Our results suggest that during long-time culture,
mesothelial cells downregulate specific sets of genes that are part of their
gene signature. Importantly, during the early phase of the culture period they
exhibit certain characteristics of stemness. However, it is questionable
whether the downregulation of gene expression observed during long-term culture
can be described as dedifferentiation in the true sense since dedifferentiation
is defined as the change of a differentiated cell to a stem or progenitor cell
. Thus, further analyses are required to determine the molecular mechanisms
that regulate the differences between human and mouse mesothelial cells in
culture, and the changes observed during the earlier and later passages.
Understanding these mechanisms will allow the development of specific culture
conditions and manipulations in order to maintain a status of stem or
progenitor characteristics in long-term cultured mesothelial cells, with the view
to exploring their potential as regenerative medicine therapies.
Mesothelial cells also mediate inflammation through the local synthesis of
hyaluronan (Yung and Chan, 2009, 2012), which is able to sequester free
radicals and initiate tissue repair responses (Yung et al., 1994, 1996, 2000;
Yung and Chan, 2007). Synthesis of hyaluronan fragments are increased by
exposure to IL-1β, IL - 6, TNF-α, TGF-β1, and platelet-derived growth factor
(PDGF; Yung et al., 1996) and can activate the inflammatory cascade in
mesothelial cells by inducing IL-8 and MCP-1 production via activation of the
NF-κB signaling pathway (Haslinger et al., 2001). In the peritoneum, induction
of these inflammatory cytokines by long-term exposure to peritoneal dialysis
(PD) fluid may promote the development of chronic peritoneal inflammation,
leading to long-term peritoneal damage and exacerbation of the fibrotic
pathway.
Although the primary target cell for pleural fibrosis is thought to be the
subpleural fibroblast, studies have shown the importance of mesothelial cells
in the pleural fibrotic response. A number of agents can induce fibrosis,
including infection, radiation, and inorganic particles such as talc and
asbestos (Dail and Hammar, 1994; Rom, 1998a,b). It is unclear how asbestos
fibers induce subpleural fibroblasts and mesothelial cells to synthesize
collagen but it is likely to be through the generation of cytokines, growth
factors, and reactive oxygen species (ROS). ROS are cytotoxic and can stimulate
fibroblasts to synthesize ECM components (Kamp and Weitzman, 1999) as well as
induce expression of genes for profibrotic mediators such as TGF-β and TNF-α
(Massague, 1996).
Unless specified otherwise in the datasheet, primary cell lines may be
re-freezed after thawing. However, primary cells are usually not good for
multiple passaging and they usually last only for up to 10-12 population
doublings. Our primary cells are usually provided at passage 2, therefore they
can be passaged for an additional 1-2 passages. For long term use, immortalized
cells are preferred.
DiscussionIn this study we have analysed the stemness and differentiation
potential of omentum-derived mouse mesothelial cells. We demonstrate the
successful long-term culture of primary mesothelial cells. However, in response
to multiple passaging, the cells shifted their expression profile, becoming
less epithelial. At early passages, cultured mesothelial cells displayed stem
or progenitor cell characteristics, as evidenced by the fact that they
exhibited clonogenic potential, expressed stem cell markers, and showed
differentiation along the osteogenic and adipogenic lineage. Furthermore, by
making use of a chimeric embryonic kidney rudiment assay, we demonstrate that
unlike MSCs, mesothelial cells do not have any noticeable adverse effects on
the ex vivo development of mouse kidney rudiments. We have previously shown that
culture of tissue explants isolated from mouse adult omentum led to the
outgrowth of mesothelial cells with typical mesothelial characteristics .
Here,
we report the long-term culture of several individually isolated mesothelial
cell cultures from mouse adult omentum for over 30 passages without evidence of
senescence. Population doubling times stabilised to around 24 hours between
passage 8 and 16. This is in contrast to human omentum-derived mesothelial
cells which have been reported to undergo premature senescence .
Characterization of the MC cultures at different passages revealed that while
the overall morphology of the cells remained similar, the molecular signature
of the cells changed (S5 Fig). In particular, mRNA levels of the key
mesothelial marker Wt1 were downregulated, but nevertheless, Wt1 protein could
be clearly identified by immunofluorescence in the nuclei of low and high
passage cells. By contrast, Msln was detectable in cells at both low and high
passages using immunofluorescence, with mRNA levels increasing significantly
with higher passages. Thus, the two mesothelial markers used in our analysis
showed opposing responses to passaging. Loss of Wt1 in the epicardium has
recently been shown not to affect expression of Msln , suggesting that the
significant increase in Msln expression we have observed is independent of Wt1
expression levels in cultured MCs. Mesothelial cells express both epithelial
and mesenchymal markers in vivo and have the ability to transdifferentiate into
myofibroblasts in response to stress or injury, through a process called
mesothelial-to-mesenchymal transition (MMT) .
Specifically, loss of Wt1 has
been reported to induce transdifferentiation of human pleural mesothelial cells
into myofibroblastic cells, suggesting that Wt1 is required for the maintenance
of mesothelial homeostasis . This is not surprising given that Wt1 has been
shown to be involved in the regulation of EMT and MET processes . The
downregulation of Cdh1 and Krt8 we observed in this study could therefore be
linked to the reduction in Wt1 expression in MCs over passages. Nevertheless,
our results suggest that long-term cultured mouse MCs remained in a status
whereby epithelial markers are lost only partially since ZO1 was still
detectable in a robust punctate pattern around the cell perimeter, and
mesenchymal markers were not significantly upregulated. Because of their
ability to proliferate over many passages, we tested the mouse mesothelial
cells for clonogenic potential. In contrast to kidney stem cells (KSCs)
isolated from mouse newborn kidneys which are clonogenic but give rise to
clonal lines with different renal phenotypes , mesothelial cells showed
properties similar to mesenchymal stem cells (MSCs), since MSCs are able to
generate clones with characteristics of the parent cell type only . The
capacity to differentiate mesenchymal stem cells towards the mesodermal
lineages, specifically adipocytes and osteocytes, has been established as an
important parameter of their stemness .
This has been exploited in studies to
demonstrate that rat and human mesothelial cells have the potential to
differentiate towards the adipogenic and osteogenic lineage . Following a
similar approach, we could show that upon appropriate stimulation, mouse
mesothelial cells adopted phenotypes and expressed genes that are indicative of
differentiation steps of adipogenesis and osteogenesis. Our analysis revealed
that the differentiation potential for osteogenesis is retained in cells up to
passage 13, while cells of higher passage failed to significantly respond.
Similarly, MCs of passage 5 could differentiate towards an adipogenic fate,
while this differentiation potential was reduced in MCs of higher passages. The
decline in the differentiation potential of long-term cultured mesothelial
cells could be reflected in the changes in expression levels of some of the
stem cell markers analysed. Therefore, our results suggest that the
differentiation potential, and in effect stemness of the mesothelial cells,
could only be maintained for a limited time under the culture conditions we
used. Since mesothelial cells showed evidence of stemness and differentiation
potential, we asked whether they had the potential to respond to a nephrogenic
environment by differentiating into kidney cells. However, since mesothelial
cells share some characteristics with MSCs, it is possible that they would have
a negative effect on ex vivo nephron development similarly to MSCs . A recently
developed ex vivo embryonic kidney rudiment assay lends itself to address these
questions since the experimental procedure involves the dissociation of
embryonic kidneys into single cells. Exogenous embryonic, adult or stem cells
are then mixed in with the embryonic kidney suspension before culture as
pelleted chimeric rudiment . Labelling the exogenous cells with lentiviral GFP,
Quantum Dots or fluorescent vital dyes allows their identification in the
chimeric rudiments, in order to determine whether cell integration into the
developing rudiment and furthermore, contribution to nephron structures has
taken place.
Using this approach, several studies have now demonstrated that
stem cells from various sources have the capacity to integrate into chimeric
kidney rudiments. In some cases this involves the contribution of exogenous
cells to developing glomeruli, comma- and S-shaped bodies . Here, we
demonstrate that mouse mesothelial cells localise inside the chimeric
rudiments, without disrupting the development of the overall kidney rudiments
and their structures. This is in clear contrast to mesenchymal stem cells,
which, despite expressing some of the key regulators of kidney development,
have been shown to disrupt nephron formation in the chimeric rudiments,
indicating that not all stem cells have the capacity to support and interact
with the developing nephron structures . Within the chimeric rudiments,
mesothelial cells were occasionally found in the nephrogenic mesenchyme, where
they showed co-expression with the nephron progenitor regulators Pax2 and Wt1
by immunofluorescence. Interestingly, Pax2 expression could not be detected in
the FACS-sorted MCs after co-culture in the chimeric rudiments, suggesting that
the number of Pax2+ MCs was very small. We also noted that mesothelial cells
aligned robustly with the basement membranes of the forming proximal tubules.
Overall, the heterogeneous distribution of MCs surrounding nascent glomeruli
and tubular structures was reflected in the up-regulation of a range of mostly
mesenchymal or EMT markers in the MCs sorted from the chimeric rudiments after
7 days of culture. Because the mesothelial cells used for the chimeric kidney
rudiment assays had been of higher passages (P22-32) due to the lentvirus
transduction protocol employed followed by expansion and FAC sorting of the
cells, it is possible that the MCGFP+ cells had reached a stage in the
long-term culture where their peak differentiation potential had been passed.
Therefore, we cannot exclude the possibility that mesothelial cells of earlier
passages would have shown a more nephrogenic differentiation response. We
conclude that clonogenicity, stem cell marker expression and differentiation
capacity observed in mesothelial cells up to passage 10–13, together provide
evidence for stem or progenitor cell characteristics in cultured mouse
mesothelial cells. This finding is in contrast to a previous report that
mesothelial cells isolated from human pericardial fluid and rat omentum could
display stem cell characteristics only up to passage 3 . Our results suggest
that during long-time culture, mesothelial cells downregulate specific sets of
genes that are part of their gene signature. Importantly, during the early
phase of the culture period they exhibit certain characteristics of stemness.
However, it is questionable whether the downregulation of gene expression
observed during long-term culture can be described as dedifferentiation in the
true sense since dedifferentiation is defined as the change of a differentiated
cell to a stem or progenitor cell . Thus, further analyses are required to
determine the molecular mechanisms that regulate the differences between human
and mouse mesothelial cells in culture, and the changes observed during the
earlier and later passages. Understanding these mechanisms will allow the
development of specific culture conditions and manipulations in order to
maintain a status of stem or progenitor characteristics in long-term cultured
mesothelial cells, with the view to exploring their potential as regenerative
medicine therapies.
Post a Comment for "Mesothelial Cells"