eGFP cells not expressing
rhodopsin at the point of transplantation. Cells expressing rhodopsin
at PN3e5 are almost certainly post-mitotic, as rhodopsin is a product
of terminal rod differentiation. Rhodopsin expression is therefore
a good marker for photoreceptor potential post-transplantation;
however, this is rapidly lost from RPC cultures. We hypothesized that
sub-optimal initial culture conditions may result in poor survival of
photoreceptor precursors. However, we have achieved a 25-fold
improvement in growth rate via extensive protocol changes, but no
increases were observed in rhodopsin expression levels or integra-
tion rates post-transplantation, regardless of whether proliferating
or differentiated RPCs were analysed. Expression patterns of marker
genes in proliferating RPCs show that they are undifferentiated;
hence, we are losing cells that have already chosen a photoreceptor
cell fate. Rapid loss of rhodopsin expression after introduction of the
cells to tissue culture indicates that the majority of post-mitotic cells
are dying within 3 days. Identifying cues by which retinal progeni-
tors are specified in vivo, and culture conditions that promote
survival in vitro after specification, but prior to injection, will be
necessary for therapeutic use of these cells in RD. However, the
improvements in isolation and growth rates described here will be
useful to anyone who might wish to investigate the therapeutic
potential of these cells for disorders such as glaucoma, Leber’s
hereditary optic neuropathy, or retinoschisis, where the defect does
not lie within the photoreceptor layer.
2. Materials and methods
2.1. Retinal dissociation and FACS analysis
For FACS experiments, we used postnatal PN3e5 rhodopsin-eGFP
(Rho-eGFP; Chan et al., 2004) heterozygote mice as donors for FACS
and subsequent transplantation. These mice express a human Rho-
eGFP fusion protein that is visible in rod outer segments following
transplantation. Heterozygotes were used in our experiments as
homozygotes show symptoms of retinal degeneration (Chan et al.,
2004). Retinas were dissected and placed in 1 ml HBSS (Lonza). The
ciliary margin was removed from all retinas prior to dissociation.
Retinal cells were analysed by FACS as previously described (Palfi
et al., 2006). Following FACS analysis, cells were spun at 2000 rpm
for 5 min and resuspended such that the approximate concentration
of cells was 200,000 per 3 ml (cell count obtained from FACS).
Following subretinal injection (see below), residual cell samples were
counted using a haemocytometer in order to assess the actual
number and viability of cells injected (given the time elapsed, this
could vary substantially from FACS figures). For each time point, FACS
sorting was carried out 3 times and for each repetition, at least 3 eyes
were injected with positive and 3 eyes with negative cells. Unsorted
cells were also transplanted as a control. Animals were sacrificed 3
months post-transplantation, eyes were sectioned as described
below. Given the fact that eGFP, in Rho-eGFP cells, is expressed as
a rhodopsin-eGFP fusion protein, positive cells were identified via
eGFP positive, morphologically correct outer segments adjacent to
the RPE.
2.2. Animals, transplantation, cryosectioning, eGFP
transplantation cell counts
For transplantation studies involving cultured RPCs, cells were
isolated at PN3e5 from transgenic mice ubiquitously expressing
eGFP (Okabe et al., 1997). Recipients for all transplantations were
C57Bl6/J mice between 2 and 6 months of age. For tissue culture
studies, PN3e5 Rho-eGFP, eGFP, C57Bl6/J and RhoÀ/À donor mice
(Humphries et al., 1997) were used. Subretinal injections were
carried out in strict compliance with EU and Irish law (Cruelty to
Animals Act 2002) and with the ARVO statement for animal use in
ophthalmic research. Anaesthesia and subretinal injections were
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e142
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Please cite this article in press as: Mansergh, F.C., et al., Loss of photoreceptor potential from retinal progenitor cell cultures, despite
improvements in survival, Exp. Eye. Res. (2010), doi:10.1016/j.exer.2010.07.003
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3. carried out as previously described (Chadderton et al., 2009). Fixa-
tion, cryosectioning and staining were carried out as previously
described (Kiang et al., 2005). All sections were cut at 12.5 mm
thickness and were DAPI stained. Cells were counted on a fluores-
cent microscope (Zeiss, Axioplan 2); channels specific for RFP were
checked for each positive cell to ensure omission of false positives
due to autofluorescence. eGFP-positive cells were counted in
different categories, depending on retinal location and morphology.
Cells were counted as either unincorporated (balls of unintegrated
cells adjacent to the injection site, for example), retinal (having
migrated into the retina but with no evidence of integration),
integrated (visible evidence of axons, dendrites etc), or possessing
photoreceptor morphology (see above). Photoreceptor morphology
was not seen following transplantation of cultured RPCs, only after
transplantation of fresh retinal cells. In every instance, the entire eye
was sectioned and all 12.5 mm sections obtained were mounted,
DAPI stained and counted. Following sectioning and counting,
numbers of correctly integrated cells were divided by the original
number injected (haemocytometer figures used for FACS cells) and
multiplied by 100 to give the percentage of integrated cells,
photoreceptors etc. Results were graphed using GraphPad Prism 3.0.
2.3. Retinal dissociation for subsequent tissue culture
Retinas were placed in 1 ml PBS or HBSS following dissection. The
ciliary margin was removed from all retinas prior to dissociation, in
order to avoid contamination by CE cells in subsequent cultures. Old
dissociation method: Retinas were placed in 1 ml HBSS (Lonza) and
100 ml 10 mg/ml trypsin (Sigma) was added. Retinas were incubated
for 10 min at 37 C, after 5 min, 10 ml 10 mg/ml DNase1 þ100 ml
20 mg/ml trypsin inhibitor were added and samples were triturated
with a P1000 pipette (Gilson). Cells were spun for 5 min at 2000 rpm
(Thermo Microlite microcentrifuge) and cells were resuspended in
1 ml growth medium. New methods: 100 ml 0.25% trypsin/EDTA
(Lonza/Biowhittaker) or 100 ml Accutase (Sigma) were added, retinas
were incubated for 5 min at 37 C, allowed to settle to the bottom of
the tube and most of the supernatant was aspirated away. 1 ml
growth medium was then added and retinas were dissociated by
trituration with a fire polished Pasteur pipette. Retinas prepared via
mechanical dissociation followed the same procedure, but omitting
enzymatic digestion. Following dissociation, cells were counted four
times using a haemocytometer.
2.4. Tissue culture
2.4.1. Media, supplements, growth factors
Neurobasal medium, B27 and B27 without Vitamin A (B27 À RA)
were obtained from Invitrogen; DMEM/F12, embryonic and post-
natal stem cell media were from Sigma. Growth medium for RPCs
was composed of DMEM/F12 supplemented with 1Â N2, 1Â B27,
1Â B27 À RA or a combination thereof. 1Â L-glutamine (Lonza), 1Â
penicillin/streptomycin (Lonza), 5 mg/ml heparin (Sigma), 20 ng/ml
fibroblast growth factor 2 (FGF2) and 20 ng/ml epidermal growth
factor (EGF) were also added. RPCs were grown in Sarstedt T25
flasks; other plastics were less conducive to growth. Neurobasal
and other media were supplemented as for DMEM/F12.
2.4.2. Differentiation
Cells were differentiated in vitro by plating cells on laminin
coated T25s or poly-L-lysine and laminin coated glass cover slips.
After 2e4 days, EGF was withdrawn from the medium for 5 days,
followed by use of final growth medium supplemented with
B27 þ RA and no growth factors for 5e7 days. Glial differentiation
can be enhanced by adding 1% fetal calf serum (FCS) to the final
growth medium.
2.4.3. Substrates
Collagen, fibronectin, laminin, poly-L-ornithine, poly-L-lysine,
poly-D-lysine and vitronectin were obtained from Sigma, while
gelatin was obtained from Millipore. All were applied at 0.1% for 2 h
at room temperature or overnight at 4 C. Flasks were rinsed in 1Â
PBS (Lonza) twice before addition of media and cells.
2.4.4. Dissociation
Cells were placed in T25 flasks at a noted cell density in 5 ml
growth medium and incubated at 37 C and 5% CO2. After initial
plating, the medium was replaced completely after 5e7 days
initially, then every 2e7 days depending on density.
2.4.5. Passaging
Cells were passaged when 80e90% confluent. 0.5 ml 0.25%
trypsin/EDTA (Lonza) or Accutase (Sigma) were added to each T25
flask, following removal of medium and rinsing with 1Â PBS. Flasks
were incubated for 5e10 min until cell monolayers lifted off. Cells
were resuspended in 3 ml DMEM/F12, counted four times and spun
at 1000 rpm. The supernatant was removed, pellets were resus-
pended in growth medium and replated, frozen, or treated with TRI
reagent (Sigma).
2.4.6. Calculations
Initial cell density and date of plating were noted for each flask.
The number of days to reach 80e90% confluence was also noted.
Cells were counted at each passage (p) and the rate of cell growth
was calculated as follows:
Increase in cell no: per day
¼
ððCell no: at pN þ 1Þ À ðCell no: at pNÞÞ
No: of days between pN þ 1 and pN
2.4.7. Freezing
Cells were resuspended in 1 ml freeze medium (7.5% glucose,
10%BSA or 1% B27 À RA,10% DMSO, made up in DMEM/F12 medium
(Sigma)), placed in a Mr Frosty and frozen at À70 C. Thawed cells
were placed in 5 ml DMEM/F12 and spun before replating.
2.5. RNA extraction
Cell pellets were resuspended in 1 ml TRI reagent (Sigma) and
triturated using a P1000, while retinas were homogenized in 1 ml
TRI reagent using a Dounce homogenizer (Fisher). RNA was
prepared according to the manufacturer’s protocol. RNA samples
were assayed for concentration and quality using a Nanodrop
ND1000 (NanoDrop Technologies) spectrophotometer.
2.6. RT-PCR
Reverse transcription was carried out as previously described
(Mansergh et al., 2009). Primers were obtained from Sigma-Genosys
(see Table 1). PCRs were carried out using Crimson Taq and buffer
(NEB) according to the manufacturer’s instructions. A “no RT” control
corresponding to each sample was included. Housekeeping genes
(beta-actin, Gapdh, 18S rRNA) were also tested by DNA based Q-PCR
to ensure that CT values for each gene were within a similar range
(1.5CT difference from an average obtained for all samples).
2.7. Q-PCR
HPLC purified primers were obtained for one step real-time
Q-PCR (Sigma-Genosys). 5 Q4ml of each RNA sample was treated with
DNAfree (Ambion) in a volume of 30 ml according to the
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e14 3
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Please cite this article in press as: Mansergh, F.C., et al., Loss of photoreceptor potential from retinal progenitor cell cultures, despite
improvements in survival, Exp. Eye. Res. (2010), doi:10.1016/j.exer.2010.07.003
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4. manufacturer’s protocol. Q-PCRs were carried out using either RNA
or DNA with the appropriate one step or two step QuantiTect kits
(Qiagen) as described previously (O’Reilly et al., 2007). Slopes were
obtained for all Q-PCR primers used with respect to beta-actin. All
were 0.1; allowing use of the DDCt method to calculate fold
changes.
2.8. Immunocytochemistry
Cells were allowed to attach to poly-L-lysine and laminin coated
10e13 mm diameter cover slips placed in 12 or 24 well tissue
culture plates, in growth medium. For differentiated cells, differ-
entiation medium was applied after 24 h, followed after 5 days by
final differentiation medium, for a further 5 days. Cells were treated
with 4% PFA for 10 min, rinsed with 1Â PBS 4 times, then treated
with blocking solution (0.1% Triton, 10% donkey serum, 1% BSA) for
15 min. Primary antibodies GFAP (1/1000 dilution, Sigma), nestin,
(1/100 dilution, DSHB), b-III-tubulin (1/4000 dilution, Convance Q5),
Pax6 (1/500 dilution, Convance), glutamine synthetase (1/250, BD
Biosciences), rhodopsin (1/100, Chemicon) and synaptophysin
(1/300, Sigma) were added at the stated dilutions for 30e60 min at
room temperature. 3 washes of 1Â PBS were carried out, followed
by one of 3% BSA. Secondary antibody (1/100e1/1000 Cy3 anti-
mouse, Cy3 anti-rabbit, Cy2 anti-mouse as appropriate, Jackson
Immuno-Research) was then applied for 1 h. 2Â washes of 1Â PBS,
1 wash of 1Â PBS þ DAPI and a final 1Â PBS wash were then carried
out. Slides were inverted on poly-L-lysine coated slides using
AquaPolyMount (PolySciences), dried at 4 C overnight and pho-
tographed on a fluorescent microscope (Zeiss, Axioplan 2).
3. Results
3.1. FACS analysis and transplantation
In order to investigate the identity of cells that can integrate and
give rise to photoreceptors, we used FACS sorting with primary
retinal cells from Rho-eGFP mice. Rhodopsin expressing, eGFP-
positive cells from PN3e5 Rho-eGFP donor mice were separated
from non-rhodopsin expressing, eGFP-negative PN3e5 Rho-eGFP
cells from the same retinas, using FACS. PN3e5 cells expressing
rhodopsin were then transplanted into 2e6 month old C57Bl/6J
recipients separately from FACS sorted Rho-eGFP cells not
expressing rhodopsin. Animals were sacrificed after 3 months,
longer than the 2e4 week intervals used in previous studies, in
order to assess long-term graft survival. NOTE: It is possible to
count eGFP-positive photoreceptors following transplantation from
the Rho-eGFP-negative pool, despite the fact that re-analysis of the
FACS pool immediately post-sorting indicates that it is indeed 99%
negative. The presence of positive cells in the recipient retina 3
Table 1
Primer sequences for PCRQ8 .
18Smm F: GTAACCCGTTGAACCCCATT
18Smm R: CCATCCAATCGGTAGTAGCG
Gapdh F: ACCACAGTCCATGCCATCAC
Gapdh R: TCCACCACCCTGTTGCTGTA
B-actin F: CGTGGGCCGCCCTAGGCACCA
B-actin R: TTGGCCTTAGGGTTCAGGGGG
Brn3b F: TGGTGCTTACTCTGCTGGATTCT
Brn 3b R: CCTATTTGTGTGTGTGCTCCAA
Rpf1 F: CCTACCACAGGAAGCCCAAG
Rpf1 R: TGGATGGCTCACTCCCAATAA
Thy1 F: CCTGTAGTGAGGGTGGCAGA
Thy1 R: GGATCAGGGACAGCAAGAGG
Isl1 F: CACAGAGCGGAAGAAACCAG
Isl1 R: GGGAGGAGAGGCAAACGTAA
Atoh7 F: CCAGGACAAGAAGCTGTCCAA
Atoh7 R: CCCATAGGGCTCAGGGTCTA
Bsn F: ATCCCTGGCCCTTATGTTGA
Bsn R: GTTCACCCTGCCCAAAGAAC
Nr2e3 F: TTGGGAAATTGCTCCTCCTG
Nr2e3 R: CCTGTGGACACTTGGCACTC
Arr3 F: CTGGATGGCAAACTCAAGCA
Arr3 R: AGGAGATGGCTTTGGATGGA
CcnD1 F: TCTGCTTGACTTTCCCAACC
CcnD1 R: TGGTCCCACCTTCACCTCTT
mKi67 F: CAACCATCCAGGGAAACCAG
mKi67 R: GGCATCTGTGTGGGTCCTTT
Th F: TTCGAGGAGAGGGATGGAAA
Th R: CGACGCACAGAACTGAGGAG
ChAT F: TCTGCTGTTATGGCCCTGTG
ChAT R: AGATTGCTTGGCTTGGTTGG
Gad1 F: AAGCAACTACAGGGCGGATG
Gad1 R: GGGTACTAACAGGGAGGGTGTG
Gabbr1 F: TGTGTGTGTGTTGCCCTGAC
Gabbr1 R: CAAAGTGGGACGCATGAGAA
Syp F: ATGGTTGGGAGCTGTGAGGT
Syp R: AGGGAGAGGGCAGAGAAAGG
Oct4 F: GAGCACGAGTGGAAAGCAAC
Oct4 R: CGCCGGTTACAGAACCATAC
Brachyury F: CATGTACTCTTTCTTGCTGG
Brachyury R: GGTCTCGGGAAAGCAGTGGC
KDR F: TTTGGCAAATACAACCCTTCAGA
KDR R: GCAGAAGATACTGTCACCACC
Fgf5 F: TGTGTCTCAGGGGATTGTAGG
Fgf5 R: AGCTGTTTTCTTGGAATCTCTCC
GscF: CAGATGCTGCCCTACATGAAC
GscR: TCTGGGTACTTCGTCTCCTGG
Gapdh F F: ACCACAGTCCATGCCATCAC
Gapdh R: TCCACCACCCTGTTGCTGTA
B-actin F: CGTGGGCCGCCCTAGGCACCA
B-actin R: TTGGCCTTAGGGTTCAGGGGG
Nodal F: TTCAAGCCTGTTGGGCTCTAC
Nodal R: TCCGGTCACGTCCACATCTT
Mash1 F: CCACGGTCTTTGCTTCTGTTT
Mash1 R: TGGGGATGGCAGTTGTAAGA
Gfap F: AAAACCGCATCACCATTCCT
Gfap R: ACGTCCTTGTGCTCCTGCTT
Dcx F: GGCCAAGAGTTTCTGCCAAG
Dcx R: TAATGCAGGGATCAGGGACA
Nest F: ATGGGAGGATGGAGAATGGA
Nest R: GTGCCAGAGGGGCAGTTTCT
Chx10 F: AAGGAGCCATGTTGGACTGAA
Chx10 R: GCCTGGGAATACAGGAGCAG
Sox2 F: CTAGACTCCGGGCGATGAAA
Sox2 R: TGCCTTAAACAAGACCACGAAA
Otx2 F: GGTCCATCAACCAGCAACCT
Otx2 R: ACACCGGATCACCTCTGCTT
Pax 6 F: GAGAAATGGCGGTTAGAAGCA
Pax 6 R: CAACCACATGAGCAACACAGA
Mitf F: GATGGACGATGCCCTCTCAC
Mitf R: CTGGGCTACTGATAAAGCACGAA
mGluR6 F: CCGTGAATTGTCTTGTTGCTG
mGluR6 R: CCACCTTTCATGTTGGTGCT
Cnga1 F: TTGGGAGAAAGAGTCGTCTGG
Cnga1 R: GAACATCGGTGGGGAAGAAA
Crx F: CTCCAGACACACCAGGAAAGG
Crx R: GTGGGAGTGCAACAGGGTTT
Nrl F: CAGCAGTTGATTGTTTGCCTAATC
Nrl R: TGAGACCTGGAGGACAGCTACA
Recov F: CAGAAAAGCGGGCTGAGAAG
Recov R: TTACCCAGCAATCCCCAAAG
Rho F: TGTGGGGACAAACAGTCCAG
Rho R: GGCTCCATCCCATTCTTTTG
Q-PCR HPLC purified primers
Qactin F: CCACCATGTACCCAGGCATT
Qactin R: ACAGTGAGGCCAGGATGGAG
QNrl F: ATGCAAGTGGATTGGAGGAG
QNrl R: CATGGCAACTGTGAGACCTG
QCrx F: TCTTCCGTAAAGGTGCTGAGA
QCrx R: TGCTGGGATTATGACCATTGA
QRho F: CTGAGGGCATGCAATGTTCA
QRho R: CATAGCAGAAGAAGATGACG
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e144
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5. months later occurs as a result of the fact that rhodopsin is
increasingly expressed in rod photoreceptors as rod cells mature.
We have found that some cells that were Rho-eGFP negative at the
point of FACS analysis at PN3e5 go on to express rhodopsin later
(and therefore also the Rho-eGFP fusion protein). These can then be
visualized when counting. Indeed, the purpose of this experiment
was to determine whether the Rho-eGFP-positive cells that end up
with photoreceptor morphology post-transplantation were pre-
specified as rods (and therefore Rho-eGFPþve) at PN3e5 or
whether the rod specification came later, after transplantation of
cells that were Rho-eGFP negative at the point of FACS sorting, but
became Rho-eGFP positive after transplantation. Our findings
indicate the former to be the case, but not exclusively so; some Rho-
eGFP-positive photoreceptor morphology is obtained after trans-
plantation of cells that are Rho-eGFP negative at PN3e5.
At both PN3 and PN5, Rho-eGFP positive, sorted cells integrated
at a higher frequency than Rho-eGFP-negative sorted cells and this
difference was more significant in PN5 cells than PN3. t-Tests gave
values of p ¼ 0.165 for PN3, p ¼ 0.050 for PN5, and p ¼ 0.014 for
both data sets combined (Fig. 1). Integration rates are lower than
the 0.6% value reported previously (MacLaren et al., 2006; Bartsch
et al., 2008), probably as a result of negative impacts on cell
survival of harsh enzymatic digestion followed by FACS analysis, or
the longer period of time elapsed between injection and sacrifice.
We conclude that rhodopsin expression is a marker of the ability of
primary retinal cells to form photoreceptors post-transplantation.
3.2. Transplantation of cultured RPCs
In contrast, transplantation of cultured RPCs resulted in lower
percentages of integrated cells and no photoreceptor morphology.
Multiple injections of proliferating and differentiated RPCs, passages
1e3, derived from PN2e5 eGFP transgenic mice, were transplanted
into 2e6 month old C57Bl/6J recipients. We used lower passage cells
as there is evidence that karyotypic instability can occur in neonatal
RPCs after passage 9 (Djojosubroto et al., 2009). Transplanted RPCs
survived in the retina at a rate of 0.022%, while integrated cells were
only 0.005% of cells transplanted initially. None gave photoreceptor
morphology (Fig. 1). This contrasts with approximately 0.2% cells
with photoreceptor morphology for freshly dissociated retinal cells
(see Fig. 1A, B). No consistent differences in survival or integration
rates between proliferating and differentiated RPCs were noted; both
were uniformly low. Integration occurs primarily in the inner plexi-
form and ganglion cell layers; a majority of integrated cells are GFAP
positive (Fig. 1CeE).
3.3. Optimisation of cell culture conditions
Previous protocols resulted in high cell death rates in the first
few days following initial plating of dissociated cells. In order to
obtain confluent cells from RPCs within 3e4 weeks, plating
densities of 1e2 Â 105
cells/cm2
had been used (2.5e5 million cells
per T25). Existing protocols recommend the digestion of retinas for
10e20 min in trypsin, collagenase, hyaluronidase, kynerunic acid or
combinations thereof (Klassen et al., 2004; Canola et al., 2007). This
results in a single cell suspension, however, the effect on the cells
may be excessively harsh, given the levels of cell death.
Previously we had incubated retinas with trypsin for 20 min at
37 C, with addition of DNase1 after 5 min and trypsin inhibitor after
20 min. Samples were triturated using a p1000 pipette, spun and
resuspended in growth medium. This protocol yields a single cell
suspension and requires plating of a minimum 1 Â105
cells/cm2
.
Growth to 80e90% confluence takes a month, and at minimum cell
density, a majority of cultures do not survive.
We then tried 10Â dilutions of Accutase and tissue culture
formulated trypsin/EDTA to digest the retinas for 5e10 min, followed
by dissociation with a fire polished and narrowed borosilicate glass
pipette. Some samples were dissociated mechanically without any
enzymatic digestion. Comparison of the numbers of resulting cells
gained by these methods gave a p value of 0.027, which underesti-
mates the significance of this result; of 12 cultures plated using the
old method, only 4 survived to be included in data processing. All
cultures plated using minimal digestion or mechanical dissociation
alone survived (Fig. 2A). Furthermore, with optimized tissue culture
conditions (see below), plating densities of 100,000 cells per T25
(4 Â103
cells/cm2
) will reliably give rise to cultures within 10e20
days (Fig. 2B). Growth times from initial plating to 80e90% conflu-
ence varied between 19.3 days for 100,000 cells to 10.75 days for 1.5
million cells per T25. Differences in the rate of cell division caused by
initial cell density are not statistically significant.
We also tested the growth rates of RPCs derived from different
mouse strains (Fig. 2C), in order to verify that the cells being trans-
planted (eGFP, Rho-eGFP) did not behave differently from wild-type
cells. We tested rhodopsin knockout derived PN3e4 cells (RhoÀ/À,
Humphries et al., 1997), to determine whether lower percentages of
photoreceptor precursors would influence growth rates; photore-
ceptors in these mice start to degenerate from birth. Differences in
the rate of cell division (increase in cell #/day) caused by strain
difference, genetic modification or the presence of eGFP are not
statistically significant. Notably, RPC growth is not influenced by the
absence of functional rhodopsin expression, since rhodopsin
knockout cells grew at indistinguishable rates from wild type.
3.4. Cell culture media and supplements
Initial protocols used DMEM/F12 medium with 1% pen-
icillinestreptomycin and L-glutamine, supplemented with N2, FGF2,
EGF and heparin. We decided to test the effect of replacing N2
supplement with B27 supplement, or B27 minus Vitamin A (also
known as retinoic acid, RA). RA is a potent morphogen and is used to
effect neural differentiation in ES cells (Bain et al., 1995). Derivatives
of RA (all-trans-retinol and 11-cis-retinal) are vital components in
the visual transduction cycle. We reasoned that B27 À RA might
better support proliferation of stem cells and prevent differentiation,
allowing better growth in culture. We also tested the addition of fetal
calf serum (FCS), which is known to bias neural stem cells towards
a glial cell fate, but which often promotes cell survival. Finally,
DMEM/F12 was replaced variously with neurobasal medium, post-
natal neural stem cell medium or embryonic neural stem cell
medium (Sigma). In each case, media were supplemented with N2 or
B27, with EGF, FGF2 and heparin as usual (Fig. 2D).
Postnatal and embryonic stem cell media did not support the
growth of RPCs. Neurobasal medium permitted growth, but at
a slower rate thanwith DMEM/F12. In terms of increased survival and
growth, DMEM/F12 with B27 À RA were by far the best combination
(N2 vs B27 À RA; p ¼ 0.0062). Predictably, B27 þ RA slowed growth
in comparison to N2 supplemented media and increased the likeli-
hood of spontaneous differentiation. Addition of FCS caused a rapid
morphological transition such that flat fibroblast type cells were
observed after a number of days; subsequent passages resulted in
a decrease in cell number. We also tested various combinations of
N2B27, N2B27 À RA, and N2B27 where BSA had been omitted from
the N2 supplement. None were superior to B27 À RA alone.
3.5. Cell attachment
We tested the influence of cell attachment on growth. Neural stem
cells can be grown as neurospheres, which contain mixtures of
differentiated and undifferentiated cells. However, RPCs show
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e14 5
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6. a strong preference for attachment to tissue culture flasks and even
tend to attach to bacterial Petri-dishes. Concern was expressed that
attached cells may be biased towards a glial lineage and growth as
a monolayer may preclude the culture of photoreceptor precursors.
We therefore tried to establish “retinospheres” in culture in ultra-low
attachment plates (Corning). To encourage initial sphere formation,
we used mechanical dissociation alone to disrupt the cells and left
them in larger clumps than is typical. While the cells could be grown
as spheres using these methods, cell numbers declined over time,
indicating that cell renewal is not adequately supported under these
Fig. 1. Transplantation. (A) Cell integration and survival rates, 3 months post-transplantation (FACS sorted Rho-eGFP derived photoreceptors, columns 1e6 and cultured eGFP RPCs,
column 7). Cells counted 3 months post-transplantation. Rho-eGFP cells that were eGFP positive at the point of FACS analysis (PN3e5, columns 4e6), show a higher integration rate
than Rho-eGFP cells that were eGFP negative at the point of FACS analysis (columns 1e3). Counts were obtained from transplantation of Rho-eGFP cells that were eGFP negative at
the point of FACS sorting, but began to express rhodopsin, and therefore the eGFP fusion protein, subsequently. The number of integrated cells is expressed as a percentage of those
injected. For comparison, an integration rate is also given for transplanted cultured RPCs, but in the case of RPCs, none integrated as photoreceptors (Column 7, grey). (B) Primary
eGFP mouse retinal cells transplanted into adult C57Bl6/J wild-type mouse retina. (CeE) Integrated cells derived from RPCs. C and D were photographed at the same magnification
as B. E (higher magnification) shows integrated cells counterstained with GFAP after counting. (FeI) FACS sorting of C57Bl6/J and Rho-eGFP cells, days 1 and 4. Note shift in Rho-
eGFP expression at day 4 in comparison to day 1, denoting upregulation of rhodopsin (and therefore eGFP) expression.
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e146
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7. Effects of Substrate on RPC Proliferation
U
nc
tao
ed,
-72B
A
Rn
U
c
tao
e
2
N,d
NF
LLP
L
DP
P
N
R
O
G
EL
L
MA
VN
LL
O
C
MAL+
NF
F
L
DP+
N
NF+LL
O
C
AL
M
LLP+
L
LE
G+
MA
0.0×10-00
2.5×1005
5.0×1005
7.5×1005
1.0×1006
1.3×1006
1.5×1006
mean p0
mean p1
mean p2
lleCniesaercnI
yaDreprebmuN
Effects of Mouse Strain
on RPC Proliferation
c57 eGFP rho-eGFP Rho -/-
0.0×10-00
2.0×1005
4.0×1005
6.0×1005
8.0×1005
1.0×1006
1.2×1006
Passage 0
Passage 1
Passage 2
Mouse Strain of Origin
lleCniesaercnI
yaDreprebmuN
Effect of Initial Cell Seeding
Density on RPC Proliferation
0 1 2
0.0×10
2.0×10
4.0×10
6.0×10
8.0×10
1.0×10
1.2×10
1.4×10
1.6×10
1 x 105
2.5 x 105
0.5 x 106
1 x 106
1.5 x 106
Passage Number
yaDreprebmuNlleCniesaercnI
Effects of Dissociation
Method on RPC Proliferation
O
ld
ohte
M
d
yrT
sp
in/E
T
D
A
Ac
esatuc
M
e
cinahc
al
taicossi
D
ion
-50000
0
50000
100000
150000
lleCniesaercnI
yaDreprebmuN
A CB
Effects of Medium Composition on RPC Proliferation
R+72B
A
A
R-72B
2
N
A
R+72B2
N
N
2
2B
7
A
R-N
2B2
7
R-
ASB-A
2B,B
N
7
A
R-B
72
R-
A
+
%1
FC
S
B
A
R-72
+
S
CF
%5
N
2
+
%1
S
CF
N
5
+2
%
FC
S
0.0×10-00
1.0×1005
2.0×1005
3.0×1005
4.0×1005
5.0×1005
6.0×1005
7.0×1005 mean p0
mean p1
mean p2
IesaercninCell
yaDreprebmuN
D
E
Fig. 2. Effects of protocol improvements and mouse strain of origin on survival and growth rates of proliferating RPCs.
(A) Effects of dissociation method on RPC growth rate. 4 Â 104
cells/cm2
were plated per T25 flask, cells were grown to 80e90% confluence. The increase in cell number per day was
calculated as follows: (final cell # À initial cell #)/# days between plating and passaging. (B) Influence of initial cell plating density on RPC growth. Cells were grown using optimized
culture conditions and plated on FN-coated flasks. Growth times from initial plating to 80e90% confluence were calculated as in A above. (C) Influence of mouse strain on RPC
growth. RPCs were derived from wild type (C57Bl6/J), eGFP transgenic mice (for routine transplantation of RPCs), Rho-eGFP knock-in mice (FACS and transplantation) and rhodopsin
knockout mice (rhoÀ/À) There are no statistically significant differences between wild-type derived RPCs and those from any of the other strains. This means that they can
legitimately be used interchangeably (t-tests; C57 vs eGFP,p0, p ¼ 0.39, C57 vs Rho-eGFP, p0, p ¼ 0.20, C57 vs RhoÀ/À, p0, p ¼ 0.11). (D) Effects of different combinations of media
and growth supplements on increase in cell number per day. p ¼ passage number; effects for p0, p1 and p2 cells are given. Cells were harvested and counted once 80e90%
confluence was achieved; in the case of p0, this typically took 10e20 days from initial plating. Intervals from plating to confluence were typically 3e7 days in subsequent passages.
Growth medium ¼ DMEM/F12 (Sigma), unless otherwise stated. NB ¼ neurobasal medium. FCS ¼ fetal calf serum, N2, B27 and B27 À RA are media supplements. N2B27 ¼ N2 þ B27
supplement. N2B27 À RA ¼ N2 þ B27 À RA. N2B27 À RA À BSA ¼ N2 without BSA and B27 À RA. All samples were grown in the presence of FGF2, EGF and heparin. Note maintenance
of higher growth rate with B27 À RA beyond p0. (E) Influence of cell substrate on RPC growth. Untreated ¼ Sarstedt T25 flasks with no further coatings applied. N2 ¼ untreated
flasks where RPCs were grown with N2 (all other samples in this graph were grown supplemented with B27 À RA). Fn ¼ fibronectin, pll ¼ poly-L-lysine, pdl ¼ poly-D-lysine,
porn ¼ poly-L-ornithine, gel ¼ gelatin, lam ¼ laminin, Vn ¼ vitronectin, coll ¼ collagen.
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e14 7
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8. conditions. Growth of retinospheres was tested with a number of
different media and supplements. No combination gave rise to robust
proliferation; plating of single cell suspensions resulted in cell death.
We tested the influence of substrate on monolayer growth. Cells
were grown in optimized RPC growth medium, in uncoated T25
flasks and in those coated with one of the following; poly-L-lysine
(PLL), poly-D-lysine (PDL), poly-L-ornithine (PORN), gelatin (GEL),
laminin (LAM), fibronectin (FN), vitronectin (VN) and collagen
(COLL) (Fig. 2E). Fibronectin provided the best growth advantage at
p0 (B27 À RA vs fn þ B27 À RA; p ¼ 0.001; N2 vs fn þ B27 À RA;
p ¼ 0.0009), but this was only significant between initial plating
and passage 0 (p0). Fibronectin solutions could only be reused
a maximum of 3 times, before efficacy diminished.
3.6. Real-time Q-PCR, immunohistochemistry, and use of Rho-eGFP
transgenic cells to track rhodopsin expression
Using improved growth conditions, we used real-time Q-PCR to
compare rhodopsin levels in cells grown under conditions outlined
above, with those grown using older methods. All conditions were
tested; a selection of results is shown in Fig. 3. Rhodopsin levels
dropped dramatically when comparing fresh retinal tissue with
passage 0 confluent plates (which had undergone 10e20 days
growth post-isolation to reach confluence); furthermore,
rhodopsin levels dropped again in subsequent passages, often to
undetectable levels. This drop was so dramatic that we had to
analyse results using the DDCt method; standard curves did not
encompass the wide range of rhodopsin levels detected. This drop
was noted in “retinosphere” cultures (results not shown), prolif-
erating adherent RPCs, and cells differentiated in vitro. Notably, the
presence of retinoic acid in B27 supplement (B27 þ RA, Fig. 3A) did
not stimulate rhodopsin expression, despite prior evidence that RA
can do this in both dissociated retinal cells, retinal explants
cultured without the RPE, and differentiating mouse ES cell cultures
(Soederpalm et al., 2000; Osakada et al., 2008).
We then studied rhodopsin levels in the days immediately after
placing the cells in culture. Dramatic reductions in rhodopsin
expression levels were detected shortly after initial plating. We
plated cells at a density of 2e6 Â 104
cells/cm2
on fibronectin-coated
T25 flasks. We harvested the cells on days 1e3, 4e6, 6e9, 9e12, and
12-confluence. Cells were counted, their RNA extracted and
rhodopsin levels determined by real-time Q-PCR. We also plated
transgenic Rho-eGFP cells on poly-L-lysine and fibronectin-coated
glass slips and tracked the reduction in rhodopsin levels via Rho-eGFP
fluorescence (Fig. 4) and separately, via immunohistochemistry, at
the intervals stated above. Cell counts indicated an initial dramatic
drop in the number of eGFP-positive cells, followed by the slow
growth of eGFP-negative cells to confluence from day 3 to day 10e20.
Q-PCR, counting of Rho-eGFP-positive transgenic cells and immu-
nohistochemistry all separately indicate that rhodopsin-positive cells
are largely lost from the cultures over the first 3 days (Fig. 4). More-
over, rhodopsin-positive rod precursors were not necessary for
establishment of healthy RPC cultures; RPCs from RhoÀ/À mice had
growth rates indistinguishable from wild-type cells (Fig. 2C).
3.7. Gene expression profiles
We used RT-PCR and immunocytochemistry to further charac-
terize RPCs. Previous studies have suggested some ganglion cell
potential post-transplantation (Canola et al., 2007) or suggested that
RPCs are radial/Mueller glia. We amplified RNA from PN3e5 retinas
and passage 0e2 RPCs derived from C57Bl6/J, Rho-eGFP, EGFP and
RhoÀ/À mice (Fig. 5). While differences between whole retinas and
RPCs were dramatic, within the RPC samples, passage number, mouse
strain of origin, presence/absence of eGFP and the developmental
stage of isolation (PN3e5) had no consistent effect (Fig. 5). Genes
associated with photoreceptor differentiation (rhodopsin, recoverin,
Cnga1, Nrl, Crx, N2re3) were downregulated in RPCs. Similarly, genes
associated with ganglion cells (Chd5, Islet1, Atoh7) were also
downregulated, with the exception of Thy1. Some genes associated
with eye specification (Pax6, Otx, Chx10) and neurotransmission
(Th, ChAT) were also downregulated. Notably, Mitf, Gad1 and Gabbr1
remained at similar levels to those seen in retinal samples. Genes
associated with neural stem cells (nestin, sox2, mash1) and cell cycle
associated genes such as Ki67 and Ccd1 were upregulated in RPCs,
particularly in later passages. Gfap, the signature marker of glial cells,
was expressed at similar levels in retina and in RPCs.
3.8. Cell morphology and immunocytochemistry
RPCs form a characteristic fusiform morphology, indicative of
neural progenitors, after approximately 1 week in culture; this is
maintained in subsequent passages (Fig. 6A). Despite plating of
Effects of Medium Composition on
Rhodopsin Expression Levels
72B
0p
A
R+
0p
A
R-72B
0p
2
N
FN
p0
0p
MAL
0p
LE
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OP
0p
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B,
72B
-
p
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-
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CF
%1+
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CF
%5+
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R-72B
0.000
0.005
0.010
0.015
trwegnahCdloF
aniteR4NP
Effects of Differentiation on
Rho mRNA Levels
gnitarefilorP
D
nereffi
t
ya
D
5(
gnitai
s
)F
GE-
noitaitnereffi
DlaniF
0.00
0.05
0.10
0.15
egnahCdloF
aniteR4NPtrw
B
A
Fig. 3. Real-time Q-PCR of rhodopsin levels in selected RPC cultures.
(A) Rho mRNA levels in selected proliferating RPC cultures once confluent (p0; 10e20
days post-plating). Fold change is expressed with respect to (wrt) Rho expression
levels obtained from PN4 retina (PN4 retina ¼ 1). Selected samples are shown above;
Q-PCR readings for all varying growth conditions tested were obtained (see Figs. 2e4),
for passage (p) 0e2 in each case. Only p0 is shown here; levels of Rho decline further
thereafter in subsequent passages. p0 ¼ initial growth period from plating to conflu-
ence, mRNA samples taken at confluence, typically 10e20 days post-plating. B27 and
N2 are media supplements, ÀRA or þ RA denotes the presence or absence of retinoic
acid in the B27 supplement, FCS ¼ fetal calf serum, NB ¼ neurobasal medium, used
instead of the default DMEM/F12 in one case above. Fn ¼ fibronectin, lam ¼ laminin,
gel ¼ gelatine, porn ¼ poly-L-ornithine. (B) Comparison of Rho mRNA levels in prolif-
erating, differentiating and finally differentiated RPCs, passage 1. No significant
differences in Rho levels were noted between proliferating and differentiated cells.
Some differences in Rho expression levels were noted between sets of experimental
repeats carried out at different times (note difference between B27 À RA and prolif-
erating in graphs A and B). Values graphed here are skewed upwards as PCRs showing
no rhodopsin expression at all could not be included in calculations.
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e148
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improvements in survival, Exp. Eye. Res. (2010), doi:10.1016/j.exer.2010.07.003
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9. retinal cells, many of which have already adopted differing cell
fates, the uniform morphology would indicate that tissue culture
conditions select for a single cell type. Notably, we have been
successful in isolating RPCs from ages E14.5 to postnatal day 46
inclusive (results not shown). The morphology of cultured cells
does not vary according to the developmental age from which the
cells were derived, although RPCs proliferate a lot more slowly
initially when isolated after PN6. Cells grow faster between initial
plating and first passage if plated on fibronectin, where a slightly
flatter morphology is noted (Fig. 6B). Cells can survive as “retino-
spheres”, however, cell numbers decline with time, indicative of
a failure to proliferate and/or higher rates of cell death (Fig. 6C).
Staining of proliferating cells (Fig. 6DeL) showed low staining for
b-III-tubulin (6D) and Gfap (6E) and stronger staining with Pax6,
nestin and glutamine synthetase (6FeH). RDS/peripherin and
rhodopsin antibodies (6I, J) are negative, similarly, Rho-eGFP
derived RPCs (6K) are usually eGFP negative, but staining of the
occasional cell is sometimes seen in some cultures, correlating with
PCR results (Fig. 5). Differentiated RPCs show strong glutamine
synthetase staining (6Q) and strong staining of some, but not all,
cells for B-TubIII (6M), and Gfap (6N). Addition of 1% serum during
final differentiation results in 80e90% Gfap staining (6T).
Differentiated RPCs show negligible staining for Rds/peripherin,
Rho (6R,S) and synaptophysin (not shown); current differentiation
methods do not favour photoreceptor or photoreceptor precursor
formation.
4. Discussion
FACS analysis and subsequent transplantation indicate that
rhodopsin is a good marker for integration potential and generation
of morphologically mature photoreceptors post-transplantation.
These data also support the conclusion that integrating cells with
photoreceptor morphology, derived from fresh retinal material, are
post-mitotic; rhodopsin is a tightly regulated gene that is only
expressed in maturing rod photoreceptors and has been used as
a marker of terminal rod differentiation (Humphries et al., 1997).
Some rhodopsin-negative cells do integrate; the likelihood is that
these are photoreceptor precursors that have just been specified,
are not yet expressing enough rhodopsin to be sorted as positives,
but increase expression of the Rho-eGFP reporter transgene after
transplantation. FACS also does not give 100% pure separation;
negatives are typically 99% pure while positives are only 80e90%
positive.
Fig. 4. Decreasing Rho mRNA and protein levels in proliferating RPCs after initial dissociation and plating. A) Cell counts taken at daily intervals following initial plating of 1 Â106
cells per T25. There is a dramatic decrease in cell number in the initial 3 days, followed by a gradual increase in cell number as surviving cells attach and divide. B) Real-time
rhodopsin Q-PCR of samples taken at daily intervals post-plating of 1 Â106
cells per T25. Rho mRNA levels drop dramatically in the first 2 days. C) Levels of Rho protein levels
assessed from counting of DAPI-stained transgenic Rho-eGFP cells that were still expressing eGFP, expressed as a % of the total Dapi-stained cells present at daily intervals
post-plating (unbroken line). Cells were also counterstained using antibodies against rhodopsin (Chemicon MAB5356), labelled with Cy3 (short dashed line). Combined results from
Rho-eGFP, eGFPþve and rho-immunocytochemistry are also presented (long dashed line). D). Photomicrograph of DAPI-stained Rho-eGFP cells, 1 day post-plating, E ¼ DAPI-stained
Rho-eGFP cells, 7 days post-plating. F ¼ DAPI-stained Rho-eGFP cells, 10 days post-plating, G ¼ DAPI-stained Rho-eGFP cells, 15 days post-plating (80e90% confluent). Note green
eGFP-positive cells in the middle of the Rho-eGFP cell clumps 24 h after plating (D), which disappear by 7 days and do not re-appear subsequently (EeG).
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e14 9
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improvements in survival, Exp. Eye. Res. (2010), doi:10.1016/j.exer.2010.07.003
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10. F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e1410
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11. Expansion of RPCs in culture eliminates photoreceptor
morphology after transplantation. This could occur for a number of
reasons hypothetically: the photoreceptor precursors might undergo
cell death, the post-mitotic nature of the cells might mean that they
are “diluted out” of the cultures as other cells divide, or their cell fate
might be altered by the tissue culture environment. We succeeded in
improving survival and/or growth rate of RPCs in culture 25-fold,
however, photoreceptor precursors are still lost shortly after plating.
Using cell counts, real-time Q-PCR, semi-quantitative PCR, immu-
nohistochemistry and tracking of reporter expressing Rho-eGFP
transgenic cells in the days after plating, we note that sharp reduc-
tions in cell numbers within the first 3 days post-plating coincide
with loss of rhodopsin expression, while culture conditions that
promote slower RPC growth do not contain higher proportions of
rhodopsin-positive cells. We conclude therefore that the mechanism
for the loss of photoreceptor precursors from cell culture is cell death
in the first 3 days after introduction of the cells to tissue culture, not
transdifferentiation or dilution.
As previously hypothesized (MacLaren et al., 2006; West et al.,
2009), photoreceptor precursors are post-mitotic, cannot adapt to
tissue culture conditions and are dying shortly after plating. This
conclusion is supported by separate culturing of FACS sorted Rho-
eGFP positive and negative cells (results not shown). Negative cells
(99% pure) establish more easily in culture than positives (80e90%
pure). However, once cultures are established, morphology of
positive and negative cultures is identical, and rhodopsin levels are
low to non-existent, as is typical for RPCs derived from unsorted
cells. Notably, less severe dissociation conditions do not improve
rhodopsin levels at p0, suggesting that the problem is not merely
one of fragility. There is an interesting shift in cell behaviour
between initial plating and establishment of the cells as prolifer-
ating RPCs. Plating the cells as small clumps initially (small enough
to be counted, but not a single cell suspension), is the biggest
contributor to the increased survival rates noted in this study. These
cells grow rapidly if plated at a reasonably low density
(4 Â103
cells/cm2
). We have also found that, where cells were
plated at too high a density initially, passage before day 10 results in
an increased risk of cell death. However, after confluence is ach-
ieved (10e20 days), use of 0.25% trypsin or 1Â Accutase for 5 min
and trituration with a 5 or 10 ml pipette will reliably generate
a single cell suspension that can be replated at 4 Â103
cells/cm2
or
more without significant trauma to the cells. A process of adapta-
tion to tissue culture conditions is obviously causing large changes
in cellecell and cellesubstrate attachment behaviour.
Previous analyses considered the possibility that RPCs could
have ganglion cell potential and/or represent immature radial/
Mueller glia (Angénieux et al., 2006; Canola et al., 2007). RPCs
migrated to the ganglion cell layer and expressed ganglion cell
markers when transplanted into models of RD (Canola et al., 2007).
However, ganglion cells are amongst the first to have their cell fate
determined, they are “born” during embryogenesis, while rods and
Mueller glia are locked into their respective cell fates during the
first few days after birth (Andreazzoli, 2009; MacLaren et al., 2006).
Mueller glia can regenerate the injured retina in fish, amphibians
and even to some extent in chick. They have a limited potential,
even in mammals, to give rise to cells with some of the character-
istics of retinal neurons (Andreazzoli, 2009; Karl et al., 2008).
Furthermore, glial cells have the capacity in RD to proliferate,
possibly in an attempt to ameliorate some of the pathogenic effects,
resulting in gliosis (West et al., 2009). Our transplantation results
also show RPC integration primarily in the inner plexiform and
ganglion cell layers (Fig. 1CeE), correlating with previous results. A
majority of cells are GFAP positive.
Results of RT-PCR and immunohistochemistry on a panel of
genes indicate that products of terminally differentiated retinal
cells, whether they are ganglion cells, other retinal neurons or
photoreceptors, are largely downregulated, as would be expected
in progenitor cells. GFAP, a glial marker, maintains expression levels
in culture similar to those seen in PN3e5 retina, but is not upre-
gulated. Interestingly, markers of neural stem cells/progenitors
such as nestin, Sox2 and Mash1 are upregulated slightly, along with
cell cycle markers such as Ccd1 and Ki67.
While we cannot rule out glial identity, it is also possible that
RPCs represent a pool of retinal progenitors, which are capable of
establishing themselves in culture. It may be the case that building
a retina occurs from a pool of unspecified stem cells, according to
positioning within the retina and mechanical cues provided by the
intercellular matrix and cellecell junctions (Andreazzoli, 2009).
Removing cells from this structure may allow the division and
establishment in culture of only those that are not yet specified.
Without the correct mechanical stimuli or positional cues, it may be
difficult to specify the desired cell fate in these cells. However, they
may yet be shown to be capable of generating the desired retinal
cell types, given correct mechanical cues or as yet unidentified
signals. Interestingly, the more robust cell growth achieved by
protocol changes described in this paper may assist in resolving
some of the differing results obtained with RPCs to date. We have
noted that sub-optimally isolated RPCs go through a slow growing
initial stage in which it is obvious morphologically that some
differentiation has occurred. Optimization of the culture conditions
ensures that this no longer occurs, allowing, possibly, more
consistent gene expression results to be obtained from different
isolates of RPCs. This may in turn resolve the question of whether
differences in potential between embryonic and postnatally iso-
lated RPCs actually exist (Yang, 2004).
RPCs are certainly capable of generating abundant glial cells if
allowed to differentiate via sequential withdrawal of growth factors
and application of 1% FCS in B27 þ RA containing media. This does
not by itself indicate that these cells are solely glial precursors
though, just that we can positively influence glial differentiation.
Even after treatment with FCS, some beta-III-tubulin positive, GFAP
negative cells remain (Fig. 6T). Some migration of RPCs to the
ganglion cell layer after transplantation may perhaps indicate
a degree of neuronal multipotentiality (Canola et al., 2007, Fig.1D, E).
Investigation of these cells in treatment of retinal disorders such as
LHON or glaucoma, where the defect does not reside within the
photoreceptor cell layer, might be warranted. Better differentiation
protocols with improved cell survival and further characterization of
resulting cell populations, in vitro and post-transplantation, would
be helpful in investigating the true potential of RPCs.
RPCs can be cultured from late embryonic stages until approxi-
mately PN6, before they become more difficult (but not impossible)
to grow. These cells probably represent an undifferentiated cell pool
from which, in vivo, unknown mechanical, hormonal or positional
cues are used to sequentially differentiate the diverse cell types found
in adult retina. Notably, cells acquire a uniform morphology during
Fig. 5. Semi-quantitative PCR analysis of gene expression profiles from freshly dissociated PN3e5 retina (left hand column) and cultured proliferating RPCs originally derived from
PN3 C57Bl6/J mouse retinas at passages 0, 1 and 2 (p0, p1 and p2, middle right hand column). p0 ¼ approx 10e20 days post-isolation, p1 ¼3e7 days after p0, p2 ¼ 3e7 days after p1
respectively. Three housekeeping genes are shown, giving equal levels of expression in all samples. However, in order to check that levels of these housekeeping genes were indeed
roughly equal, they were also amplified by Q-PCR from the same samples; variation was indeed minimal (see Materials and methods). All amplifications were carried out using an
annealing temperature of 62 C, cycle numbers are indicated beside the gene name.
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e14 11
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improvements in survival, Exp. Eye. Res. (2010), doi:10.1016/j.exer.2010.07.003
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12. the initial period of growth post-plating; this morphology does not
change with subsequent passages, suggesting expansion of a uniform
stem cell type. Photoreceptor precursor cells are a post-mitotic cell
populationwithin the neonatal retina which are capable of becoming
mature photoreceptors post-transplantation. These cells die off
rapidly when placed in a tissue culture environment. This is prob-
lematic, as therapeutic strategies for RD may need to focus on
expansion of undifferentiated progenitors (currently possible) and
then application of unidentified triggers in order to obtain photore-
ceptor precursors. Differentiation of RPCs in vitro does not currently
result in significant increases in rhodopsin levels, but does give rise to
high levels of cell death (which may indicate death of cells choosing
a photoreceptor fate at the point of differentiation). In support of this
point, attempts by this group to transfect various cell lines with
rhodopsin in the past has not met with success; cells expressing the
transgene tend to die (S. M-W, unpublished results). Unless occurring
in a very structured environment, rhodopsin expression may well be
very toxic to the cell.
We therefore need to identify the triggers that promote terminal
differentiation of rod and cone photoreceptor cells in vivo. We may
then be able to adapt tissue culture protocols such that these new
triggers can be applied in vitro and the resulting cells can be
persuaded to survive for as long as it takes to inject them into
a recipient. Identification of the key points in retinal development
will require detailed analysis of the transcriptome and epigenetic
structure of differentiating retinal cells in vivo, in conjunction with
further use of transgenics such as Crx-, Nrl- or Rho-eGFP mice, where
cells can be sorted prior to transplantation, microarray analysis or
proteomics. Development of robust methods for photoreceptor
specification in vitro are likely to be useful in improving methods for
ES and iPS cell differentiation as well as RPCs.
Uncited references
Meshorer et al., 2006; Qiu et al., 2004.
Acknowledgements
This work was funded by Fighting Blindness Ireland (in collab-
oration with the Fighting Blindness Vision Research Institute),
Fig. 6. Live RPCs (AeC), antibody stained proliferating RPCs (DeL) and stained differentiated RPCs (MeU).
Live cells: RPCs grown on tissue culture coated T25s alone (A), T25s coated with fibronectin (B), and spheres grown on low adherence plates are shown (C). Cells acquire char-
acteristic (and uniform) morphology after 10e14 days in culture. This does not change during subsequent passaging.
Immunocytochemistry: proliferating cells (red font: DeL) shown were plated after 2e3 passages and stained after 3e4 days of growth on glass slips. Differentiated cells were plated
in growth medium for 2 days, then differentiated for 10 days via sequential withdrawal of growth factors, before staining (see Materials and methods) (blue font, MeU).
DeL: proliferating RPCs; beta-III-tubulin (D), GFAP (E), nestin (F), Pax6 (G), glutamine synthetase (H), rhodopsin (I), RDS/peripherin (J), Rho-eGFP cells showing rare eGFPþve cells
(K), PBS control (L).
MeU: beta-III-tubulin (M), GFAP (N), nestin (O), Pax6 (P), glutamine synthetase (Q), rhodopsin (R), RDS/peripherin (S), cells differentiated in the presence of 1% FCS, showing GFAP
(red) and beta-III-tubulin (green) staining (T), PBS control (U).
Rho-eGFP cells usually show no eGFP-positive cells, but very occasionally 1e2% are seen, correlating with real-time Q-PCR results, which show low to non-existent Rho levels (K).
The majority of proliferating RPCs are nestin/Pax6 positive and following differentiation with 1% FCS are Gfap or b-III-tubulin positive. Differentiation without serum gives rise to
lower percentages of glia (N). Nuclei are counterstained with DAPI.
F.C. Mansergh et al. / Experimental Eye Research xxx (2010) 1e1412
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Please cite this article in press as: Mansergh, F.C., et al., Loss of photoreceptor potential from retinal progenitor cell cultures, despite
improvements in survival, Exp. Eye. Res. (2010), doi:10.1016/j.exer.2010.07.003
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13. Health Research Board Ireland (HRB), Science Foundation Ireland
(SFI) and the Deutsche Forschungsgemeinschaft (DFG). We would
like to thank Dr. Alfonso Blanco of the Conway Institute, UCD, for
assistance with FACS sorting, Chris Egan for use of CHD5 primers
and Drs Matt Campbell and Michael Wride for constructive
comments with regard to this manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.exer.2010.07.003.
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