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Localization of candidate stem and progenitor cell markers within the human cornea limbus and bulbar conjunctiva in vivo and in cell culture.

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THE ANATOMICAL RECORD PART A 288A:921–931 (2006)
Localization of Candidate Stem and
Progenitor Cell Markers Within the
Human Cornea, Limbus, and Bulbar
Conjunctiva In Vivo and in Cell Culture
SANDY GIAN VASCOTTO1 AND MAY GRIFFITH1,2*
University of Ottawa Eye Institute, Ottawa, Ontario, Canada
2
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,
Ontario, Canada
1
ABSTRACT
Corneal diseases are some of the most prevalent causes of blindness
worldwide. While the most common treatment for corneal blindness is the
transplantation of cadaver corneas, expanded limbal stem cells are finding
recent application. Unknown, however, is the identity of the actual repopulating stem cell fraction utilized in both treatments and the critical factors
governing successful engraftment and repopulation. In order to localize potential stem cell populations in vivo, we have immunohistochemically mapped a
battery of candidate stem and progenitor cell markers including c-Kit and
other growth factor receptors, nuclear markers including ⌬Np63, as well as
adhesion factors across the cornea and distal sclera. Cell populations that
differentially and specifically stained for some of these markers include the
basal and superficial limbal/conjunctival epithelium and scattered cells within
the substantia propria of the bulbar conjunctiva. We have also determined that
the culture of differentiated cornea epithelial cells as dissociated and explant
cultures induces the expression of several markers previously characterized as
candidate limbal stem cell markers. This study provides a foundation to explore candidate corneal stem cell populations. As well, we show that expression
of traditional stem cell markers may not be reliable indicator of stem cell
content during limbal stem cell expansion in vitro and could contribute to the
variable success rates of corneal stem cell transplantation. Anat Rec Part A,
288A:921–931, 2006.
Key words: stem cell; progenitor cell; limbus; bulbar conjunctiva; immunohistochemistry; cell culture
The cornea represents the primary protection, focusing,
and transmission interface for light entering the eye. The
cornea is a relatively simple structure comprising three
cellular layers that include a stratified nonkeratinizing
epithelium, extracellular matrix (ECM)-rich stroma, and
single endothelial layer. Damage to the cornea by disease
or injury is the second most common cause of vision loss or
blindness worldwide (Whitcher et al., 2001). Corneal vision loss is most commonly treated by the grafting of donor
cadaver corneas, but is ineffective for indications where
the corneal surface is damaged to the extent that the stem
cells, which usually repopulate the epithelium, are absent.
These include autoimmune conditions and chemical and
thermal burns (Tsubota et al., 1999). In some cases, corneal stem cells, which are believed to lie within the limbus
of the cornea, are grafted in explant or following expansion in cell culture to resurface the cornea in disorders
where epithelial stem cells are considered damaged or
Grant sponsor: Canadian Stem Cell Network.
*Correspondence to: May Griffith, University of Ottawa Eye
Institute, Ottawa Hospital-General Campus, 501 Smyth Road,
Ottawa K1H 8L6, Canada. Fax: 613-739-6070.
E-mail: mgriffith@ohri.ca
Received 5 October 2005; Accepted 19 April 2006
DOI 10.1002/ar.a.20346
Published online 15 June 2006 in Wiley InterScience
(www.interscience.wiley.com).
922
VASCOTTO AND GRIFFITH
deficient (Tsubota et al., 1999; Kinoshita et al., 2004).
Results of corneal stem cell transplantations vary depending on the type of transplant (direct graft or expanded
culture cultures) and from center to center (Reinhard et
al., 2004; Spelsberg et al., 2004; Sangwan et al., 2005). The
survival of grafted donor cells recovered posttransplantation is also variable (Wollensak and Green, 1999; Spelsberg et al., 2004). In all cases, source material includes all
cell layers of the limbal epithelium, the superficial stroma
of the limbus, and the superficial bulbar conjunctiva.
While corneal stem and progenitor cell transplantation is
becoming widespread, details about the specific repopulating cell fraction employed in both treatments and the
critical factors governing successful engraftment and repopulation are unknown (Shimmura and Tsubota, 2002;
Espana et al., 2004a).
Although there is no definitive corneal stem cell marker,
recent studies have identified putative stem cell populations outside of the eye epithelium within the conjunctiva,
fornix, and ciliary margin (Wirtschafter et al., 1999; Coles
et al., 2004; Nagasaki and Zhao, 2005). The p63 transcription factor family (especially ⌬Np63␣) and ATP binding
cassette transporter G2 (ABCG2) are considered to be the
most reliable markers for corneal stem cells and early
transiently amplifying cells (TACs), although some evidence questions their validity (Chen et al., 2004; Espana
et al., 2004b; Triel et al., 2004; Watanabe et al., 2004; de
Paiva et al., 2005; Kawasaki et al., 2005). Alpha9 and ␤1
integrins, keratin 19 (K19), ␣-enolase, and epidermal
growth factor receptor (EGFR) are believed to mark immature corneal epithelial cells (Chen et al., 2004; Joseph
et al., 2004; Wolosin et al., 2004; Pajoohesh-Ganji et al.,
2005). Immunolocalization of p63 and K19 have also been
used to confirm the presence of stem cells within the
cultures prior to transplantation to ensure long-term engraftment (Harkin et al., 2004). Culturing in vitro, however, can induce cells to modify their phenotype and express more primitive markers that may thereby
exaggerate the contribution of stem and progenitor cells
(Bohnke et al., 1998; Diaz-Romero et al., 2005). This may
confound evaluations of the culture composition, particularly when trying to use this as a prognostic indicator.
The objective of this study was, therefore, to map potential stem cell populations across the intact human cornea,
limbus, and adjacent bulbar conjunctiva using a candidate
marker approach. These are the regions that would be
included in typical limbal stem cell grafts. This approach
has identified three marker-rich populations within the
cornea that could conceivably contribute to clinical corneal
repopulation and include cells entrapped within the limbal stroma and substantia propria. The contribution of
nonepithelioid cells from enzymatically dissociated explants was examined morphometrically to determine
whether a nonepithelial contribution could be maintained
during limbal stem cell expansion. Finally, to examine for
potential changes in epithelial cells expanded in culture,
the specificity of these markers was also examined in both
explanted corneas and primary monolayer cells. In addition to identifying morphologically distinct cell populations within primary monolayer cultures, we show that
the majority of epithelioid cells in whole limbal and bulbar
conjunctival explant cultures or in dissociated cell culture
express markers that are classically believed to indicate
limbal stem cells. Together, in addition to providing candidate markers and populations for further analysis, this
study highlights some of the difficulties in transferring
expression profiles between in vivo and in vitro systems.
MATERIALS AND METHODS
Corneas
Postmortem human corneas stored in Optisol, obtained
from the Eye Bank of Canada (Toronto, Canada), were
either processed for immunohistochemistry or dissected
for culture. Twelve corneas examined ranged from 21 to 74
years of age (median, 58 years) and were processed within
a mean of 14.8 ⫾ 9.1 hr postmortem.
Culture
A 2 mm band of the superficial tissue from the limbusbulbar conjunctiva interface was excised and digested for
15 min in trypsin-EDTA at 37°C to release cells. Cells
were plated onto 1% gelatin-coated culture dishes and
supplemented with Keratinocyte Serum-Free Medium
(KSFM; Invitrogen, Carlsbad, CA) containing 100 U/ml
penicillin and 100 ␮g/ml streptomycin incubated at 37°C
in a humidified environment with 5% CO2. Cultures were
evaluated at 48 hr postplating, and morphological profiles
were obtained for adherent cells. All experiments were
performed using unpassaged primary cells.
For explant cultures, residual donor rims from transplantations containing the limbal and bulbar conjunctival
areas were cut radially into 4 mm pieces and explanted in
KSFM for 72 hr at 37°C in 5% CO2 with rocking at 20 rpm.
Immunohistochemistry
Corneal and explant samples were fixed in 4% paraformaldehyde in 0.1 M PBS for 10 hr at 4°C and processed for
cryosectioning. Ten micron sections were incubated for 1
hr in a blocking solution [4% fetal bovine serum (FBS), 1%
BSA in tris-buffered saline, pH 7.4 (TBS)] to prevent nonspecific binding. They were then incubated overnight at
4°C in appropriate primary antibody (Table 1) diluted in
blocking solution containing 0.1% Triton X-100. After
washing in TBS, sections were reacted with their respective Alexafluor-conjugated secondary at 1:400 for 1 hr at
room temperature. The sections were then counterstained
with DAPI (Sigma, Mississauga, Canada) to localize cell
nuclei, mounted, and visualized by fluorescent microscopy. Where rhodamine-conjugated secondary antibodies
were used, the resulting images were pseudocolored green
to be consistent with FITC-stained images. CD31 was
used to delineate vascular endothelium from putative
stem cell populations. AE1/AE3 antibodies were used to
delineate epithelial cells. All staining was verified on at
least three independent corneas.
Primary cell cultures were expanded to ⬃ 50% confluence (approximately 10 days) and fixed in 4% paraformaldehyde. After quenching for endogenous peroxidase with
3% hydrogen peroxide, immunostaining was performed as
per the corneal sections using fluorochrome conjugated or
biotinylated secondary antibodies. The latter were in turn
reacted with Streptavidin Horseradish Peroxidase (Amersham Biosciences, Baie D’urfe, Canada) and visualized by
reaction with diaminobenxidine substrate (Roche, Laval,
Canada). All cell counts and morphometric data were obtained from images derived from a Zeiss inverted microscope and processed using Axiovision (Zeiss, Hallbergmoos, Germany) and Northern Eclipse (EMPIX,
Mississauga, Canada) software.
923
CANDIDATE STEM AND PROGENITOR CELL MARKERS
TABLE 1. Antibodies used for immunohistochemistry
Primary Antibodies
Abbrev.
Antigen:
Marker of:
Supplier
Clone
General epithelial and epidermal
cells
Epidermal and breast epithelial
stem cells, basal limbal cells
Self-renewing cells
MP Biomedicals, Irvine,
CA, USA
Sigma, Oakville, ON,
CA
Calbiochem, La Jolla,
CA, USA
Calbiochem, La Jolla,
CA, USA
BD PharMingen,
Mississauga, ON,
Canada
DSHB, Iowa City, IA,
USA
BD PharMingen,
Mississauga, ON,
Canada
BD PharMingen,
Mississauga, ON,
Canada
Calbiochem, La Jolla,
CA, USA
BD PharMingen,
Mississauga, ON,
Canada
Chemicon, Temecula,
CA, USA
Cymbus Biotech, Hants,
UK
Santa Cruz Biotech,
Santa Cruz, CA, USA
AE1/AE3
1,2
A53-B/A2
3,2,4
Ab-1
5
Ab-1
6,4
GoH3
7
P5D2
8,9,4
439-9B
8
WM59
10
EA-5
11
IM7
12,13
KIT4
14,15,16
EGFR1
17,18
H-100
19
5D3
20,21,6
AE1/3
Cytokeratins 1/3
K19
Cytokeratin 19
Tel
Telomerase Subunit
⌬Np63
Epithelial stem cells
CD49f
N-terminal-truncated
Protein 63
␣6 Integrin Subunit
CD29
␤1 Integrin Subunit
CD104
␤4 Integrin Subunit
Epidermal and hair follicle stem
cells, basal limbal cells
Mouse epidermal stem cells
CD31
Platelet Endothelial Cell
Adhesion Protein 1
Vascular endothelial cells
CD40
Basal limbal epithelium
CD44
Tumor Necrosis Factor
Receptor Family 5
Hyaluronate Receptor
CD117
c-Kit
EGFR
Epithelial Growth
Factor Receptor
Transforming Growth
Factor Beta Receptor
1
ATP Binding Cassette
Transporter G2
Cardiac, hematopietic, and
neural stem cells
Neural stem cells and basal
epithelial stem cells
Basal limbal epithelial cells
TGFBRI
ABCG2
Epidermal stem cells
Basal epithelial and prostate
progenitor cells
Limbal stem cells
R&D Systems, Hornby,
ON, Canada
Reference
Secondary Antibodies
AlexaFluor 594 goat anti-rabbit IgG
AlexaFluor 488 goat anti-mouse IgG
AlexaFluor 488 goat anti-rat IgG
Biotinylated goat anti-mouse IgG
Biotinylated goat anti-rabbit IgG
Molecular Probes, Eugene, OR, USA
Molecular Probes, Eugene, OR, USA
Molecular Probes, Eugene, OR, USA
Amersham Biosciences, B’aie D’urfe, QC, Canada
Amersham Biosciences, B’aie D’urfe, QC, Canada
1
Kiritoshi et al., 1991 2Kivela and Uusitalo, 1998 3Helczynska et al., 2003 4Chen et al,. 2004 5Miura et al., 2004 6De paiva et
al., 2005 7Li et al., 1998 8Watt, 2002 9Zhou et al., 2004 10Toti et al., 2002 11Iwata et al., 2002 12Tran et al., 2002 13Zhu et al.,
1997 14Beltrami et al., 2003 15Das et al., 2004 16Erlandsson, 2004 17Tarasenko et al., 2004 18Liu et al., 2001 19Joyce and Zieske,
1997 20Budak, et al. 2005 21Watanabe et al., 2004
RESULTS
Immunostaining of Intact Cornea
Human corneas were immunostained for candidate
markers and their distribution within the stroma and
epithelium examined to identify markers that are specific
to the limbus and distal bulbar conjunctival region. Figure
1 indicates the regional definitions of the cornea utilized
within this study. Table 2 summarizes the expression of
various markers within the intact human cornea, explants, and cultured cells.
As expected, the differentiated epithelial cell layers
were consistently labeled by the pan-keratin AE1/3-positive control antibody (data not shown). Of the candidate
stem and progenitor cell markers examined, only the antiK19 antibody stained exclusively occasional superficial
epithelial cells within the limbus and bulbar conjunctiva
(Fig. 2A, arrows). Antibodies against integrins ␣6, ␤1, and
␤4 labeled basal epithelial cells in the central cornea
through and including the bulbar conjunctiva, as well as
occasional wing cells (Fig. 2B, C, D, arrows). Antibodies
against receptors for EGF and TGF-␤ demonstrated similar staining pattern, but tended to stain more than the
occasional wing cell (Fig. 2E and F, arrows). Markers that
were confined to the limbal and bulbar conjunctival compartments demonstrated heterogeneous profiles within
the epithelium and stroma. Basal epithelial cells in the
limbus and bulbar conjunctiva were labeled strongly by
anti-TNF-R5 and hyaluronate receptor (CD44) antibodies
(Fig. 2G and H, arrows). However, the latter antibody also
labeled most stromal cells (Fig. 2G and H, arrowheads).
Telomerase immunostaining was localized primarily to
occasional cells within the stroma throughout the cornea,
within the substantia propria, and occasional cells within
the superficial epithelium (Fig. 2I, arrowheads and arrows
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AE1/3
K19
Tel
P63
CD49f
CD29
CD104
CD40
CD44
CD117
EGFR
TGFBRI
ABCG2
Abbreviations: Super - superficial cell layer, Int - wing/intermediate cells, Str - lamellar stroma, SP - substantia propria, Vasc - vasculature, Det - Detaching fraction
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Epi
Str
Epi
SP
Basal Matrix Vasc
Int
Epithelium
Super
Str
Basal
Int
Epithelium
Super
Str
Basal
Int
Epithelium
Super
Abbrev.
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Other
Epi
Det
SP
Str
Det
Epi
Bulbar Conjunctiva
Cornea Explant
Limbus
Center
Bulbar Conjunctiva
To investigate whether the expression of candidate stem
and progenitor cell markers in the intact cornea remains
constant under cell culture conditions, the same markers
were evaluated in human cornea following explant culture. Immunohistochemical results are summarized in
Table 2. Spherical cells that were detaching from the
conjunctival epithelium were labeled with the AE1/3 antibody as well as K19, TGF␤RI, and TNF-R5 (data not
shown; Fig. 3A, F, and G, inset). However, a small number
of detaching cells also stained positively for EGFR,
ABCG2, telomerase, and c-Kit (Fig. 3E, I, K, and L, inset).
Attached epithelial cells in the cornea, limbus, and bulbar
conjunctiva were stained with antibodies to the integrins,
⌬Np63, TGF␤RI, TNF-R5, and hyaluronate receptor
(CD44; Fig. 3B–D, F, G, J, and H, arrows). Relatively
fewer cells within the epithelium stained positively for
EGFR, telomerase, ABCG2, and c-Kit (Fig. 3E, I, K, and L,
arrows). Rare bulbar conjunctival epithelial cells stained
for antibodies to K19 (Fig. 3A, arrows). Cornea stromal
cells also stained very strongly with antibodies for
TGF␤RI, TNF-R5, and hyaluronate receptor, while some
cells within the substantia propria stained for c-Kit (Fig.
3F–H and L, arrowheads). Occasional cells within the
substantia propria that did not appear to represent vasculature also stained for TGF␤RI, TNF-R5, and telomerase, while the vasculature stained positive for the integrin
subunits (Fig. 3B–D, F, G, and I, arrowheads) in addition
to CD31.
Intact Cornea
Immunostaining of Corneal Explants
Limbus
respectively). Vascular endothelial cells were localized by
antibodies to CD31 and the integrins (data not shown; Fig.
2B–D, arrowheads). Reported stem cell markers, ⌬Np63
and ABCG2, stained rare basal epithelial cells in the
limbus and bulbar conjunctiva (Fig. 2J and K, arrows).
Finally, anti-TNF-R5, and c-Kit antibodies stained subpopulations of cells within the substantia propria of the
bulbar conjunctiva that were not associated with vasculature (Fig. 2G, J, and L, arrowheads).
Center
Fig. 1. DAPI overlain section through a representative human cornea
in phase contrast, showing the demarcations of the central button,
limbus, and bulbar conjunctiva. The limbus is bound centrally by the
cornea and peripherally by the bulbar conjunctiva. Its inferior boundary
is the corneal stroma and the beginning of the undulated palisades of
Vogt. The substantia propria of the bulbar conjunctiva represents the
spongy ECM that overlies the scleral lamellar stroma and is comprised of
vasculature and keratocytes. E denotes the epithelium and is comprised
of the superficial epithelial layer (arrow), intermediate or wing cell layer (i),
and intensely DAPI-stained basal cell layer (b). LS, lamellar stroma; V,
vasculature; SP, substantia propria. Scale bar ⫽ 100 ␮m.
Primary Culture
VASCOTTO AND GRIFFITH
TABLE 2. Summary of candidate stem cell marker localization across the intact cornea, explanted samples and dissociated primary cell cultures
924
CANDIDATE STEM AND PROGENITOR CELL MARKERS
925
Fig. 2. Localization of candidate stem and progenitor cell markers
within representative donor corneas. Panels include the central cornea
(top), limbus (middle), and bulbar conjunctiva (bottom). A–L: DAPIstained nuclei (blue) overlain immunofluorescent staining (green) for K19,
␣6, ␤1, and ␤4 integrins (CD49f, CD29, and CD104, respectively), EGFR,
TGF␤RI, TNF-R5 (CD40), hyaluronate receptor (CD44), telomerase (Tel),
⌬Np63, ABCG2, and c-Kit (CD117), respectively. Arrows and arrowheads indicate positively stained cells within the epithelium and the
underlying matrix, respectively. Inset images represent secondary antibody controls. Scale bar ⫽ 25 ␮m.
Composition and Immunostaining of Limbal
and Bulbar Conjunctival Cell Culture
661.5 ⫾ 234.2 ␮m2; L:W 27.54 ⫾ 10.99) and represented
less than 2% of the adherent fraction (Fig. 4B, arrow). The
remaining distinguishable morphologies consistently observed included very large flattened cells (SA 5,806.95 ⫾
2,079.58 ␮m2; L:W 1.58 ⫾ 0.42), small roughened mesenchymal cells (SA 883.21 ⫾ 308.29 ␮m2; L:W 2.54 ⫾ 0.82),
small smoothened mesenchymal cells (SA 624.31 ⫾ 257.47
␮m2; L:W 3.08 ⫾ 1.04), and fibroblastic cells (SA
1,790.632 ⫾ 876.24 ␮m2; L:W 17.89 ⫾ 10.28; Fig. 4C–E).
All morphological subpopulations could be clonally expanded in culture and thereby suggest the presence of
subpopulations within extracted human limbus/bulbar
conjunctiva.
The localization of markers within adherent cells in
primary culture is summarized in Table 2. The majority of
antibodies that stained the basal and superficial epithelial
cells within the intact cornea were found expressed by the
majority of the adherent epithelioid cells in culture (Fig.
5). This population was also labeled by the AE1/3 antibody
In most cases during limbal stem cell expansion, the
epithelium including underlying tissue is removed, enzymatically dissociated, and cultured or explanted intact in
vitro. To investigate the composition of cells that could be
released during this process, and whether the apparent
induced expression of markers identified in explant culture is context-specific, the epithelium and underlying
tissues were lightly dissociated with trypsin, cultured,
and morphometrically and immunocytologically examined. Approximately 60% of the cells derived from the
bulbar conjunctiva and limbal epithelium adhered to the
gelatin-coated dishes (Fig. 4A). Approximately 93% of adherent cells demonstrated a round epithelioid morphology
with a mean surface area (SA) of 701.1 ⫾ 244.2 ␮m2 and
a length:width ratio (L:W) of 1.24 ⫾ 0.21 (Fig. 4A and B).
Epithelioid cells with extended processes were morphologically distinct from the rounded epithelioid cells (SA
926
VASCOTTO AND GRIFFITH
Fig. 3. Immunofluorescent localization of candidate stem and progenitor cell markers within explants of human cornea (top panel), limbus
(middle), and bulbar conjunctiva (bottom). Insets show cells that have
detached from the explant. A–L: DAPI overlain immunofluorescent staining for K19, ␣6, ␤1, and ␤4 integrins (CD49f, CD29, and CD104, respectively), EGFR, TGF␤RI, TNF-R5 (CD40), hyaluronate receptor (CD44),
telomerase (Tel), ⌬Np63, ABCG2, and c-Kit (CD117), respectively. Blue
indicates DAPI-stained nuclei. Arrows and arrowheads indicate positively stained cells within the epithelium and the underlying matrix,
respectively. Large inset image (A) represents secondary antibody controls. Scale bar ⫽ 25 ␮m.
that recognizes epithelial cells as well as cytoplasmic
K19 and nuclear telomerase and ⌬Np63 antibodies
(data not shown; Fig. 5A, I, and J). Most epithelial cells
also stained for the ␣6, ␤1, and ␤4 integrin adhesion
molecules (Fig. 5B–D). As well, most stained positively
for the growth factor receptors including EGFR,
TGF␤RI, TNF-R5, and c-Kit (Fig. 5E, G, and L). There
was some variability in the intensity of staining of individual epithelioid cells within the primary cultures,
such as ABCG2 (Fig. 5K), that likely represents inherent differences in cell organization (i.e., flattened versus
rounded), and degree of differentiation. Some, but not
all, adherent nonepithelioid cells stained positively for
markers, including ␣6 and ␤4 integrins, CD44, EGFR,
TNF-R5, and c-Kit, indicating that this fraction is heterogeneous. Nonepithelioid cells did not stain for the
pan-keratin AE1/3 or K19. Cells loosely adhered to the
surface of epithelial cells and referred to as the detaching fraction strongly stained for K19 and AE1/3, and
growth factor receptors including EGFR, TGFBRI, and
ABCG2. Similarly, they demonstrated mixed staining
for the integrins and TNF-R5 (Table 2).
CANDIDATE STEM AND PROGENITOR CELL MARKERS
927
Fig. 4. Morphological composition of primary cells derived from the extracted interface of the limbus and bulbar conjunctiva of human donor
corneas. A: Frequency of each class of cells within primary cultures. All counts were performed on at least three samples from at least three corneal
preparations. Values are represented as mean ⫾ SD. Cells were classified as (B) epithelioid, extended epithelioid (arrows), or (C–E) other (including
fibroblastic, large flat, flattened mesenchymal, and mesenchymal). Scale bar ⫽ 10 ␮m.
DISCUSSION
To date, corneal stem cells are believed to reside primarily within the limbal epithelium and are being exploited for transplantation as limbal grafts or after expansion in culture (Shimmura and Tsubota, 2002; Sun and
Lavker, 2004). Using a range of antibodies against markers of corneal and other adult-derived stem cells, progenitor cells, and differentiated cells, we mapped distinct cell
populations to the basal corneal epithelium, the basal and
superficial limbal and conjunctiva epithelium, and scattered cells within the substantia propria of the bulbar
conjunctiva of donor quality human corneas used for
transplantation. The putative stem cell populations identified confirm previous reports (Pellegrini et al., 1999;
Nagasaki and Zhao, 2005). Basal cells across the cornea
and adjacent epithelium indiscriminately stained for ␤1
integrin and EGFR, while basal cells within the limbus
and bulbar conjunctiva demonstrated regional specificity
in their localization of hyaluronate receptor and TGF␤RI.
Similarly, most basal limbal and conjunctiva cells localized TNF-R5, a marker for cells with proliferative potential (Iwata et al., 2002). In a more restricted fashion, K19
staining localized to only a few basal cells in agreement
with some reports, though others have localized K19 expression to all layers of the corneal and conjunctival epithelium (Risse Marsh et al., 2002; Chen et al., 2004; Joseph et al., 2004). Most of these cells therefore likely
represent transiently amplifying progenitor cells. Only
rare basal epithelial cells within the limbus and bulbar
conjunctiva stained positively for ⌬Np63. ⌬Np63 is a p63
isoform that is believed to mark stem cell subpopulations
(Chen et al., 2004; Koster et al., 2004). While p63 broadly
marks basal epithelial cells in the limbus and bulbar
conjunctiva, ⌬Np63 appears to have a more restricted
pattern (Pellegrini et al., 2001; Moore et al., 2002; Di Iorio
et al., 2005). ABCG2 staining showed a similar pattern
within the intact limbus and bulbar conjunctiva. This
supports the contention that both ⌬Np63 and ABCG2
mark more primitive corneal cells.
The colocalization of both stem and transiently amplifying cell markers within the same layer suggests that the
epithelial stem and progenitor cell distribution within
both the basal limbal and bulbar conjunctival epithelia is
heterogeneous, similiar to that of intestinal epithelial
crypts (Clatworthy and Subramanian, 2001). The recent
identification of analogous crypts within the corneal limbal epithelium supports this observation (Dua et al.,
2005).
The documentation of corneal stromal stem or progenitor cells is an area of recent focus. In this study, we found
cells within the substantia propria of the limbus and bulbar conjunctiva that were labeled by markers of potential
progenitor cells (CD44, TNF-R5) as well as the occasional
stem cell marker (c-Kit, telomerase). In contrast to the
epithelium, the integrin staining within the bulbar conjunctiva represents associated vasculature and not discrete candidate populations (Pajoohesh-Ganji et al., 2005).
Overall, our observations of distinct subpopulations
within the substantia propria of the limbus and bulbar
conjunctiva support the contention for the existence of
corneal and bulbar conjunctival stromal stem cells (Li and
Tseng, 1997; Espana et al., 2003; Uchida et al., 2005).
Other markers such as TGF␤RI and hyaluronate receptor
most likely labeled the differentiated stromal cell populations (Asari et al., 1996; Li et al., 1999; Wilson et al.,
2001). The possibility that the corneal stem cell is represented within the substantia propria and contributes to
the stromal keratocyte population or migrates to contribute to the limbal epithelium is an intriguing possibility.
This potential is supported by the demonstration that
928
VASCOTTO AND GRIFFITH
Fig. 5. Pairs of images shodwing immunolocalization of candidate
stem and progenitor cell markers within cultured dissociated cells from
the interface of the limbus and bulbar conjunctiva (left images) and each
corresponding phase contrast image (right image). A–L: Immunofluorescent staining (green) of primary cells with antibodies against K19, ␣6, ␤1,
and ␤4 integrins (CD49f, CD29, and CD104, respectively), EGFR,
TGF␤RI, TNF-R5 (CD40), hyaluronate receptor (CD44), telomerase (Tel),
⌬Np63, ABCG2, and c-Kit (CD117), respectively. Blue indicates DAPIstained nuclei. Inset images represent secondary antibody controls.
Scale bar ⫽ 20 ␮m.
bone marrow can contribute hemopoietic stem cell marker-positive cells to the corneal stroma in mouse models
(Nakamura et al., 2005; Sosnova et al., 2005). Similarly,
multipotent fibroblasts have been isolated from human
corneolimbal tissue that may be induced to differentiate
into corneal epithelial cells in vitro (Dravida et al., 2005).
In this context, limited populations within the substantia
propria may represent interesting candidates, including
c-Kit-positive cells or those expressing TNF-R5.
We also immunolocalized ⌬Np63 and K19 to discrete
cells within the superficial layer of the limbal and bulbar
conjunctival epithelium, suggesting the presence of distinct cell populations. K19 localization to the superficial
epithelium of the peripheral region and reexpression
within proliferating primary cells suggest that they may
be less differentiated (Kivela and Uusitalo, 1998; Fu et al.,
2001; Helczynska et al., 2003; Harkin et al., 2004). Recent
evidence from Di Iorio et al. (2005) suggests that not all
CANDIDATE STEM AND PROGENITOR CELL MARKERS
⌬Np63 isoforms are stem cell markers, but instead may
have a role in epithelial differentiation. The presence,
however, of ⌬Np63 along with localization of telomerase,
K19, and c-Kit also to the limbal and bulbar conjunctival
superficial layer suggests that this layer may contain
some cells with proliferative potential. Alternatively, it
may be a population that is re-expressing some candidate
stem cell markers. The latter possibility is discussed below.
Most dissociated limbal and bulbar conjunctival epithelial cells that adhered in culture showed an epithelioid
phenotype. These cells were stained by a range of antibodies that included markers of progenitor and stem cells,
such as K19 and ⌬Np63. Since these antibodies localized
only to rare cells within the superficial or basal cells of the
intact corneal limbus and bulbar conjunctival epithelium,
the labeling of cells in vitro likely represents de novo
expression of epitopes that was induced by culturing.
Many basal cell markers localized to the majority of cells
covering the surface of explanted tissues including the
integrins, c-Kit, and TNF-R5. The variability in integrin
staining has been very well profiled across human corneal
epithelium (Schlotzer-Schrehard and Kruse, 2005). In the
cornea, ␤1, ␤4, and ␣6 integrins have been implicated in
adhesion to the basement membrane, migration, and
maintenance of a less differentiated phenotype (Watt,
2002; Wang et al., 2003; Zhou et al., 2004). Recently,
Pajoohesh-Ganji et al. (2005) identified a correlation between ␤1 and ␤4 localization and slow epithelial cell cycling in the mouse model. In the current human study, all
three were identified in the basal epithelial cells across
the stable intact cornea, suggesting a relationship more to
the basement membrane than differentiation state. The
corneal basement membrane is comprised of laminin, fibronectin, tenascin, and collagen, all of which are ligands
for these integrins (Tuori et al., 1996, 1997; Watt, 2002).
Similarly, this composition changes spatially across the
cornea, limbus, and bulbar conjunctiva and in response to
wounding (Ljubimov et al., 1995, 1998). The pattern of
integrin localization that we identified in the intact tissues and the very active expression within the epithelium
of the explanted corneas and dissociated cell culture suggest that these are poor candidate limbal stem cell markers. Their expression more likely correlates to cell migration and changes in adhesion associated with active
proliferation. Similarly, TNF-R5 is a member of the TNF
receptor family that can affect cytokine production, cell
adhesion, and immune recruitment (Aldinucci et al., 2002;
Ardjomand et al., 2003). In accordance with previously
published data, TNF-R5 is localized to the basal cells of
the human sclera limbus and likely represents proliferative potential (Iwata et al., 2002). The epithelial layer in
explanted corneas also strongly stained for this marker
supporting this possibility. Lastly, the expression TNF-R5
in the majority of primary cells indicates that it is a factor
inducible by culture conditions.
The induction of epitope expression by dissociated cells
in cell culture is supported by our observations of increased immunolocalization of stem cell markers (⌬Np63,
ABCG2), in particular within corneal, limbal, and bulbar
conjunctival explants and in detaching cells derived from
these explants. The culture-induced expression of stem
cell markers in the corneal and conjunctival-derived cells
has been correlated with the loss of the differentiated
phenotype (Kivela and Uusitalo, 1998; Harkin et al.,
929
2004). The expression of K19 and ␤1 integrin and p63 are
inducible under cell culture conditions and are associated
with expanded proliferative capacity (Pellegrini et al.,
2001; Risse Marsh et al., 2002; Harkin et al., 2004; Joseph
et al., 2004; Kim et al., 2004). In other systems, the generation of ␤1 integrin and K19 cell islands in the spinous
and granular layers of the human epidermis is believed to
be the product of dedifferentiation, as is K19 expression
within human mammary ductal cells and hepatocytes
(Blaheta et al., 1998; Fu et al., 2001; Helczynska et al.,
2003). Similarly, adult rabbit corneal epithelial cells can
be reprogrammed to generate fully differentiated epidermis, hair follicles, and sweat glands (Ferraris et al., 2000).
Further exploration, however, is needed to determine if
re-expression of primordial markers indicate dedifferentiation of corneal epithelial cells. Should dedifferentiation
be a factor, this could lead the way to expanding progenitor cell pool for clinical use by preculturing corneal biopsies (Gan et al., 1998; Harkin et al., 2004; Kim et al.,
2004). A conclusive evaluation of the correlation between
the expression of stem cell markers within such cultures
and the successful engraftment following transplantation
into a patient remain to be performed.
Using an immunohistological approach, we localized a
series of potential stem cell markers to the superficial and
basal limbal and conjunctival epithelium, as well as substantia propria. All of these regions are represented in the
clinic during limbal stem cell transplantation. Many of
these antigens, including the integrins and c-Kit, represent potential markers for fractioning specific subpopulations and subsequent evaluation either alone or in combination. Investigations are underway to purify and identify
the differentiation potential of some of these populations.
We also show that the expression of traditional stem cell
markers may not be reliable indicators for stem cells in
cell cultures and selection for marker expressing cells
could contribute to the variable success rates of corneal
stem cell transplantation.
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