To further explore the cell population derived with our culture system,
we transplanted expanded HLE on AM as a composite subcutaneous graft in
immunocompromised mice to promote stratification and differentiation.
In addition to the nude gene, which results in an absence of
thymus-derived T-cells, these mice have two other mutations important
in regulating the function of the immune system. They are X-linked
immune defect (xid), which affects the maturation of T-independent
B-lymphocytes, and beige (bg), in which the homozygote is devoid of
natural killer cells that are cytotoxic to tumor cells in vitro. In
both conditions a nicely stratified epithelium was found with a
relatively small and compact basal cell layer. The presence of the
devitalized amniotic epithelium could not be discerned underneath the
stratified HLE any longer, suggesting their partial or complete
disintegration, as proposed by others.
18 Our results
showed that the basal layer of the resultant stratified epithelium on
intact AM did not express Cx43, in contrast to HLE on EDTA-treated AM
(Figs. 6C 6D) . In addition, we found that keratin K3 was absent in the
basal layer on intact AM, whereas the basal layer in the EDTA-treated
group expressed keratin K3
(Figs. 6E 6F) . Moreover, BrdU
label-retaining nuclei were found in the basal layer of the epithelium
on intact AM, but not at all in EDTA-treated AM cultures
(Figs. 6G 6H) . That the amniotic epithelium and fibroblasts are devitalized and
do not have any proliferative activity after the present method of
preservation
18 shows that the basal BrdU-labeled cells are
derived from HLE. Collectively, these data indicate that a rapid cell
cycle and positive expression of Cx43 and keratin K3 were promoted when
HLE grew directly on the amniotic basement membrane, strongly
supporting the notion that this culture condition promotes TAC
differentiation. This interpretation, however, cannot be extrapolated
to the method used by Koizumi et al.
15 22 in which 3T3
fibroblasts feeder layers are routinely included. Nevertheless, in
another report,
13 they mentioned that corneal
differentiation is promoted, but failed to provide evidence that limbal
epithelial progenitor cells are actually preserved. Future studies are
needed to show that denudation of the amniotic epithelium to expose the
amniotic basement membrane is an important microenvironmental “cue”
in promoting TAC differentiation. This hypothesis can be tested by
transplanting ex vivo expanded HLE in a rabbit model of limbal SC
deficiency that we have recently reported.
23 If this were
the case, we may understand why migration of the offspring of limbal
SCs to the corneal basement membrane signifies the beginning of TAC
differentiation. Future studies should also be extended to
investigating which amniotic basement membrane component(s)—laminin-1,
laminin-5, collagen VII, and fibronectin
24 —is responsible
for upregulation of Cx43 expression and thus, TAC differentiation. In
this regard, previous studies have shown that rat hepatocyte
cultures
25 26 and human epidermal keratinocyte
cultures
27 show an increase in gap junction synthesis and
GJIC when exposed to certain extracellular matrix components (e.g.,
glycosaminoglycans, proteoglycans, and laminin-5).