Biomedical Engineering Reference
In-Depth Information
nuclei of keratinocytes in areas including the skin, cervix, prostate and cornea [
10
].
p63 was found to be highly expressed by limbal basal cells but also by young
transit amplifying cells [
11
]. It is therefore not regarded as a truly specific stem
cell marker but a marker of cells with proliferative potential. Many other markers
have been suggested and include integrins a9 and b1, importin 13, OCT4, cyto-
keratin 15, N-cadherin [
9
,
12
-
16
], however, all are disputed as true limbal stem
cell markers. The absence of cornea specific differentiation markers cytokeratins 3
and 12 [
17
,
18
] from the limbal basal epithelium indicates the basal cells are the
least differentiated of the corneal epithelial cells [
12
]. LESCs can also be identified
by their functional ability to form colonies, colony forming efficiency (CFE), when
plated at a low density on a mouse embryonic 3T3 fibroblast feeder layer
(Fig.
3.1
c). Colony forming cells are heterogeneous in their capacity for growth
and the growth potential of cells can be estimated from the colony type that results
[
19
]. Holoclones are large with a smooth perimeter and are usually formed of
small cells, which may concentrate at its perimeter. These cells have the highest
growth potential and are likely to represent the LESCs. Paraclones are small,
highly irregular and terminal and meroclones are typically in transition between
the two with a wrinkled appearance suggesting a level of heterogeneity within the
colony. Meroclones will eventually become terminal but reach a larger size than
the paraclones. The number of holoclones within a given population of cells can be
used to assess the percentage of stem-like cells within a population. As no
definitive marker or test for LESCs exists, a battery of stem and differentiated
markers and tests like the CFE assay must be used to characterise the phenotype of
any corneal and limbal cells under investigation, until a suitable marker is found.
Stem cell populations throughout the body have been found to reside in
specialised protective microenvironments, termed stem cell niches (reviewed in
[
20
-
22
]). Although stem cells exhibit intrinsic properties that influence their
behaviour, evidence suggests that cellular and extracellular components of the
niche contribute to the regulation of a stem cell population to sustain their pro-
liferative potential while at the same time preventing over-exuberant proliferation.
The niches are not only home to stem cells but also a diverse range of differen-
tiated cell types which secrete extracellular matrix components and soluble factors
to contribute to the maintenance of the neighbouring stem cell population.
The LESC niche specifically has distinct anatomy that is thought to provide a
physically protective environment for the stem cells. The limbal palisades of Vogt
are a series of radially orientated fibrovascular ridges that were presumed to be the
niche structures in the limbus [
23
]. However, it is now clear that the palisades are
only one element of a more complex niche system as two novel niche structures
have recently been described by Shortt et al. namely the limbal crypts and the focal
stromal projections [
24
]. The limbal crypts are distinct invaginations of epithelial
cells into the underlying stroma that extend from the peripheral cornea into the
limbus. Scanning electron microscopy reveals the crypts are polarised, opening
onto the central cornea as opposed to the conjunctival surface (Fig.
3.1
d and e).
Additionally, the surrounding stroma is highly cellularised with keratocytes and
there is a distinct vascular supply closely associated with the crypts. Dua and
