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TISSUE-SPECIFIC STEM CELLS
Comparative Analysis of Progenitor Cells Isolated from the Iris,
Pars Plana, and Ciliary Body of the Adult Porcine Eye
ANGUS MACNEIL,a RACHAEL A. PEARSON,a,b ROBERT E. MACLAREN,a,c ALEXANDER J. SMITH,a
JANE C. SOWDEN,b ROBIN R. ALIa
a
Division of Molecular Therapy, Institute of Ophthalmology, University College London, London, United Kingdom;
Developmental Biology Unit, Institute of Child Health, University College London, London, United Kingdom;
c
Vitreoretinal Service, Moorfields Eye Hospital, London, United Kingdom
b
Key Words. Stem cell • Progenitor cell • Retinal transplantation • Neural differentiation • Cell culture • Pig • Neurosphere
ABSTRACT
Photoreceptor loss causes irreversible blindness in many
retinal diseases. The identification of suitable donor cell
populations is of considerable interest because of their potential use to replace the photoreceptors lost in disease. Stem
or progenitor cells that give rise to neurons and glia have
been identified in several regions of the brain, including the
embryonic retina and the ciliary epithelium of the adult eye,
raising the possibility of autologous transplantation. However, there has been little systematic investigation into precisely which regions of the large mammalian adult eye give
rise to such cells. Here, we show for the first time using the
porcine eye the presence of progenitor cells in additional
regions of the adult eye, including the pars plana and iris,
regions that, in the human, are readily accessible during
routine eye surgery. When cultured in the presence of
growth factors, these cells proliferate to form neurospheres
comprised of cells expressing retinal progenitor markers.
Using an adherent monolayer culture system, these cells
could be readily expanded to increase their number more
than 1 million-fold and maintain a progenitor phenotype.
When grown on the substrate laminin in the presence of
serum, cells derived from both spheres and monolayer cultures differentiated into neurons and glia. These results
suggest that a population of cells derived from the adult iris,
pars plana, and ciliary body of a large mammalian species,
the pig, has progenitor properties and neurogenic potential,
thereby providing novel sources of donor cells for transplantation studies. STEM CELLS 2007;25:2430 –2438
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Retinal degenerative diseases are the leading causes of untreatable blindness. The most common causes, age-related macular
degeneration and retinitis pigmentosa, result in the loss of photoreceptor cells. One attractive therapeutic strategy is the transplantation of stem or progenitor cells into the diseased eye in
order to replace the photoreceptor cells lost over the course of
the disease. We have recently shown that cell transplantation
can restore visual function in the diseased mouse retina, provided that the correct ontogenetic stage of donor cell is used [1].
A subsequent and significant challenge is to find suitable
sources of donor stem or progenitor cells and to find ways to
manipulate them in vitro to the appropriate developmental stage
prior to transplantation.
Transplantation studies conducted in small mammals have
used either fetal or early postnatal tissue or embryonic stem cells
as sources of donor cells [1– 4]. Ethical considerations are likely
to make it difficult to use fetal tissue as a source of donor cells
in humans, and thus an adult source would be preferable. Recent
research has suggested that progenitor cells with stem-like properties of self-renewal and multipotentiality might be isolated
from the adult human eye [5], raising the theoretical possibility
of autotransplantation if these cells could be expanded and
induced to differentiate into photoreceptors. Autotransplantation
could circumvent the need for long-term immunosuppression,
which is required to prevent rejection of transplants originating
from a nonautologous source.
Two populations of proliferative cells have been described
in the adult mammalian eye: retinal stem cells and retinal
progenitor cells [6 –9]. During retinal histogenesis, progenitor
cells are capable of a finite number of divisions to generate the
full complement of retinal neurons and glia. By contrast, stem
cells theoretically persist during adult life and retain properties
of multipotentiality and self-renewal. However, there is limited
evidence of either specific cell markers or assays that permit the
distinction between these two cell types [10, 11].
In contrast to mammals, lower vertebrates continue to generate new retinal neurons throughout life from the ciliary marginal zone (CMZ), which is the circumferential region anterior
to the neural retina [12–15]. The progeny derived from these
CMZ cells comprises various cell morphologies and laminar
positions, including retinal pigment epithelium (RPE), photoreceptors, and inner retinal neurons, indicating that CMZ progenitors are multipotent [16]. Until recently, it was thought that the
adult mammalian retina lacked proliferative or regenerative
capacity. However, in vitro experiments suggest that the ciliary
epithelium, part of the ciliary body, a structure analogous to the
lower vertebrate CMZ, contains a population of progenitor cells
Correspondence: Robin R. Ali, Ph.D., Division of Molecular Therapy, Institute of Ophthalmology, University College London, 11-43
Bath Street, London, EC1V 9EL U.K. Telephone: ⫹44 (0) 2076086817; Fax: ⫹44 (0) 2076086991; e-mail: [email protected] Received
January 12, 2007; accepted for publication June 18, 2007; first published online in STEM CELLS EXPRESS June 28, 2007. ©AlphaMed
Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0035
STEM CELLS 2007;25:2430 –2438 www.StemCells.com
MacNeil, Pearson, MacLaren et al.
[6, 7], but their function in vivo is still unclear [10]. Although
mitotically quiescent during adult life, when cultured in vitro
these cells demonstrate characteristics typical of stem cells,
including multipotentiality and self-renewal [6, 7]. Cells from
the ciliary body, but not the sensory neural retina, have been
shown to generate neurospheres that express nestin, a marker for
somatic neural progenitor cells [7]. Following incubation under
differentiation conditions, these cells may differentiate into both
neuronal and glial phenotypes, indicating that a proportion of cells
in the ciliary body are multipotent. Although there is some evidence to suggest that these cells differentiate into cells that are
immunopositive for specific retinal markers [5], it is unlikely that
these represent normal, fully differentiated retinal neurons [17].
Previous studies in lower vertebrates and small mammals
have indicated that cells other than those derived from the
ciliary epithelium may be potential sources of adult progenitor
cells, capable of differentiating into neural phenotypes [18 –20].
Furthermore, during development, some cell types are capable
of dedifferentiation; following surgical removal of the retina
from chick embryos and implantation of slow release fibroblast
growth factor (FGF) beads, the remaining RPE dedifferentiates to
form an inverted retina in vivo [21]. It therefore seems likely that
a number of cell types within the adult mammalian eye are potential sources of cells with progenitor cell properties. However, there
has so far been little systematic investigation into exactly which of
the many regions of the adult eye give rise to these cells.
We sought to determine the potential for harvesting adultderived progenitor cells from different regions of the large
mammalian eye, particularly those most readily accessible during routine eye surgery, such as the iris and pars plana. The
porcine eye provides an excellent model, since its morphology
is similar to the human eye and the anatomical regions can be
clearly defined and accurately dissected. Previous studies have
demonstrated that progenitor cells can be derived from the
porcine brain and propagated in culture [22–24] and demonstrate many similarities with their human counterparts [25]. The
genetic similarities between pig and human have already been
utilized in the generation of porcine heart valves for transplantation into human subjects [26].
We demonstrate that neurospheres containing progenitor
cells could be derived from not only the ciliary body but also the
iris and pars plana, whereas the anterior neural retina failed to
give rise to cells with such characteristics. Cells derived from
each of the adult porcine ciliary body, iris, and pars plana could
be similarly expanded in culture, maintaining a proliferative
phenotype, and we show that these cells can be differentiated
into cells that express either neuronal or glial markers.
MATERIALS
AND
METHODS
Animals
Male Landrace pigs were reared according to British farming regulations and were approximately 2 years of age at the time of
slaughter. Eyes were obtained from the slaughter house and placed
immediately on ice prior to dissection. This occurred within 2 hours
of slaughter. Eyes used for control reverse transcription-polymerase
chain reaction (RT-PCR) analysis were obtained at the time of
slaughter, the lens removed, and the eyes placed in RNA-later
(Ambion; Applied Biosystems, Warrington, U.K., http://www.
appliedbiosystems.com) for transportation.
Dissection Procedure
Eyes were dissected in Earl’s balanced salt solution (Sigma-Aldrich,
Poole, U.K., http://www.sigmaaldrich.com). Eyes were hemisected
and the posterior half of the eye removed. Taking the anterior half,
samples from each of the anterior neural retina, pars plana, ciliary
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2431
body, and the iris were carefully dissected free from all surrounding
tissue.
In Vitro Cultures
Primary Neurosphere Cultures. Tissue samples were dissociated
according to the manufacturer’s instructions using a papain-based
dissociation system (Worthington Biochemical, Lakewood, NJ,
http://www.worthington-biochem.com) (supplemental online Methods). Dissociated cells were resuspended in Dulbecco’s modified
Eagle’s medium-F12/Glutamax (Invitrogen, Paisley, U.K., http://
www.invitrogen.com) containing N2 supplement (1:100; Invitrogen), penicillin-streptomycin solution (1:100; Invitrogen), epidermal growth factor (EGF) (20 ng/ml; Peprotech, London, http://
www.peprotech.com), FGF-2 (20 ng/ml; Peprotech EC), and
heparin (2 ␮g/ml). Cells were plated at a density of 10 –20 cells per
microliter in untreated 24-well tissue culture plates. Plating at this
density is reported to lead to the formation of clonally derived
neurospheres [5, 7]. Fresh growth factors were added every other
day, and medium was exchanged every 5–7 days.
Monolayer Cultures. Spheres isolated from each region at 7 days
in vitro were plated in plastic 24-well tissue culture plates. Spheres
were cultured in the presence of 5% fetal calf serum (FCS) and
mitogens for an additional 7 days. During this time, spheres became
adherent to the culture dish, and cells spread out and migrated away
from the sphere to form monolayers. Monolayer cultures were split
every 7 days using trypsin-EDTA solution (Sigma-Aldrich) and
replated into fresh plates. The number of cells was determined using
a hemocytometer to count the number of viable cells in a 20 ␮l
sample of cell suspension immediately following passaging and
prior to replating.
Differentiation Cultures. To assess the differentiation potential
of cells within neurospheres, individual spheres were plated in
24-well plates on a substrate of poly-L-ornithine (10 ␮g/ml)/laminin
(100 ng/ml) (both Sigma-Aldrich) in medium supplemented with
10% FCS. Medium was changed every 3– 4 days, and the cells were
allowed to migrate and differentiate over the course of 21 days.
During the first 3 days of differentiation, FGF-2 (20 ng/ml) was
added to each well.
Characterization of Neurospheres
Neurosphere Counts. At 7 days in vitro, cultures were assessed
for neurosphere number. Neurospheres were defined as free-floating, with a diameter of ⬎40 ␮m and a clearly defined outer
boundary. This feature readily distinguishes neurospheres from any
smaller aggregations of cells, which have uneven boundaries. All
spheres in a given well were counted. Results are expressed as
number of neurospheres per 50,000 cells. To assess size and pigmentation, neurospheres were visualized using an inverted Leica
DMIL microscope (Leica, Heerbrugg, Switzerland, http://www.
leica.com) fitted with a camera and image capture system and analyzed off-line. Neurospheres were selected at random from the image
set, which had previously been recoded to ensure blind assessment.
Neurosphere diameter was measured twice, the second measurement
perpendicular to the first, and an average of the two was calculated.
Pigmentation Scoring. Pigmentation was assessed using the images captured for size analysis and Image-Pro software. Black was
given a pixel value of 0, whereas white had a value of 255. Image-Pro
software calculates the average pixel intensity of a given region. The
regions were selected manually to encompass the whole neurosphere,
as determined by drawing around the perimeter of the sphere. Neurospheres were selected at random from the masked image set.
Immunohistochemistry
Proliferating Neurospheres. Spheres cultured for 7 days in vitro
were placed on poly-L-lysine coated glass slides (BDH; VWR,
Leicestershire, U.K., http://uk.vwr.com) and allowed to settle prior
to fixation with 4% paraformaldehyde (PFA) for 20 minutes at room
temperature (RT). Spheres were preblocked in Tris-buffered saline
(TBS) containing normal goat serum (1%), bovine serum albumin
(1%), and 0.5% Triton X-100 for 2 hours at RT before being
Porcine Eye Adult Progenitor Cells
2432
Figure 1. Comparison of neurospheres
from different regions of the eye. (A): Schematic showing the anatomical locations of
the CB, I, PP, AR, and RPE. (B): Representative images of neurospheres generated
from dissociated cells of the CB, PP, and iris
after 7 days in tissue culture. Scale bars ⫽ 50
␮m. (C): A comparison of the average number of neurospheres generated from 50,000
cells taken from each region of nine eyes.
(D): Average size of neurospheres generated
from each region (N ⫽ 9 eyes). (E): Pigmentation levels differ among neurospheres derived from the iris, pars plana, and ciliary
body. Histogram shows average pigmentation levels of neurospheres cultured for 7
days in vitro. Abbreviations: AR, anterior
retina; CB, ciliary body; I, iris; ns, neurospheres; PP, pars plana; RPE, retinal pigment epithelium.
incubated with primary antibody overnight at 4°C. After rinsing
three times for 30 minutes each time (3 ⫻ 30 m) with TBS, spheres
were incubated with secondary antibody for 4 hours at RT, rinsed
(3 ⫻ 30 m), and counterstained with Hoechst 33342. Negative
controls omitted the primary antibody.
Proliferating Monolayers. Monolayer cultures were fixed at 7
days in vitro with 4% PFA for 10 minutes at RT. Cells were
preblocked in TBS containing normal goat serum (1%), bovine
serum albumin (1%), and 0.1% Triton X-100 for 1 hour at RT
before being incubated with primary antibody overnight at 4°C.
After rinsing 3 ⫻ 30 m with TBS, cells were incubated with
secondary antibody for 2 hours at RT, rinsed (3 ⫻ 30 m), and
counterstained with Hoechst 33342. Negative controls omitted the
primary antibody.
Differentiation Potential. Plates were fixed in 4% PFA. Cells
were preblocked, as above, before being incubated with primary
antibody overnight at 4°C. After rinsing 3 ⫻ 10 m with TBS, cells
were incubated with secondary antibody for 2 hours at RT, rinsed
(3 ⫻ 10 m), and counterstained with Hoechst 33342. Negative
controls omitted the primary antibody. Positive controls comprised
staining of frozen porcine retinal sections. The percentage of cells
positive for a given marker was determined by counting cells with
a signal greater than background from more than three regions per
well and more than three independent experiments.
Antibodies. The following antibodies were used: Pax6 (mouse;
1:100; Developmental Studies Hybridoma Bank, Iowa City, IA,
http://www.uiowa.edu/⬃dshbwww) and Sox2 (rabbit; 1:200; Abcam, Cambridge, U.K., http://www.abcam.com) for stem/progenitor
cells, ␤-III tubulin (mouse; 1:1,000; Promega, Madison, WI, http://
www.promega.com) and NeuN (goat; 1:100; Chemicon, Temecula,
CA, http://www.chemicon.com) for neurons, Brn-3b (goat; 1:100;
Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.
com) for ganglion cells, Rho4D2 (rabbit; 1:100; kind gift from R.
Molday) and recoverin (rabbit; 1:100; Chemicon) for photoreceptors, protein kinase C (PKC) (mouse; 1:100; Sigma-Aldrich) for rod
bipolar cells, and RPE65 (mouse; 1:100) for RPE cells. The appropriate Alexa-tagged (Molecular Probes, Eugene, OR, http://probes.
invitrogen.com) secondary antibodies were used. Staining was visualized using either a Zeiss (LSM 510; Carl Zeiss, Jena, Germany,
http://www.zeiss.com) or Leica (SP2) confocal microscope.
Hoechst 33342 was included in the final wash to label all cell nuclei.
The percentage of cells that were BrdU-positive was determined by
counting the number of BrdU-labeled and Hoechst-labeled cells in
more than five randomly selected fields of view from each well and
more than six wells per region. The results presented are from N ⫽ 3
(passage 1) and N ⫽ 4 (passage 11) independent experiments.
Confocal Microscopy
Neurospheres were mounted on glass coverslips and imaged using
an inverted confocal microscope (Zeiss LSM510). The fluorescence
of Hoechst 33342, Alexa-488, and Alexa-546 were excited with the
350-nm line of the UV laser, the 488-nm line of the argon laser, and
the 543-nm line of the HeNe laser, respectively. Images show
projections of multiple single confocal sections taken at approximately 5–10 ␮m steps.
RT-PCR
RNA was isolated from fresh tissue or cells using the TRIzol reagent
(Invitrogen) in accordance with the manufacturer’s instructions. cDNA
was generated from 1 ␮g of RNA using the QuantiTect reverse transcription kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) following the manufacturer’s instructions. Two hundred ng of cDNA was
used in each polymerase chain reaction (PCR). Porcine retinal cDNA
or porcine genomic DNA (200 ng per reaction) were used as positive
or negative controls. Previously published primers that amplify specific
target porcine sequences were used according the conditions used in the
original publication [27]. In each case, PCR primers were designed to
flank at least one intron.
Statistics
Results are presented as the mean ⫾ SEM. Where appropriate, N
indicates the number of eyes and n the number of neurospheres
investigated. The different regions were compared using a one-way
analysis of variance (ANOVA) test with Dunnett’s correction for
multiple comparisons, where appropriate.
RESULTS
Incorporation of 5-Bromo-2ⴕ-deoxyuridine In Vitro
Proliferative Potential of Different Regions of the
Porcine Eye
At 7 days in vitro, monolayer cells were pulse labeled with 5-bromo2⬘-deoxyuridine (BrdU) (0.5 ␮M) for 4 hours. The plates were
fixed in 4% PFA for 20 minutes before processing for BrdU
immunohistochemistry. Briefly, cells were exposed to 2 M HCl for
30 minutes at 30°C to denature cellular DNA. HCl was neutralized
by application of 0.1 M Na-borate prior to rinsing with phosphatebuffered saline. Rat anti-BrdU (1:500; Abcam) and then Alexa-546
goat anti-rat (1:250; Molecular Probes) were used for BrdU staining.
The ability to form clonal spheres, whereby a stem cell undergoes proliferation to form a free-floating neurosphere containing
stem/progenitor cells, is well documented and provides an indication of the number of endogenous stem-like cells in a given
tissue [5, 7, 28, 29]. To examine the different regions of the
porcine eye, primary cells from the iris, ciliary body, pars plana,
and anterior neural retina (Fig. 1A) were isolated and dissoci-
MacNeil, Pearson, MacLaren et al.
2433
Figure 2. Stem cell potential of neurospheres from different regions of the porcine eye. Confocal projection images of neurospheres immunostained
for the presence of Sox2, Pax6, and ␤-III-tubulin expression. Neurospheres were generated from dissociated cells of the ciliary body, iris, or pars plana
and grown for 1 week in serum-free medium containing fibroblast growth factor and epidermal growth factor. Nuclei were labeled with
4,6-diamidino-2-phenylindole. Scale bars ⫽ 10 ␮m.
ated into single cells. Free-floating neurospheres could be easily
derived from single dissociated cells of the adult porcine iris,
ciliary body, and pars plana but not the anterior neural retina
(Fig. 1B). Neurospheres were visible at 3 days in vitro, and the
number and size of neurospheres was assessed at 7 days in vitro.
Significantly, the number of neurospheres isolated from both
the iris and the pars plana was approximately two- and
threefold greater, respectively, than the number isolated from
the ciliary body (N ⫽ 9; Fig. 1C). Neurosphere size provides
an indirect measure of proliferation; neurospheres from all
three regions were of a similar size, with no significant
differences among groups (Fig. 1D). Only rarely were
sphere-like structures derived from the anterior retina. These
were extremely small and did not grow with additional culturing (data not shown).
Spheres could be partially dissociated and grew to form
secondary neurospheres, but passaging did not lead to expansion of cell number. Thus, neurospheres from each of the
three regions had limited self-renewal potency, consistent
with previous reports of studies using neurospheres derived
from either the chicken iris [19] or rat ciliary body [30].
Previous studies have suggested that the cells that give rise to
neurospheres in the murine ciliary epithelium are pigmented
and that pigmentation is lost during the proliferation of the
neurospheres [7]. Here, we found that, typically, neurospheres derived from the ciliary body and pars plana contained a core of pigmented cells, which were surrounded by
nonpigmented cells. By contrast, neurospheres derived from
the iris were highly pigmented and failed to depigment even
during prolonged culture periods. To assess pigmentation,
neurospheres were quantified using a linear scale from black
(0) to white (255) and showed a significant difference in the
pigmentation of neurospheres derived from the iris (average
37) compared with those derived from the ciliary body (98)
and pars plana (98) (Fig. 1E; N ⬎ 9 for each region; one-way
ANOVA test p ⬍ .0001).
Cellular Composition of Porcine Retinal
Neurospheres
Since the above data indicated some phenotypic differences
between neurospheres derived from the different regions of
the porcine eye, we examined the cellular make-up of the
neurospheres using immunocytochemistry and a variety of
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Table 1. Expression of additional retinal progenitor markers in
neurosphere cultures
Region Dach1
CB
PP
Iris
⫹
⫹
⫹
Lhx2
Hes1
Six3
GFAP
DCX
Vim
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫹
⫹
⫹
Neurospheres were cultured for 1 week in serum-free conditions
before the extraction of RNA for reverse transcription-polymerase
chain reaction analysis. Neurospheres from each region expressed
markers for eye field and retinal progenitor cell; however, no
expression of doublecortin, a marker of more differentiated
neuronal cells, was observed.
Abbreviations: CB, ciliary body; DCX, doublecortin; GFAP, glial
fibrillary acidic protein; PP, pars plana; Vim, vimentin.
progenitor cell markers. At 7 days in vitro, neurospheres
from all three regions (iris, ciliary body, and pars plana)
expressed the stem/progenitor cell marker Sox2 and the neural progenitor marker Pax6 (Fig. 2). Occasionally, small
numbers of cells, usually at the outer edges of the spheres,
labeled for the neuronal marker ␤-III-tubulin, suggesting that
some differentiation may occur even under proliferative culture conditions (Fig. 2).
RT-PCR analysis was used to confirm the presence of
additional markers of retinal and neural progenitor cells (Table
1). Neurosphere cultures were shown to express RNA encoding
Hes1, which is expressed in immature central nervous system
progenitors in the pig [25] and the eye field genes Dach1, Lhx2,
and Six3. Lhx2 and Dach1 are expressed in the forebrain, optic
vesicle, and developing neural retina of the mouse [31–33].
During eye development, Six3 is initially expressed throughout
the optic vesicle before becoming restricted to the neural retina
and lens [34]. The glial-associated intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin were
also detectable in spheres from all regions. RT-PCR failed to
detect expression of doublecortin, indicating a lack of immature
neuronal cells within spheres in serum-free culture. Together,
these data indicate that the majority of cells within spheres from
each region were progenitor-like cells expressing markers characteristic of immature retinal cells.
2434
Figure 3. Expansion of porcine retinal stem/progenitor cells in monolayer cultures. (A): Plot of the average total cell number as a function of
cell passage number (N ⫽ 3). Neurospheres were grown for 1 week in
serum-free medium then transferred to monolayer medium and passaged
every 7 days. (B): Percentage of BrdU-positive cells detected in monolayer cultures from each region at passage 1 (N ⫽ 3) and passage 11
(N ⫽ 4) following a 4-hour pulse label. Abbreviations: BrdU, 5-bromo2⬘-deoxyuridine; CB, ciliary body; PP, pars plana.
Expansion of Adult Porcine Progenitor Cells in
Monolayer Cultures
Given the limited self-renewal potential of neurospheres, we
assessed the use of adherent monolayer cultures for expanding
cell number. Neurospheres derived from each of the three regions grew readily as monolayers. Whereas monolayer cells
could be maintained for 1–2 passages in the absence of serum,
sustained expansion only occurred in the presence of low serum
(4%). Cells from the ciliary body increased in number from
2.0 ⫻ 104 to 5.8 ⫻ 109 over nine passages, iris cells expanded
from 2.0 ⫻ 104 to 3.9 ⫻ 108 cells, and cells from the pars plana
grew in number from 2.0 ⫻ 104 to 4.3 ⫻ 107 cells over the same
period (Fig. 3A). Cells from each region continued to proliferate
throughout the period studied and could be passaged 25 times,
although they failed to expand significantly after this time (data
not shown). The growth rate of monolayer cultures from all
three regions was also investigated by incubation in the presence
of BrdU. At passage 1, cells derived from the pars plana and
iris showed proliferation rates equivalent to those derived
from the ciliary body, as determined by BrdU uptake (N ⫽ 3;
Fig. 3B). By passage 11, the percentage of BrdU uptake for
cells from each region had increased approximately threefold
(N ⫽ 4; Fig. 3B).
A common concern regarding the use of stem cells cultured
for long periods in cellular therapies is the potential for immortalization and tumorogenesis. Typically, immortalized cells
overcome contact inhibition and continue to divide, having
reached confluency. By contrast, the cells described here ceased
to divide upon achieving confluency and required regular passaging to maintain expansion. Furthermore, our finding of novel
progenitor populations within the porcine eye is based on the
analysis of more than ten independently derived cell cultures
and so is highly unlikely to be due to immortalization events.
Expansion of cell number was not seen beyond passage 25, also
indicating that these cells are not immortal. Finally, proliferating
cells cease to divide upon removal of mitogens and the addition
Porcine Eye Adult Progenitor Cells
Figure 4. Stem cell potential of monolayer cultures from different
regions of the porcine eye. Representative immunohistochemistry of
monolayer cultures (passages 4 and 11) from the CB, PP, and iris grown
in the presence of fibroblast growth factor and epidermal growth factor.
Cultures were stained for the presence of Pax6 (red), Sox2 (red), and
␤-III-tubulin (green). Nuclei were labeled with 4,6-diamidino-2-phenylindole. Scale bars ⫽ 100 ␮m. Abbreviations: CB, ciliary body; P,
passage; PP, pars plana.
of serum (10%). Such patterns of behavior are typical for
primary progenitor cell populations grown in monolayer cultures [35, 36] and suggest that these cells senesce after moderate
expansion.
Cellular Composition of Porcine Retinal Monolayer
Cultures
To determine whether monolayer cultures derived from the
different regions of the eye retain the ability to express the
progenitor markers found in primary neurospheres, we used
immunohistochemistry to look for expression of Sox2 and Pax6
in passage 4 monolayer cultures (Fig. 4). Staining confirmed the
presence of Sox2 and Pax6 expression in cultures from all three
regions. Many of the cells staining with Sox2 and Pax6 were
located within densely packed regions within each well and
were surrounded by cells with lower levels of Sox2 and Pax6
expression (Fig. 4). Analysis of cells from later passages (passage 11) revealed that Sox2 expression was detectable in 70% of
cells of pars plana origin, 55% of ciliary body cells, and 20% of
iris derived cells. However, we were unable to demonstrate
robust Pax6 immunostaining in these cells (data not shown). To
determine the prevalence of more mature progenitor cells within
the passage 11 monolayer cultures, immunohistochemistry for
␤-III-tubulin was performed (Figs. 4, 6A). Positive staining was
present in 9%, 15%, and 47% of ciliary body, pars plana, and
iris cells, respectively.
Together, these findings indicate that the cells within monolayer cultures derived from the porcine ciliary body, pars plana,
and iris have some self-renewal potency, similar to that reported
for cells from the rat ciliary body [30, 37] and chicken iris [19].
Both monolayer culture cells and sphere cultures from all three
regions exhibited morphological characteristics consistent with
MacNeil, Pearson, MacLaren et al.
2435
from all three eye regions and appeared very specific to individual cells within each culture (Fig. 5). These findings indicate
that progenitor cells from the porcine iris, pars plana, and ciliary
body may be expanded and are each capable of yielding neuronal cells. However, under the conditions used here, differentiated cells failed to show convincing immunoreactivity for
rhodopsin, phosducin, recoverin, PKC, or RPE65 (data not
shown), suggesting, as has been described in other species [17],
that fully differentiated retinal cell types were not produced.
Differentiated primary neurospheres and differentiated cells
from late passage (P12) monolayer cultures were also examined by
RT-PCR. Expression of the eye field markers Dach1, Lhx2, and
Six3 was found in all cultures (Table 2). However, in contrast to
undifferentiated neurosphere cultures grown without serum but in
the presence of mitogens, differentiating cells from both late passage monolayer cultures and neurosphere cultures were positive for
doublecortin, a marker of immature neuronal cells. The glialassociated markers GFAP and vimentin were also detectable. Together, these results suggest that progenitor cells derived from the
iris, pars plana, and ciliary body of the adult porcine eye are able to
generate new neurons and glial cells in culture.
DISCUSSION
Figure 5. Differentiation potential of porcine retinal stem cells. Primary neurospheres (P0) from each region were cultured on laminincoated tissue culture plates under differentiation conditions for 21 days.
Cells migrated from the neurospheres to form a monolayer, which was
stained for the presence of ␤-III-tubulin. Nuclei were labeled with
4,6-diamidino-2-phenylindole (DAPI). Dissociated monolayer cells
(P11) were cultured on laminin-coated tissue culture plates under differentiation conditions for 21 days and then stained for the presence of
␤-III-tubulin (green) or NeuN (red). Nuclei were labeled with DAPI.
Light microscope images show neuronal morphology of differentiated
cells from each region. Scale bar ⫽ 100 ␮m. Abbreviations: CB, ciliary
body; P, passage; PP, pars plana.
other mammalian neural stem/progenitor cultures, including
analogous cells from humans [7]. However, the decreased expression of Pax6 staining and prevalence of the neuronal marker
␤-III-tubulin at later passages suggest that significant differentiation occurs in tissue culture.
Differentiation Potential of Adult Porcine Progenitor
Cells
To assess the neurogenic potential of progenitor cells derived
from the adult ciliary body, pars plana, and iris, neurospheres
from each region were plated on laminin-coated tissue culture
plates in the presence of 10% serum and FGF-2 (20 ng/ml).
During this time, cells migrated out of the neurospheres and
proliferated. After 3 days, the medium was replaced with serumcontaining mitogen-free medium. By 4 weeks, ⬎90% of the
cells were immunopositive for the neuronal marker ␤-III-tubulin
and had morphologies consistent with those of immature neurons (Fig. 5). Similarly, cells grown and passaged as adherent
monolayers could be induced to differentiate and express neuronal markers. Dissociated monolayer cells (passage 11) taken
from each region were plated on laminin-coated tissue culture
plates under differentiation conditions as described above. At 4
weeks, Sox2 expression was no longer detectable (Fig. 6B). We
observed robust expression of ␤-III-tubulin in 28%– 40% of
cells (Figs. 5, 6A). To further confirm the neuronal phenotype of
these differentiating cells, immunostaining for NeuN was also
performed. NeuN staining was prevalent in samples derived
www.StemCells.com
Here we have shown that the iris and pars plana regions of the adult
porcine eye yield cells that are capable of significant proliferative
expansion in EGF/FGF-2 supplemented media in vitro in a manner
virtually indistinguishable from those derived from the ciliary
body. Robust passaging and expansion could be achieved with
monolayer cultures but could not be readily established with cultures of free-floating neurospheres, possibly because cells in the
latter configuration are not easily dissociated. Proliferating cells
from both sphere and monolayer cultures expressed the developmentally regulated transcription factors Sox2 and Pax 6 together
with eye field and retinal markers including Lhx2, Dach1, and
Six3. Finally, with serum-induced differentiation, cells expressed
the glial markers GFAP and vimentin or the neuronal markers
␤-III-tubulin, doublecortin, and NeuN, although no photoreceptor
specific markers could be elicited using these protocols. Single
cells isolated from all three regions of the porcine anterior uvea (but
not retina) can therefore undergo clonal expansion for up to 25
passages in vitro and subsequently express neuronal features after
induced differentiation with serum based medium. Such cells are
readily accessible during ophthalmic surgery and may provide
alternative sources of donor cells for retinal or neuronal autotransplantation.
A population of stem-like cells has been described for the CMZ
of lower vertebrates and the mammalian equivalent, the ciliary
epithelium. However, there has been little systematic study of the
different regions of a larger mammalian eye. The populations of
cells that we isolated from the adult porcine iris, pars plana, and
ciliary body displayed a number of properties similar to those
described previously for stem/progenitor cells in the pigmented
ciliary margin in smaller mammals where these regions are less
easy to define [6, 7]. Neurospheres derived from all three regions
expressed the stem cell marker Sox2 and the progenitor marker
Pax6. The number of neurospheres generated from both the iris and
the pars plana was 2–3 times greater than that isolated from the
ciliary body, suggesting that these regions may be particularly rich
in stem/progenitor cells. Individual neurospheres from each of the
three regions were similar in size, indicating that they have comparable expansion potential when grown as neurospheres. In contrast to other regions of the porcine eye, the anterior retina of the
adult pig lacks progenitor cells capable of expansion under the
conditions used in this study.
Porcine Eye Adult Progenitor Cells
2436
Figure 6. Prevalence of Sox2 and ␤-IIItubulin expression under proliferating and
differentiation conditions. Passage 11 cells
from each region were grown as either proliferating monolayers or incubated for 4
weeks under differentiation conditions. (A):
Percentage of ␤-III-tubulin positive cells in
monolayer and differentiation cultures. (B):
Percentage of Sox2 positive cells in monolayer and differentiation cultures. Abbreviations: CB, ciliary body; PP, pars plana.
Table 2. Expression of additional markers in differentiated
neurosphere and monolayer cultures
Region
Differentiated
Differentiated
Differentiated
Differentiated
Differentiated
Differentiated
Dach1 Lhx2 Hes1 Six3 GFAP DCX Vim
CB ns
PP ns
iris ns
CB p12 cells
PP p12 cells
iris p12 cells
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
To assess the differentiation potential of neurospheres or late
passage monolayer cultures (P11), cells were plated on laminincoated tissue culture wells under differentiation conditions
(Materials and Methods). Reverse transcription-polymerase chain
reaction indicated that differentiating neurospheres and late
passage monolayer cultures express doublecortin, a marker for
immature neuronal cells, in addition to markers for retinal
progenitor cells and glial cells (GFAP and vimentin).
Abbreviations: CB, ciliary body; DCX, doublecortin; GFAP, glial
fibrillary acidic protein; ns, neurosphere; PP, pars plana; Vim,
vimentin.
To utilize cells for autologous transfer, large numbers of
cells may be needed. Neurospheres generated from the ciliary
body and iris of other species have previously been shown to
have limited potential for secondary neurosphere formation
and passaging [5]. Similarly, we also found limited secondary
sphere formation from these regions, together with the pars
plana, in the porcine eye. However, it was possible to achieve
rapid and significant expansion of neurosphere-derived progenitor cells using adherent monolayer cultures in the presence of FGF-2 and EGF, conditions that led to an approximately 1 million-fold increase in cell numbers. Neurospheres
from the ciliary body, pars plana, and iris could be efficiently
expanded, forming depigmented monolayers.
We have recently demonstrated that, in contrast to previously
held opinion, the optimal donor cell for retinal transplantation is not
an uncommitted progenitor or stem cell but rather one that has
already committed to the photoreceptor fate [1]. For stem and
progenitor cells from either embryonic or adult sources to be viable
donor cells, it will be necessary to determine ways in which to
generate such committed precursor cells in vitro. Wu et al. [38]
reported a priming procedure using FGF-2 to treat fetal human
neural stem cells in vitro before further differentiation that allowed
them to obtain cholinergic neurons in vitro. Recently, Merhi-Soussi
et al. [39] used a modified protocol to generate significant numbers
of murine stem cells committed to the neuronal lineage. Here, we
used a similar technique, priming cells with FGF in the presence of
serum, followed by the complete withdrawal of growth factors. We
found that, upon differentiation, cells from all three regions of the
porcine eye, in both neurosphere and monolayer cultures, showed
robust expression of the neuronal marker ␤-III-tubulin and exhibited the morphology of immature neurons. Such morphology was
consistent with the relatively lower levels of labeling for NeuN (a
more mature neuronal marker) observed in differentiated monolayer culture.
Previous reports have indicated that the addition of exogenous FGF-2 to dissociated P0 rat retinal cells grown as monolayers caused a marked increase in the number of cells that
express rhodopsin [40]. However, this effect was apparently
absent at more immature developmental stages (embryonic day
16) [41], a finding replicated in the mouse retina [39]. Lineagetracing studies of single progenitor cells in vivo have shown that
multipotent progenitors give rise to all the cell types of the
retina, including photoreceptors [42]. When placed in culture,
these cells may also have the capacity to differentiate and
express genes specific to mature cell types, such as photoreceptors [39]. After long-term expansion, these cells maintain the
capacity to generate cells committed to the photoreceptor pathway. However, the proportion of photoreceptors generated in
vitro is very low compared with the expected numbers generated
in vivo [39]. Similarly, despite assessing a wide array of retinalspecific markers, we did not observe any convincing immunoreactivity for retinal neurons, including photoreceptors, suggesting that our culture conditions maintain a large number of cells
in a primitive neuronal precursor stage. The loss of robust Pax6
staining from monolayer cultures at passage 11 may indicate a
loss of retinal identity after prolonged expansion of cells in
tissue culture. Such findings are in accordance with previous
reports using smaller mammals, which have also found that
progenitor cells derived from the iris or ciliary margin fail to
differentiate into retinal-specific neurons [20]. Furthermore, expression of retinal-specific markers such as the photoreceptor
pigment rhodopsin could only be obtained following viral transduction of the photoreceptor transcription factor Crx [20]. Other
reports have suggested the presence of retinal markers in differentiated stem/progenitor cell cultures, as determined by immunohistochemistry [5, 19]. However, there is increasing evidence to suggest that such positive staining is unlikely to
represent true differentiation into mature retinal phenotypes
[17]. Indeed, undifferentiated cells have been reported to not
only express differentiated cell markers, but can also express
markers corresponding to more than one lineage [43, 44]. Such
findings highlight the difficulties in assessing the potential of
stem and progenitor cell populations and the need for cautious
and thorough assessments.
A promising strategy for directing the differentiation of a
population of progenitor cells is to utilize viral gene transfer to
express key transcription factors. Studies using rat and primate
iris tissue have been successful in generating cells with a photoreceptor-like phenotype following transduction with Crx and
Crx/NeuroD, respectively [45]. Further studies will be needed to
establish whether similar techniques may be used to guide the
differentiation of adult-derived iris, ciliary body, and pars plana
progenitor cells that have undergone multiple passages and
expansion in tissue culture. Ideally, by using gene transfer, the
appropriate combination of factors could be delivered to gener-
MacNeil, Pearson, MacLaren et al.
2437
ate functional photoreceptor precursor cells with the potential
for integration into a recipient retina.
SUMMARY
The results presented here suggest that a population of cells
derived from each of the adult porcine iris, pars plana, and
ciliary body have retinal progenitor cell characteristics and
neurogenic potential. Further studies are needed to understand more about the factors that induce cell-specific differentiation. The ability to generate large numbers of neural
precursor cells in a controlled manner from an accessible
region of a human might provide a valuable source of donor
cells for autotransplantation.
ACKNOWLEDGMENTS
We would like to thank P. Buch for technical assistance, R.
Molday for providing antibodies, and members of R.R.A.’s lab
for helpful discussions. This work was supported by Grants
from the Medical Research Council U.K. (G03000341), the
Royal Blind Asylum & School, the Scottish National Institute
for the War Blinded, and the Special Trustees of Moorfields Eye
Hospital. R.E.M. is a clinician scientist supported by the Health
Foundation.
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CONFLICTS
The authors indicate no potential conflicts of interest.
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