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The ocular surface consists of two distinct types of epithelial cells; conjunctival and
corneal. Although anatomically continuous, these epithelia comprise two distinct cell
populations. Corneal stem cells are located at the limbus. The microenvironment of the
limbus is important in maintaining “stemness” of the stem cells and also acts as a barrier
to conjunctival epithelial cells preventing them from migration onto the corneal surface.
Damage to the limbus results in varying degrees of limbal stem cell deficiency with
characteristic clinical features including conjunctivalization of the cornea. Regenerative
management of corneal conjunctivalization utilizing stem cells comprises of two
approaches; limbal auto- or allografts by using existing stem cells and induction and
regeneration of ocular tissues from embryonic stem cells. Herein, we review stem cells
and limbal stem cells in particular, types of epithelial cells in the cornea, markers of
corneal epithelial cells in different stages, as well as the current approach to corneal
epithelial regeneration.
Key words: Stem Cells; Limbus Corneae; Epithelium, Corneal
J Ophthalmic Vis Res 2009; 4 (1): 40-58.
Correspondence to: Marzieh Ebrahimi, PhD. Department of Stem Cells, Cell Science Research Center, Royan
Institute, ACECR, Tehran, Iran, PO Box 19395-4644, Tehran, Iran; e-mail: ebrahimi@royaninstitute.org
Received: July 29, 2008 Accepted: October 11, 2008
primarily by aligned bundles of collagen ter- below, enable stem cells to accomplish their
med lamellae which are stacked in an ortho- important task to maintain tissue integrity
gonal pattern.5 throughout the body.
The corneal endothelium functions to main- 1) Error-free proliferation: Error-free mito-
tain the stroma in a relatively dehydrated state. sis is essential since any genetic error at the
This function depends on the presence of a ba- level of the stem cell will continuously and
rrier formed by focal tight junctions preventing permanently pass on to the whole clone of cells,
fluid flow and on a pumping action provided resulting in abnormal differentiation and cellu-
by Na+/K+ ATPase and Mg++ dependent bicar- lar dysfunction. To minimize any error produ-
bonate enzymes present in the lateral mem- ced during stem cell mitosis, several protective
branes of the endothelial cells.6 mechanisms have been developed. First, stem
cells are relatively quiescent during the state of
CORNEAL ORGANOGENESIS IN HUMANS steady growth. They leave the job of active
DNA synthesis and cell number amplification
The development of cornea as a tissue initiates to transient amplifying cells (TACs). So that
by derivation from the ectoderm overlying the even if an error is made at the TAC level it will
crystalline lens as early as five weeks in the be self-limited because all cells except stem
human embryo. In brief, a primitive two-cell cells have a limited lifespan. In cell kinetic
layer thick epithelium is first apparent at about terms, stem cells have a longer cell cycle time
five weeks which is contiguous with the surface (180 vs 90 hours) and a shorter S-phase dura-
ectoderm. During the next one to two weeks, tion (2–3 vs 9–21 hours) as compared to TACs
the epithelium stratifies to 3 to 4 cell layers, the in the case of skin epidermis.9 Secondly, Potten
lens completes its formation and detaches from et al10 demonstrated that there is asymmetrical
the ectoderm, and the eyelids form and fuse. DNA segregation during stem cell mitosis,
Almost immediately after separation of the lens suggesting that stem cells retain their original
from the corneal epithelium, waves of neural genetic message during mitosis, passing only
crest cells migrate into the space between the the new copy onto TACs.
lens and epithelium. These cells become the cor- 2) Poor differentiation: It has been empha-
neal endothelium and the stromal keratocytes. sized that the concept of ‘‘stemness’’ excludes
Corneal development continues gradually until further differentiation as a necessary property.
the time of eyelid opening, which is associated Therefore, it has long been recognized that the
with major developmental changes.7 cytoplasm of stem cells appears primitive and
At the time of birth, approximately 20% of contains few, if any, differentiation products. If
epithelial and stromal cells and 12% of endo- differentiation is envisioned as reprogramming
thelial cells are actively progressing through of the genome, then the process of differen-
the cell cycle. By the time of eyelid opening, the tiation also means removal of cells from the
number of cells actively proliferating in both stem cell population. Therefore, a stem cell
the stroma and endothelium has decreased to having responded to a differentiation stimulus
nearly zero. This low level of proliferation will is out of the stem cell population. Two possible
be maintained throughout life. In contrast, the mechanisms can explain how a differentiation
level of proliferation increases noticeably in the event is induced. First, a full mitotic cell cycle is
corneal epithelium and peaks after eyelid needed to produce an asymmetrical cell divi-
opening with almost 75% of the basal cells sion into two different daughter cells. One will
actively proliferating. The burst of proliferation remain a stem cell (self-renewal), while the
correlates well with the stratification of the other is destined for cellular differentiation.
epithelium.8 Second, a full cell cycle may not occur; instead,
differentiation stimuli affect stem cells at the G0
STEM CELLS IN THE EYE state, in which most stem cells are during
steady state growth.10,11 Affected cells will be
Several unique inherent properties, discussed removed from the stem cell population by vir-
kers20 demonstrated that basal cells of the sub-population of the total tissue and have
limbal epithelium are normally slow cycling in been estimated to make up from 0.5% or less to
nature, but can be made to cycle much more 10% of the total cell population located in
rapidly when the central corneal epithelium is palisades of Vogt (Fig. 1).7,25,26
damaged.
3) Immunohistochemical studies: Initial THE LIMBUS AS LIMBAL STEM CELL NICHE
immunohistochemical data suggested that lim-
bal basal cells have the lowest level of differen- What maintains the stemness of a stem cell is
tiation among all corneal epithelial cells. Kera- not well understood. In addition to charac-
tins are a group of water-insoluble cytoskeletal teristics inherent to stem cells, extrinsic influ-
proteins that form the desmosome-associated ences from surrounding microenvironment
10-nm intermediate filament in almost all epi- may also play a role.27 Schofield28 suggested for
thelia.21 Perhaps the most important keratin the first time that this property of stem cells is
type for ocular surface epithelia is the K3-K12 maintained by extrinsic factors in their mic-
pair, which is synthesized by corneal and some roenvironment. Upon stem cell division, one
oral mucosal epithelium but only in small daughter cell remains a stem cell and returns to
amounts in conjunctival epithelia.19,22 the niche to replenish the stem cell pool while
4) High proliferative capacity of limbal the other becomes a TAC that will eventually
epithelium in cultures: Limbal basal epithelial terminally differentiate.29 The microenviron-
cells have higher proliferative potential in cell ment of the limbus differs from that of the
cultures than central and peripheral corneal cornea; one of the most striking differences is
epithelial cells. Limbal basal cells respond to the presence of blood supply at the limbus. The
central corneal wounds and to tumor-promo- blood vessels form an undulating network in
ting agents by undergoing greater proliferation the palisades of Vogt. This arrangement allows
than central corneal epithelial cells, which ter- close approximation between blood vessels and
minate proliferation-initiating differentiation.14 the epithelium, potentially providing increased
5) Corneal conjunctivalization: When the levels of nutrients and blood-borne cytokines to
limbal epithelium is partially or completely re- the cells at the limbus.1
moved, a spectrum of corneal surface abnor- The limbal basement membrane differs
malities occur which are characterized by con- from that of the cornea in that it undulates with
junctival epithelial ingrowth (conjunctivaliza- pegs of stroma extending upward interconnec-
tion), vascularization, and chronic inflamma- ted with anchoring fibrils linked to the base-
tion, which can be explained by limbal stem cell ment membrane. This could provide resident
deficiency.23 stem cells with an adherence niche, protecting
6) Corneal epithelial neoplasm: The limbal them from injury and movement within their
location of corneal epithelial stem cells could microenvironment. Corneal basement mem-
account for the relative preponderance of lim- brane is composed of collagen type IV (α3,
bal neoplasms and the scarcity of corneal epi- α4, and α5 chains), collagen type VII, laminin
thelial tumors.3 (Ln-α1, Ln-α3, Ln-β1, Ln-β3, Ln-γ1 and Lnγ2
7) Corneal regeneration: A mathematical chains), fibronectin, and heparan sulfate pro-
analysis of the kinetics of maintenance of cor- teoglycans. Limbal basement membrane po-
neal epithelial mass confirms that the corneal ssesses additional laminin α2 and β2 chains
epithelium can be maintained by centripetal together with α1 and α2 chains of collagen type
migration of epithelial cells originating from IV but no collagen type XII and collagen type
the limbus without contribution by adjacent IV (α3, α4, and α5 chains). Although not pro-
conjunctiva.24 ven, the differences in the composition of the
The above sources of evidence point to the basement membrane in limbal and corneal epi-
1.5-2 mm wide area of basal layer in the limbal thelium might be at least partially responsible
epithelium as the region harboring stem cells for different cell phenotypes and proliferative
for corneal epithelium. These cells are a small behaviors of these two distinct cell poulations.29
Future studies are needed to clarify the role of Transient Amplifying Cells (TACs)
stromal microenvironment in the regulation of
stem cell function. It is most likely that limbal basal epithelium
Another difference between the limbus and consists not only of stem cells but also of TACs
the cornea is the expression of several proteins which are located in the basal layer of the
at higher concentrations in the basal cells limbus and peripheral corneal epithelium. They
of limbal epithelium as compared to central have an important role in wound healing.29
corneal epithelium, such as cytochrome oxi- TACs in the limbus and peripheral cornea
dase,30,31 Na+/K+ ATPase and carbonic anhyd- display some characteristics of stem cells such
rase.26 It is not clear whether any of these pro- as a long life, slow cycling with low mitotic
teins is involved in the maintenance and regu- activity, and less differentiation in the normal
lation of stem cells. There seem to be regional steady state. In contrast, TACs in the cornea
differences in the distribution and concentra- have a short lifespan, are rapid cycling, and can
tion of various regulatory factors, such as reti- amplify cell mass effectively through limited
noic acid in human limbal and corneal epithe- rounds of mitosis. At a critical point, TACs stop
lium, as well as in the underlying stroma and mitosis and differentiate into corneal supra-
fibroblasts26 which affect stemness properties. basal post-mitotic cells and terminally differen-
Other important differences include different tiated cells (TDCs).34,35 Although the exact me-
expressions of cytokines in the limbus and cor- chanism remains unknown, many studies have
nea. Three types of cytokines have been deter- indicated that the control of mitotic kinetics in
mined in limbus and cornea: TACs for limbal stem cells is different from that
• Type I cytokines: These include interleukin 1β, for corneal cells. The presence of TACs in the
which is produced by both limbal and corneal limbus and cornea provides advantages inclu-
fibroblasts and is increased following injury ding: (1) amplifying each stem cell division and
to the corneal epithelium. minimizing the need for stem cell proliferation
• Type II cytokines: Including transforming and conserving stem cell energy, (2) mini-
growth factor β1, produced by both limbal mizing the chance for introducing replicative
and corneal fibroblasts. DNA errors into the stem cell population, and
• Type III cytokines: Keratinocyte growth factor (3) providing new cells that are much closer to
(KGF) and hepatocyte growth factor (HGF) the terminally differentiated functional cellular
are released by fibroblasts. KGF, pre- compartment, for example, the epithelium that
dominantly produced by limbal fibroblast, covers the central cornea.12
causes proliferation of limbal epithelial cells
and HGF, produced by corneal fibroblast, Terminally Differentiated Cells (TDCs) and
causes migration of epithelial cells onto the Post-Mitotic Cells (PMCs)
corneal surface.32,33
The division of TACs results in non-dividing
TYPES AND FUNCTIONS OF CORNEAL PMCs, which differentiate and migrate toward
EPITHELIAL CELLS the central cornea and specifically take on
the final corneal cell phenotype as TDCs. These
Stem Cells cells have no capacity for self-renewal and
no proliferative potential. In normal healthy
Stem cells are few in number but have high tissues they are continuously being shed and
capacity for self-renewal and large proliferative replaced because of their limited lifespan. They
potential. In healthy tissues, they have a ten- are located in the superficial part of the central
dency to remain quiescent. They divide and cornea and have many tight junctions.29 Figure
undergo differentiation to form TACs when 2 demonstrates cell contents of the limbal stem
stimulated and are located in the basal layer of cell niche.
the limbal epithelium.29
CORNEAL AND LIMBAL MARKERS in the epithelium but should also allow for iso-
lation, enrichment, and molecular characteriza-
The ideal stem cell marker should not only be tion of viable stem cells. To date several pu-
able to pinpoint the location of stem cells with- tative stem cell markers have been proposed,
2) Aldehyde dehydrogenase (ALDH) and major cytosolic proteins in the corneal epithe-
transketolase (TKT) have been identified as lium, but are completely absent (ALDH) or
only minimally expressed (TKT) in the murine known to be involved in tumor suppression
limbus.41,42 The corneal zone expresses a dram- and morphogenesis, and belongs to a family
atic accumulation of large quantities of globular that includes p53 and p73.55,56 P63 is consistent-
metabolic enzymes, in particular ALDH and ly expressed in basal cells of stratified epithelia
TKT.43 The expression of these enzymes may be on the nucleous, and is essential for epithelial
correlated with corneal transparency and its development and differentiation.57 Originally,
refractive properties. In the rabbit, the copious epithelial cells expressing p63 were found
expression of ALDH class 1 is limited to the specifically in the basal layer of the limbus but
corneal domain.44 The magnitude of changes not in the corneal epithelium in humans.55
in phenotype at the limbo-corneal demarcation 6) Cytokeratines (CK) together with micro-
has also been suggested to be due to differences filaments and microtubules form the cytoskele-
in shape, size and intracellular complexity of ton of all vertebrate cells.58 There are a total of
basal cells in both domains in rodents and approximately 30 keratins that can be divided
humans.45 into an acidic (type I) and neutral-to-basic (type
3) Lectin staining of corneal sections has II) subfamilies. It is notable that each basic
yielded unique insights to changes occurring keratin tends to co-express with a particular
within basal cells in their transition across the acidic keratin, forming a so-called keratin pair.
limbal-corneal margin in the rabbit.46,47 Limbal In addition, each keratin pair tends to be ex-
epithelial cells have been shown to express on pressed in a tissue-restricted and differentia-
their cell surface unsialylated galactose resi- tion-dependent fashion.16 Using a monoclonal
dues that are recognized by peanut lectin and antibody (AE5) it was demonstrated that K3 is
lack any sialic acid bound through α-2,3 bonds. expressed suprabasally in limbal epithelium
Differentiation of the cells causes sialylation of but uniformly in the central corneal epithelium
these residues and the appearance of α-2,3 sialic (Fig. 6). This finding implies that the K3-posi-
acid residues, suggest the expression or activa- tive basal cells in the central corneal epithelium
tion of α-2,3-sialyltransferase. may have attained a more advanced state of
4) Gap junctions are communicating cell- differentiation than the K3-negative basal cells
cell junctions consisting of six transmembrane of the limbal epithelium. Also, immunohisto-
proteins called connexins (Cx). The gap junc- chemistry has demonstrated that cytokeratine
tion channels allow diffusion of ions, low mo- CK3/12 identifies more differentiated cells in
lecular weight metabolites and second messen- the corneal epithelium. This CK dimmer is
gers between cells and thus, determine the ex- therefore absent in limbal stem cells and early
tent of cell metabolic synchrony or cooperation TACs in the limbal epithelium.19
within a population (Fig. 3 A, B).48-50 The pre- 7) PAX6 plays critical roles in mature
sence of Cx26, Cx30, Cx43 and Cx50 gap junc- tissue.59 In humans, heterozygous loss of PAX6
tions have been confirmed at the protein level function results in multiple ocular develop-
by immunohistology in the corneal epithelium mental defects, the major one being aniridia.7
of the human, rat and rabbit.51,52 Cx43 and Cx50 The corneal epithelium of PAX6 +/+/PAX6 -/-
are abundantly expressed in the corneal epi- mouse chimeras, exhibits a decrease in the ex-
thelium with Cx50 being expressed throughout pression of adherence proteins and keratin K12,
all layers and Cx43 being mainly confined to and shows conjunctival invasion consistent
the basal cell layer (Fig. 4).37,47 In contrast, both with limbal cell deficiency.60,61 Eventually,
connexins were absent at the limbal basal layer PAX6 -/- cells are excluded from the corneal
in human, mouse, chicken, and neonatal rabbit epithelium, pointing to the acute dependence
eyes, whereas suprabasal limbal cells showed of the phenotype on PAX6 expression.62 Basal
slightly positive membranous staining (Fig. 3 cells in the limbal epithelium of the rat eye
C).49,51,53,54 have been reported to express the transcription
5) P63, a transcription factor expressed in factor PAX6, which is present in the developing
the nucleus, has been suggested as a new mar- central nervous system and plays a central
ker for limbal stem cells (Fig. 5). This protein is role in eye development.63 PAX6 has also
been shown to be strongly expressed in the monkey eyes, and might be necessary for the
nuclei of all cells within the corneal, limbal, and maintenance or proliferation of corneal stem
conjunctival epithelia of adult mouse and cells.64
Microvillus
A B C
Tight Junction
Adherence
Junction
Gap Junction
Desmosome
Figure 3 Types of cell junctions (A), electron-microscopic location of cell junctions in cells (B),48-50 and immuno-
staining of connexin-43 in limbal stem cell culture (C).54
limbus. In contrast, basal cells of human ocular some basal epithelial cells in the human
surface epithelia showed preferential mem- limbus.70,71
brane associated immunoreactivity for P-cad-
herin with small clusters of negative cells FACTORS REGULATING LIMBAL STEM
among positive cells in the limbal basal epi- CELL PROLIFERATION
thelium (Fig. 7). Beta-catenin is a central com-
ponent of the cadherin cell adhesion complex Many regulatory factors affect the proliferation
and, as an essential molecule in the Wnt sig- of limbal stem cells in both fetal and adult life.
naling pathway, a key regulator of epithelial 1) Growth factor receptors (GFRs): GFRs
differentiation and proliferation. Therefore, it consists of epithelial (EGFR), keratinocyte
has been shown to be essential for the main- (KGFR) and hepatocyte (HGFR) which prefer-
tenance of keratinocyte stem cells.68 In rat and entially localize to cell membranes of limbal
rabbit corneas, β-catenin was strongly positive basal cells. Undifferentiated cells in the limbal
in the basal layer of the limbal epithelium, but basal epithelium have been originally reported
relatively weakly positive in the basal layer of to contain higher levels of GFRs than supra-
the cornea.69 basal cells in the rat cornea (Fig. 9).9,72,73 Al-
9) Integrins: Immunohistochemical studies though a strong expression of GFRs in limbal
have identified several subunits of integrins, basal cells was also confirmed in human cor-
such as integrins α2, α3, α4, α5, α6, and αv as neas, there was no clear difference in staining
well as β1, β4, and β5 to be present in the intensity between the basal cells of the limbus,
human corneal epithelium, whereas integrins cornea, and conjunctiva.40,74 It has been sug-
α1 and β3 were not detected.35 Integrins β1, β4, gested that high levels of GFRs may inhibit
α2, α3, α6, and αv were mainly expressed in the differentiation by signaling the cells to main-
basal epithelial cell layer; integrins α6 and β4, tain their proliferation potential.73
as components of hemidesmosomes, were lo- 2) ABCG2 is a member of the ATP binding
calized specifically to the basal membrane of cassette (ABC) transporters, localizes predomi-
basal cells (Fig. 8). Some integrins have been nantly to the plasma membrane and has been
suggested to be markers for epidermal stem proposed as a universal and conserved marker
cells, such as integrins β1 and α6. Integrin β1 for stem cells from a wide variety of tissues.75,76
was abundantly expressed in corneal and lim- ABCG2, also known as breast cancer resistant
bal epithelia with a much higher levels of ex- protein 1 (BCRP1), causes resistance to certain
pression in limbal basal versus limbal supra- chemotherapeutic drugs. Immunolocalization
basal cells.70 These observations suggest that experiments have consistently demonstrated
limbal basal cells deficient in Cx43, P-cadherin, ABCG2-positive cells in basal77 and also fre-
and integrins α2, α3, α6, and β4 are thought to quently in suprabasal layers of the limbal epi-
represent corneal stem cells. The lack of inter- thelium,70 but not in the corneal epithelium
cellular communication (connexins) and adhe- (Fig. 9).
sion molecules (cadherins, integrins) may be an
inherent feature of limbal stem cells reflecting LIMBAL STEM CELL DEFICIENCY (LSCD)
their need for independence and the unique-
ness of their microenvironment. LSCD may occur due to a deficient stromal
10) Transferring receptors: Different ex- microenvironment supporting the stem cells,
pressions of the transferring receptor CD71 such as aniridia, congenital erythrokeratoder-
have been observed in limbal and corneal epi- mia, keratitis associated with multiple endo-
thelia. In contrast to basal cells in the corneal crine deficiencies, neurotrophic keratopathy
epithelium which stain intensely with an anti- and chronic limbitis; or more commonly follo-
CD71 antibody, the limbal basal cells are nega- wing external insults which destroy the limbal
tive.35 However, in a more recent study, anti- stem cells such as chemical or thermal injuries,
CD71 antibody stained not only the cell mem- Stevens-Johnson syndrome (SJS), ocular cicat-
branes of most corneal epithelial cells but also ricial pemphigoid (OCP), multiple surgeries or
blems, management of dry eye and control of vested with a carrier which may be conjunctiva
systemic diseases.89 (conjunctival-limbal grafts) or cornea (Kerato-
Theoretically it is possible to restore stem limbal grafts)90 A variety of methods have been
cell function by expanding the stem cell popu- employed for preparing transplant sheets, e.g. a
lation through modulations in the microen- sheet of corneal epithelial cells alone, a sheet of
vironment, or by inducing TACs mitosis with corneal epithelial cells cultured on a polymer
the use of appropriate growth factors.79 Limbal substrate, collagen or fibrin,91 or on biomaterial
stem cells may be obtained from the fellow such as amniotic membrane.92,93 Surgical pro-
eye (autograft), a cadaver (allograft) or a living cedures used for treatment of partial or total
relative (allograft). Limbal stem cells are har- limbal stem cell deficiency are discussed below.
Amniotic Membrane Transplantation surface in patients with severe dry eye caused
by OCP and SJS. It has been reported that AMT
The human amniotic membrane is the inner- alone is sufficient to restore the corneal surface
most layer of the placenta. Histologically the in eyes with partial LSCD, suggesting that
amnion is 0.02 mm to 0.5 mm in thickness, com- AMT may help expand the remaining limbal
posed of three basic layers; the epithelial mono- epithelial stem cells in vivo.99,100
layer, the thick basement membrane and the Studies have shown that the amniotic
avascular hypocellular stromal matrix.94 The membrane contains high levels of EGF, KGF,
structural integrity, transparency and elasticity HGF, TGF (tumor growth factor), and bFGF
of amniotic basement membrane make it cur- (basic fibroblast growth factor) which are po-
rently the most widely accepted tissue replace- tentially involved in epithelial–stromal inter-
ment for ocular surface reconstruction. Am- actions of the human ocular surface including
niotic membrane is known to promote epithe- epithelialization, and modulation of prolifera-
lial cell migration, adhesion and differentia- tion and differentiation of stromal fibroblasts.101
tion. It is an ideal substrate for supporting the Therefore the amniotic epithelium might pro-
growth of epithelial progenitor cells by pro- vide cytokines, which play a crucial role in the
longing their lifespan, maintaining their cloni- microenvironmental niche of limbal progenitor
genicity and preventing epithelial cell apop- cells. In addition, the basement membrane of
tosis (Table 3).95,96 the amniotic membrane contains types IV, V,
Amniotic membrane transplantation (AMT) and VII collagen, Ln1, Ln5, and fibronectin that
was initially reported for corneal surface recon- play an important role in corneal epithelial cell
struction in a rabbit model of total limbal de- adhesion and migration.94 The stromal matrix
ficiency.97 Tsubota et al98 have used this tech- also suppresses the expression of certain in-
nique, combined with allograft limbal trans- flammatory cytokines that originate from ocu-
plantation, to effectively reconstruct the corneal lar surface epithelia, including interleukin 1α
(IL-1α), IL-2, IL-8, interferon γ, tumor necrosis tory cells infiltrating the ocular surface and
factor-β, basic fibroblast growth factor, and contains various forms of protease inhibitors
platelet derived growth factor.102 The amniotic which explain some of its anti-inflammatory
membrane attracts and sequesters inflamma- properties.103
Table 3 Amniotic membrane (AM) characteristics mimicking stem cell niche and promoting stem cell expansion96
AM characteristics Potential mechanism for ex vivo expansion of limbal stem cells
Amniotic epithelium
Cytokines (major: EGF, KGF, HGF, Prevents early contact with ECM-components
bFGF, NGF, and minor: TGF-α,
TGF-β1, TGF,β2)
Tissue inhibitors of metalloproteinases Provides cytokines affecting the cell cycle and cell survival
Thrombospondin-1
Amniotic basement memrane
Collagens IV (α chain), and VII Facilitates cell migration and ECM adhesion
Laminin 1 and 5
Fibronectin Triggers signaling pathways through integrins
Amniotic stroma
Cytokines (major: NGF, HGF, KGF, and Provides a non-inflamed microenvironment
minor: TGF-α, TGF-β1+2, EGF, bFGF)
Tissue inhibitors of metalloproteinases Provides cytokines for major signaling pathways known to be
Thrombospondin-1 involved in stromal and limbal epithelial communication
EGF, epithelial growth factor; KGF, keratinocyte growth factor; HGF, hepatocyte growth factor; bFGF, basic fibroblast growth
factor; NGF, nerve growth factor; TGF, tumor growth factor; ECM, extracellular matrix.
This technique was first introduced by Kenyon substrate for cultivating limbal epithelium and
and Tseng in 1989, and subsequently by many the third combines the mentioned methods.
others for treating patients with focal or uni- The original method of culturing limbal
lateral LSCD in different clinical settings. epithelium on amniotic membrane involves
Donor tissue can be harvested from the heal- taking a small limbal biopsy (approximately 1
thy fellow eye (conjunctival limbal autograft), mm) or explants, and culturing it in the center
from a living related donor (conjunctival limbal of the amniotic membrane (Fig. 11). The ex vivo
allograft) or from a cadaver eye (keratolimbal expanded limbal epithelium grows out from
allograft).110-112 the explant onto the amniotic membrane. When
Limbal grafting involves transplantation of the amniotic membrane is sufficiently covered
large pieces of healthy limbus from the donor. by ex vivo expanded limbal cells, it is trans-
Each of the varieties of the technique has draw- planted to the eye with LSCD (Fig. 12).117 Am-
backs; in the case of autografting and allo- niotic membrane promotes epithelialization,118
grafting from living related donors, there is a reduces inflammation and scarring,119 pre-
limit to the amount of limbal tissue that can be serves and maintains existing limbal stem cells,
harvested, due to the risk of producing iatro- and serves as a natural substrate on which
genic LSCD in the donor eye.23 Allografts from limbal stem cells can grow and proliferate. It
living related or cadaveric donors entail the also enables easier handling of the cultured
risk of tissue rejection and their survival de- limbal stem cells. It is believed that the am-
pends on aggressive systemic immunosupp- niotic membrane may act as a barrier to im-
ression113,114 which is associated with signi- mune cells, diminishing the immune response
ficant morbidity and reduction in quality of life. by inhibiting IL-1β and IL8 expression, and
may also produce anti-angiogenic proteins.119
Ex Vivo Limbal Stem Cell Transplantation Fibrin substrate has also been used to
culture limbal stem cells.91 The culture system
Recently, with the increased knowledge on may be maintained for 14-28 days and either
stem cell biology, techniques have been deve- transferred to the recipient bed or subjected to
loped to expand small biopsies of limbal tissue air-lifting in order to promote epithelial tight
in culture for subsequent transplantation, this junction formation and stratification.120
overcomes some of the main hurdles entailed
by whole limbal tissue transplants.115 The con- CONCLUSION
cept of culturing stem cells was derived from
the use of cultured human epidermal cells as The concept of limbal stem cells has greatly im-
autologous grafts in patients with burns and in proved our understanding of corneal epithelial
plastic and reconstructive surgery.116 Currently, proliferation, migration, and regeneration. This
culturing corneal epithelial stem cells is the has also contributed directly to improved me-
most exciting and promising technique in lim- dical and surgical management of a wide range
bal transplantation. It is possible to culture of ocular surface disorders. Many questions
stem cells using a small amount of tissue there- however remain. Clinically, the most important
by minimizing damage to the donor and dep- is the issue of limbal allograft rejection and the
letion of its stem cell reserve. With this tech- long term survival of limbal transplants and
nique only epithelial cells (not Langerhans’ that of improving immunosuppressive regi-
cells and blood vessels) are transplanted, mens. In terms of stem cell biology, unans-
therefore theoretically reducing the possibility wered questions include: How is “stemness” of
of rejection. stem cells maintained? Which factors regulate
There are 3 main techniques for culturing the asymmetric division of stem cells? What are
limbal epithelium. The first involves the co- the external and internal modulators influ-
culture of limbal epithelium with mitotically in- encing stem cells? What is the role of the
activated 3T3 mouse fibroblasts, the second en- microenvironment in stem cell function and
tails the use of human amniotic membrane as a regulation?
cytometry. Invest Ophthalmol Vis Sci 2003;44:5125- 60. Davis J, Duncan MK, Robison WG Jr, Piatigorsky J.
5129. Requirement for Pax6 in corneal morphogenesis: a
46. Wolosin JM, Wang Y. Alpha-2,3 sialylation role in adhesion. J Cell Sci 2003;116:2157-2167.
differentiate the limbal and corneal epithelial cell 61. Ramaesh K, Dhillon B. Ex vivo expansion of
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