Stem Cell Rev (2008) 4:159–168
DOI 10.1007/s12015-008-9029-x
Corneal Epithelial Stem Cells: Deficiency and Regulation
Genevieve A. Secker & Julie T. Daniels
Published online: 12 July 2008
# Humana Press 2008
Abstract The corneal epithelium is continuously renewed
by a population of stem cells that reside in the corneoscleral
junction, otherwise known as the limbus. These limbal
epithelial stem cells (LESC) are imperative for corneal
maintenance with deficiencies leading to in-growth of
conjunctival cells, neovascularisation of the corneal stroma
and eventual corneal opacity and visual loss. One such
disease that has traditionally been thought to be due to
LESC deficiency is aniridia, a pan-ocular congenital eye
disease due to mutations in the PAX6 gene. Corneal changes
or aniridia related keratopathy (ARK) seen in aniridia are
typical of LESC deficiency. However, the pathophysiology
behind ARK is still ill defined, with current theories
suggesting it may be caused by a deficiency in the stem
cell niche and adjacent corneal stroma, with altered wound
healing responses also playing a role (Ramaesh et al,
International Journal of Biochemistry & Cell Biology
37:547–557, 2005) or abnormal epidermal differentiation
of LESC (Li et al., The Journal of Pathology 214:9, 2008).
PAX6 is considered the master control gene for the eye and
is required for normal eye development with expression
continuing in the adult cornea, thus inferring a role for
corneal repair and regeneration (Sivak et al., Developments
in Biologicals 222:41–54, 2000). Studies of models of Pax6
deficiency, such as the small eyed (sey) mouse, should help
to reveal the intrinsic and extrinsic mechanisms involved in
normal LESC function.
Keywords Limbal epithelial stem cells . Limbus . Cornea .
Aniridia . PAX6
G. A. Secker (*) : J. T. Daniels
Cells for Sight Transplantation and Research Programme,
Ocular Repair and Regeneration Biology Unit,
Division of Pathology, UCL Institute of Ophthalmology,
11-43 Bath Street,
London EC1V 9EL, UK
e-mail: g.secker@ucl.ac.uk
Limbal Epithelial Stem Cells
All self-renewing tissue must contain a stem cell pool,
which provides an unlimited supply of proliferating cells.
This is true for the corneal epithelium with a large body of
research indicating these cells reside in the limbal basal
region and are aptly named limbal epithelial stem cells
(LESC; Fig. 1). LESC share a number of features with
other adult somatic stem cells. These include having small
cell size [1], the lack expression of differentiation markers,
such as cytokeratin 3/12 [2, 3] and high nuclear to
cytoplasmic ratio [7].
LESC are considered to be primitive cells as they are
slow cycling and therefore label retaining under normal
conditions but have the ability to be highly proliferative in
response to injury [5–7]. Stem cells have the ability to
divide asymmetrically to repopulate the stem cell pool.
Barbaro et al., recently found expression of C/EBPδ in a
subset of LESC both in vivo and in vitro, and have
suggested it is involved in the regulation of self renewal
and cell cycle length of LESC. Other pathways have been
linked to stem cell renewal, such as Notch-1. Corneal
specific inducible ablation of Notch1 demonstrated differ-
entiation of LESC into hyperplastic, keratinised skin like
epithelium [8]. Furthermore LESC express progenitor
markers, including, p63 [9], ABCG2 [10] and more
recently N-cadherin
Evidence for the Location of LESC to the Limbus
The first experimental evidence for the location of LESC to
the limbus was the movement of pigment from the limbal
region towards an epithelial defect in rabbit corneas
following wounding [11]. Some years later Davanger and
Evenson [12], observed a similar migration of pigment
from limbus to central cornea and proposed that the
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Fig. 1 A cross-sectional
diagram of the human corneal
limbus. Limbal epithelial stem
cells reside in the basal layer of
the epithelium (Ep) which
undulates at the limbus.
Daughter transient amplifying
cells divide and migrate towards
the central cornea (arrowed) to
replenish the epithelium, which
rests on Bowman’s layer (BL).
The stroma (St) of the limbal
epithelial stem cell niche is
populated with fibroblasts and
melanocytes and also has a
blood supply
Palisades of Vogt (PV) situated in the corneal limbus
provided the source of LESC [13]. This movement from
limbal to central cornea has been described as centripetal
migration. This was demonstrated by gradual replacement
of donor epithelium with host cells following lamellar
keratoplasty, by looking at the dilution of sex chromatin
using a female donor graft in a male recipient in rabbits
[14]. Furthermore, the complete removal of the limbus
results in impaired corneal function, neovascularisation and
conjunctival ingrowth [13].
As stem cells are slow cycling they divide occasionally
and therefore can be identified as label retaining cells
(LRCs) [15]. Exposing cells to DNA precursors such as
tritiated thymidine and bromodeoxyuridine followed by a
chase period of 4–8weeks, the slow cycling stem cells
retain this label whereas the more differentiated transient
amplifying cells (TAC) undergo dilution of the label
through multiple divisions. Through the use of tritiated
thymidine, Cotsarelis et al., found slow cycling or LRCs
located in the limbal basal region of the mouse cornea and
postulated that 10% of limbal basal cells were stem cells.
This population of limbal basal cells phenotypically appear
to be more primitive as they are small and round [1].
The limbal basal region has areas lacking in differenti-
ation markers. For example, the 64kDa cytokeratin 3 (CK3)
was found in all layers of the corneal epithelium and the
suprabasal layers of the limbal epithelium, however it was
absent from the limbal basal cells and the adjacent
conjunctiva [2]. A similar pattern was found with the
corneal specific 55kDa protein, cytokeratin 12 (CK12) [16].
Furthermore, there is a lack of markers such as connexin 43
[17] and involucrin [18], both associated with cells destined
for differentiation. Interestingly, the limbal basal region
expresses progenitor cell markers such as the transcription
factor p63 [9], especially the ΔNp63α isoform [19], the
ATP-binding cassette transporter Bcrp1/ABCG2 and more
recently N-cadherin [20].
Stem Cell Rev (2008) 4:159–168
In vivo and in vitro studies have found that limbal basal
cells have a higher proliferative potential when compared to
peripheral and central cornea. Large epithelial wounds in
rabbits have been shown to heal faster than smaller central
defects, implying that the proliferative capacity of the
peripheral cornea is greater than that of the central [21]. In
the human, limbal explant cultures have a greater prolifer-
ative potential when compared to central explants [22, 23].
Based on human epidermal studies [4], supporting clono-
genicity studies found cells isolated from the limbus
produced the larger holoclones (stem cell derived) com-
pared to the less clonogenic meroclones and paraclones
found elsewhere in the cornea [24]. Furthermore, LESC
proliferation is resistant to inhibition by tumour-promoting
phorbol esters [25, 26].
Supplementary to experimental studies the clinical
evidence also points toward the limbus as the location of
corneal stem cells. In normal corneal maintenance, the
limbal epithelial cells are thought to act as a barrier to the
conjunctival epithelial cells [27]. Ambati et al., has recently
shown that soluble vascular endothelial growth factor
receptor 1 (sFlt1) plays an important role in corneal
avascularity [28]. Further to this, expression of sFlt1 was
found in the corneal epithelium of normal individuals with
less seen in vascularised patients [29]. When the limbus is
non functional, the conjunctiva can invade the corneal
epithelium leading to chronic inflammation, neovascular-
isation and corneal opacity. This phenomenon is known as
limbal stem cell deficiency and can be attributed to both
hereditary and acquired conditions. Further clinical evidence
suggesting the location of LESC was shown by Kenyon and
Tseng, where they transplanted two limbal explants taken
from the contralateral healthy eye of patients onto the other
damaged eye. This resulted in re-epithelisation of the cornea
and regression of persistent epithelial defects and neo-
vascularisation [30]. This initial work has lead to the use
of expanded LESC grown on amniotic membrane [31] and
Stem Cell Rev (2008) 4:159–168
more recently the use of autologous mucosal epithelial cell
grafts [32].
The LESC Niche
The surrounding microenvironment or niche of a stem cell,
which consists of cellular and extracellular components, is
hypothesised to prevent them from differentiating and thus
determines their fate [33, 34]. Once a stem cell divides
asymmetrically and leaves its niche it enters a differentia-
tion pathway under the influence of different environmental
stimuli. Interestingly, the mechanisms by which this occurs
still remains unclear. This theory is in keeping with the
LESC niche as it differs from the remaining corneal stroma
both anatomically and functionally.
The LESC niche is thought to be located in the area of the
Palisades of Vogt (PV) which undulate providing increased
surface area. They are highly pigmented, due to presence of
melanocytes [12, 35] and infiltrated with Langerhans cells
[36] and T-lymphocytes [37]. The melanin pigmentation
shields cells from damaging ultraviolet light and the resultant
generation of reactive oxygen species [38]. When comparing
the LESC niche to the cornea, the basement membrane
contains papillae of stroma which project upwards [39] and
is also fenestrated [40] suggesting close interaction with
surrounding stromal cells. The limbal and corneal basement
membrane components also differ, with the limbal region
containing laminin-1,5 and α2β2 chains which are not found
in the cornea. Furthermore, type IV collagen α1, α2 and α5
chains are found in the limbal region whereas α3 and α5 are
located in the cornea [41, 42]. A more recent study by
Schlötzer-Schrehardt et al., found patchy immunolocalisation
of laminin γ3 chain, BM40/SPARC and tenancin C, which
was also found to co-localise with ABCG2/p63/K19-positive
cell clusters, suggesting they may be involved in retaining
cell stemness [43].
It has also been suggested the LESC niche basement
membrane may sequester and therefore modulate growth
factors and cytokines involved in LESC function allowing
for efficient and precise cellular control [44]. Interestingly,
it has been recently found that hypoxic conditions in vitro
produce larger cell colonies and a decrease in differentia-
tion markers, suggesting low oxygen levels induces
selective proliferation of undifferentiated cells [45]. Al-
though the cornea is exposed to atmospheric oxygen, the
LESC niche lies beneath a number of cell layers and
therefore the environment may have lower oxygen levels,
which help maintain the stemness of the cells.
Interestingly, beneath the basement membrane the limbal
niche is vascularised and highly innervated [46] compared to
the avascular cornea thus potentially providing extra nutrients
and growth factors to LESC. The cells residing in the stroma
161
are heterogeneous, containing limbal fibroblasts which have
been found to preferentially express secreted protein acidic
and rich in cysteine (SPARC) when compared to corneal
fibroblasts, which may contribute to LESC adhesion [47].
Furthermore, Nakamura et al. [48], identified a population of
bone marrow-derived cells located in the limbal stroma
following transplantation of GFP labelled bone marrow cells
into nude mice. This study suggested these cells are able to
migrate into the limbal stroma, however functionality was
not discussed and should be explored. The interaction
between the limbal stromal cells and LESC is not fully
understood and needs further investigation.
This specialised microenvironment also provides protec-
tion. Firstly, the LESC are located in the basal layer, therefore
harmful agents must penetrate the full thickness of the corneal
epithelium prior to reaching the stem cells. The deep
undulations of the of Vogt at the limbus provide LESC with
a protective environment away from the external surroundings
and also protect against shearing forces [49] along with the
non-uniform junctions between limbal epithelium and
stroma. Recently, Shortt et al. [39] has found limbal
epithelial crypts and focal stromal projections (FSP) are
predominantly located at the superior and inferior aspects of
the cornea, which offers further protection by the eyelid.
Limbal Epithelial Stem Cell Markers
Much effort has been made to identify potential molecular
markers to distinguish LESC from TAC located in the limbal
and corneal basal regions. As discussed below, some
progress has been made using varied approaches, however
there remains controversy with no one molecule being
definitive. Markers can be divided into two subgroups, both
negative and positive. Negative markers are absent mole-
cules from the limbal basal cells and include the cytokeratins
CK3 and CK12 [2] the gap junction protein Cx43. With
positive markers, such as p63 and ABCG2, being located in
the LESC themselves.
Pellegrini et al. [9], initially proposed the transcriptional
factor p63 as a putative LESC marker, as it was found to
distinguish human keratinocyte stem cells from their TA
progeny and was therefore suggested to be a epidermal
stem cell marker. Through clonal analysis and western
blotting, they demonstrated that p63 was expressed in
limbal holoclones with little or none being seen in
meroclones and paraclones. Immunohistochemically, p63
was located to limbal basal epithelium, supporting its role
as a LESC marker. p63 has also been localised to basal cells
of the peripheral and central cornea in humans [18, 50] and
in rats [51] thus ruling it out as a exclusive LESC marker.
However, when combining high p63 expression with
nuclear/cytoplasmic ratio a small population of cells has
162
been identified and suggested to represent LESC [52].
More recently, the isoform, ΔNp63α has been proposed to
be more specific and therefore has been suggested to be a
stem cell marker [19].
Many types of organ-specific stem cells have been
recently shown to exhibit a side population (SP) phenotype
based on their ability to efflux Hoechst 33342 dye through
the ATP-binding cassette transporter Bcrp1/ABCG2 and
therefore it has been proposed to be a universal marker for
stem cells [53]. Flow cytometric and real time RT PCR
analysis of limbal basal cells has shown these cells to
express this same SP and was therefore suggested as a
putative LESC marker [10]. In support, the protein has been
immunolocalised to the cell membrane and cytoplasm of a
population of limbal basal cells and a few suprabasal cells
[18] with ABCG2 positive cells producing higher colony
forming efficiencies than their negative counterparts [54].
Furthermore, our laboratory has localised ABCG2 to the
outer edge of holoclones where it is thought that the stem
cells reside (Fig. 2). Indeed, these studies suggest it is an
ideal candidate as a LESC marker, however further
clarification is needed.
Integrins such as α9 have been suggested as a possible
marker as it has been localised to small clusters of cells in
the limbal basal epithelium [55]. However, murine studies
have demonstrated an upregulation of α9 in wounded
corneas, suggesting it is expressed in TAC’s as opposed to
LESC [56]. One other integrin β1 has been associated with
LESC, it was originally suggested to be a keratinocyte
marker [57]. Furthermore cells that rapidly adhere to the
Fig. 2 Characteristics of limbal
epithelial stem cells in vitro.
When seeded at low density,
limbal epithelial stem cells pro-
duce large holoclone colonies
(a, stained with haematoxylin
and b, phase contrast). The cells
at the outer edge of holoclones
express ABCG2 (c, green),
ΔNp63α (d, red) a merge of the
two markers is shown in e with
DAPI staining of the nuclei
Stem Cell Rev (2008) 4:159–168
integrin β1 ligand, collagen IV, display many LESC
properties [58]. Although it is found to be expressed in
limbal and corneal cells, suggesting it is expressed by more
differentiated cells, limbal basal cells are said to be integrin
β1 bright.
N-cadherin mediates cell–cell adhesion and has been
suggested to play an important role in the maintenance of
haemopoietic stem cells, by facilitating adhesion to osteo-
blasts in the bone marrow niche [59, 60]. With this in mind,
Hayashi et al, explored the possibility of role for N-
cadherin in LESC maintenance. They found expression of
N-cadherin in a subpopulation of limbal epithelial basal
cells, suggesting they were putative stem cells. Further to
this, they also found expression in adjacent melanocytes
implying N-cadherin plays an important role in interactions
between LESC and their corresponding niche cells [20].
As the limbal epithelium is derived from the neural
ectoderm a number of neural stem cell markers have been
suggested as LESC markers. Nerve growth factor (NGF) has
been associated with the proliferation and differentiation of
human corneal epithelium with stimulation of its receptor,
TrkA, being thought to promote cell survival [61]. This high
affinity receptor is expressed in both corneal and limbal basal
cells compared with the low affinity NGF receptor, p75NTR
which is localised to suprabasal limbal and entire corneal
epithelium. As TrkA lacks specificity it cannot be considered
a LESC marker, however p75NTR could be looked upon as a
marker of differentiation. Recent in depth immunological
studies of neurotrophic factors and their receptors in the
human has found NGF, Glial cell-derived neurotrophic factor
Stem Cell Rev (2008) 4:159–168 163

(GDNF) and their corresponding receptors TrkA and GDNF
family receptor alpha (GFRα)-1 are exclusively expressed in
the limbus [62].
Intermediate filaments such as vimentin, keratin 15
(K15) and keratin 19 (K19) have been localised to basal
cells of the limbus [63].
Notch 1 is a ligand-activated transmembrane receptor,
which has been shown to maintain progenitor cells in other
systems in an undifferentiated state. Antibody staining
demonstrated cell clusters in the Palisaids of Vogt with some
co-localisation with ABCG2, thus suggesting it may be a
LESC marker [64]. However, others have suggested it plays
a role in differentiation, proliferation and corneal repair.
Using Notch 1 deficient mice, Vauclair et al, demonstrated
Notch 1 signalling is required for cell fate maintenance
during corneal epithelial wound healing linking this to
regulation of vitamin A metabolism [8]. Furthermore, Ma
et al. [65], found Notch 1 and other Notch family members
and their receptors to be expressed throughout the cornea and
suggested a role in differentiation.
Opposed to the identification of a LESC marker is the
existence of markers of corneal epithelial differentiation. The
corneal specific differentiation markers K3 and K12 are
expressed throughout the corneal epithelium but are absent
from the limbal basal cells, thus indicating that these cells are
the least differentiated and therefore may be more stem like
in nature. Cross talk between cells is facilitated by gap
junctions with connexin 43 and connexin 50 being localised
to the corneal epithelium [66]. Cx 43 is expressed by corneal
basal cells except that of the limbus, implying it is utilised by
more early TACs. The lack of intracellular communication
has been suggested to help maintain stem cells and their
niche [17] and protects the cells from damage affecting its
adjacent neighbouring cells [51]. Involucrin is produced by
all cells within stratified squamous epithelia apart from basal
cells [67] this also being true for the corneal epithelium [18].
Furthermore, larger cells from human corneal epithelial
cultures display increased expression of involucrin, this
along with immunological staining suggests it is also a
marker of differentiation.
In depth immunohistochemical panels have been
assessed in attempt to identify informative markers. These
latest findings are summarised in Table 1 and suggest when
comparing limbal basal epithelial cells to corneal basal
cells, they are positive for K19, ABCG2, vimentin, KGF-R,
metallothionein and integrin α9. Furthermore they are
found to be negative for CK3 and CK12, Cx43, E-cadherin,
involucrin and integrins α2, α6 and β4 [68]. The majority
of LESC putative markers are expressed in most limbal
basal cells and some suprabasal cells, therefore it cannot be

Table 1 Localisation of puta-

tive stem cell markers of the Limbal epithelium Corneal epithelium
corneal epithelium
Basal Suprabasal Basal Suprabasal

Positive markers
p63 +++ +/− +/− −
Δp63α ++ − − −
ABCG2 +++ +/− − −
Integrin α9 +++ +/− − −
NGF R (TrkA) +++ ++ +++ ++
α-enolase +++ + ++ +
Vimentin +++ +/− − −
Keratin 19 ++ − − −
Integrin β1 +++ ++ ++ +
N-Cadherin ++ − − −
GDNF +++ ++ − −
GFRα-1 +++ ++ − −
Keratin 15 ++ ++ − −
KGF-R +/− − − −
Negative markers
CK3/CK12 − +++ +++ +++
Cx 43 − +++ + +++
Cx 50 − +++ − +++
Involucrin − +++ + +++
NGFR(p75NTR) − +++ +++ +++
Adapted from [68] and [51] E-cadherin +/− +++ +++ +++
− No staining, +/− very weak Integrin α2 − or +++ ++ +++ +++
staining, + weak staining, ++ Integrin α6 − or +++ ++ +++ +++
moderate staining, +++ strong Integrin β4 − or +++ ++ +++ +++
staining
164 Stem Cell Rev (2008) 4:159–168

ruled out that these markers also present in early TAC.
Currently the most promising candidate markers are
ABCG2, Notch-1 and N-cadherin. ABCG2 and Notch 1
both identify small clusters of limbal basal cells, with N-
cadherin also identifying adjacent melanocytes.
LESC Deficiency
Partial or full LESC deficiency occurs as a result of damage
or disease to the LESC population, which leads to
deleterious effects on corneal wound healing and surface
integrity [50, 69]. Deficiency can arise from injuries
including chemical or thermal burns and through diseases
such as Stevens Johnson syndrome and aniridia. Character-
istics of LESC deficiency include ingrowth of the conjunc-
tival epithelium, vascularisation, chronic inflammation,
recurrent erosions and ulceration, basement membrane loss
and ingrowth of fibrotic tissue. This leads to functional
impairment and eventual visual loss [30, 70, 71]. Long term
restoration of visual function requires renewal of the
corneal epithelium, through replacement of the stem cell
population by grafting limbal auto- or allografts [30].
Traditionally, treatment of LESC deficient patients has
been achieved by grafting viable limbal tissue, from either the
fellow healthy eye or a donor eye, thus replacing the LESC
population [72]. A variety of limbal tissue transplantation
methods have been used, including cadaveric keratolimbal
allograft (KLAL), live or living related conjunctival limbal
allograft (Ir-CLAL) and limbal autograft. Each procedure
carries a risk of complication such as damage to healthy eye
with removal of autologous grafts and complications due to
long term immunosuppression with allogenic tissue, there-
fore new strategies have been developed. Once such
procedure is transplantation of cultured LESC grown on a
suitable substrate such as amnionic membrane or fibrin, with
or without growth arrested 3T3 fibroblast feeder layers [73–
77]. The mechanisms behind this method are not clearly
defined however, initial success rates are promising.
The underlying process of these abnormalities is poorly
understood and is thought to be due to stem cell failure [78–
80] however, it has been proposed that it may be due to a
deficiency in the stem cell niche and adjacent corneal stroma
[81] and more recently a downregulation of PAX6 has been
linked to abnormal epidermal differentiation of cornea
epithelial cells [82].
An important factor leading to progressive loss of vision
is aniridic-related keratopathy (ARK) [78, 83] which occurs
in 90% of patients. Initially the cornea of patients appears
normal during childhood [79, 84]. Changes occur in
patients in their early teenage years, with the disease
manifesting as a thickened irregular peripheral epithelium.
This is followed by superficial neovascularisation and if left
untreated it may result in subepithelial fibrosis and stromal
scarring (Fig. 3). Furthermore patients develop recurrent
erosions, ulcerations, chronic pain and eventual blindness
[85]. Histologically, stromal neovascularisation and infil-
tration of inflammatory cells is seen with the destruction of
Bowman’s layer. Additionally, the presence of goblet and
conjunctival cells is seen on the corneal surface [83].
Traditionally, these clinical and histological manifestations
has lead to the consensus that LESC deficiency is largely
responsible for corneal abnormalities in aniridia [83, 86].

PAX6 Insufficiency and LESC Deficiency

Aniridia

One of the causes of blindness in children with aniridia is due

to progressive ocular surface failure. The disease is caused by
PAX6 heterozygosity, which leads to a pan-ocular, bilateral
condition most prominently characterised by iris hypoplasia,
which varies from a relatively normal iris to the complete lack
of an iris. Aniridia is often associated with cataracts, corneal
vascularisation and glaucoma, with a significant number of
cases of visual morbidity being due to corneal abnormalities.
Fig. 3 Aniridia. An aniridic human eye (a) showing neovascularisa-
tion, inflammation and opacity of the cornea. WT mice have a normal
complement of Pax6 gene expression and normal eyes (b). The Pax6
heterozygous sey small eyed mouse with LESC failure is a model of
human aniridia (c)
Stem Cell Rev (2008) 4:159–168
As a LESC marker has yet to be definitively identified, a
true demonstration of LESC deficiency cannot be assumed.
Furthermore treatment for these patients involving replace-
ment of LESC, either by keratolimbal allografts or more
recently ex vivo expanded LESC grafts, provides a better
outcome than corneal transplants [77, 85, 87], this being
consistent with LESC deficiency. However, patients who
receive both limbal and corneal tissue seem to have the
better outcome, suggesting an abnormality with corneal
tissue and not just the limbus. This may be a downstream
effect of LESC deficiency or that low levels of Pax6 has a
generalised effect on the entire cornea. Alternatively, ARK
may be a consequence of an abnormal corneal epithelial/
stromal healing response as there is insufficient evidence to
indicate that the proliferative potential of LESC is impaired
[81, 88]. Recently, studies looking at the regulation of
genes downstream of Pax6 in the Pax6 mutant mouse,
suggests the pathogenesis of ARK is due a number of
mechanisms and not solely due to LESC deficiency [81].
Further studies are needed to elucidate the exact mecha-
nisms of ARK progression to allow the use of appropriate
treatments.
Heterozygous Pax6+/− mice or small eye (sey) mice,
provide an excellent model for aniridia and the progressive
nature of associated corneal abnormalities [89, 90]. As the
name suggests, mice with semidominant mutations develop
small eyes and other ocular deformities (Fig. 3.). Homo-
zygotes generate an ultimately lethal phenotype with no
eyes and nasal primordial [91, 92]. A number of sey mice
arose independently all of which are semidominant and by
examining comparative mapping studies and phenotypic
similarities to aniridia, it was suggested to be the mouse
homologue of the human disease [92]. This research led to
the discovery that the Pax6 gene was responsible for the
Sey phenotype and suggested that it was also responsible
for the human disease, aniridia [91].
The Pax6 Gene
The PAX6 gene is a hierarchical transcriptional factor
regulating a multitude of genes involved in eye develop-
ment and adult homeostasis. The PAX6 protein has been
found to be expressed in the adult eye, cerebellum and
pancreas suggesting it has an important role in maintenance
and remodelling of adult tissues [93–96]. PAX6/Pax6 is
involved in a number of regulatory roles in the adult cornea
and is essential for the expression of Cytokeratin 12,
gelatinase B (MMP-9) and cell adhesion molecules (CAM)
[89, 96, 97] and more recently it has been suggested that
knockdown of Pax6 expression promotes EGF-induced cell
proliferation [98].
Cytokeratins (CK) are structural proteins expressed by
epithelial cells, with CK12 being a specific for differentiated
165
corneal epithelial cells [99]. It has been found that CK12
expression is Pax6 dependant [99] with CK12 deficient mice
producing a fragile superficial corneal epithelium that often
detaches [100]. This phenotype is also seen in the Pax6+/−
mice [72] and when comparing them to Pax6+/+mice, there
is a decrease in CK12 staining. Further to this, aniridic
patients also have a decrease in CK12 staining [80]. This
suggests the corneal fragility seen in both Pax6+/− mice and
aniridic patients is due poor differentiation as a consequence
of lowered levels of CK12, being a downstream of altered
Pax6 expression. A recent human study has suggested that
loss of PAX6 and resultant down regulation of CK12 seen in
squamous metaplasia results in abnormal epidermal differ-
entiation as evidenced by expression of CK10 and filaggrin
both known epidermal proteins. Furthermore, due to local-
isation of CK10 expression to the suprabasal cells in the
limbal and peripheral cornea suggests PAX6 controls LESC
plasticity and a loss leads to transdifferentiation in squamous
metaplasia and could also indicate a role for the limbal stem
cell niche [101].
Sonic hedgehog, Wnt/β-catenin, TGF-β and Notch
signalling pathways have all being implicated in niche
control of stem cells, however the LESC and their niche is
still relatively unchartered. It has recently been shown that
mice lacking in expression of Dkk2, a Wnt pathway
inhibitor, display epidermal differentiation on the ocular
surface. The lack of Dkk2, leads to increased Wnt/β-
catenin signalling in the limbal stroma, demonstrating the
importance of limbal niche control over LESC differentia-
tion during development. Pax6 expression is lost in the
corneal epithelial cells of these mice, suggesting it is
downstream of Dkk2 [102]. Furthermore a small population
of progenitor corneal fibroblast or keratocytes has been
identified as being Pax6 positive [103]. Down regulation of
Pax6 in these cells could lead to altered LESC control.
Deficiencies in PAX6 resulting in impaired corneal epithe-
lial function and eventual LESC failure may be due to
altered niche development.
Summary
Limbal epithelial stem cell health is imperative to normal
corneal function with deficiencies leading to conjunctival-
isation, neovascularisation and eventual visual loss due to
corneal opacity. One such disease that has been considered
to be a consequence of LESC deficiency is aniridia, which
is caused by mutations in the PAX6 gene. A better
understanding of PAX6 regulation of LESCs through the
use of animal models has helped to gain insight into LESC
characteristics and function. However, LESC control is still
relatively uncharted and therefore a better understanding of
regulation will improve disease prevention, progression and
treatment in the future.
166
Acknowledgements This work was supported by the ERANDA
Foundation (GAS), the Special Trustees of Moorfields Eye Hospital
and the NIHR BMRC for Ophthalmology (JTD). Thank you to
Mr. Alex Shortt for the clinical data.
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