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The International Journal of Biochemistry & Cell Biology 36 (2004) 621642
Review
Mesothelial progenitor cells and their potentialin tissue engineering
Sarah E. Herricka,, Steven E. Mutsaers b
a School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UKb Asthma&Allergy Research Institute, Department of Medicine, University of Western Australia, Nedlands, Australia
Received 16 September 2003; received in revised form 3 November 2003; accepted 4 November 2003
Abstract
Themesothelium consists of a single layer of flattened mesothelial cells that lines serosal cavities and the majority of internal
organs, playing important roles in maintaining normal serosal integrity and function. A mesothelial stem cell has not been
identified, but evidence from numerous studies suggests that a progenitor mesothelial cell exists. Although mesothelial cells
are of a mesodermal origin, they express characteristics of both epithelial and mesenchymal phenotypes. In addition, following
injury, new mesothelium regenerates via centripetal ingrowth of cells from the wound edge and from a free-floating population
of cells present in the serosal fluid, the origin of which is currently unknown. Recent findings have shown that mesothelial
cells can undergo an epithelial to mesenchymal transition, and transform into myofibroblasts and possibly smooth muscle
cells, suggesting plasticity in nature. Further evidence for a mesothelial progenitor comes from tissue engineering applications
where mesothelial cells seeded onto tubular constructs have been used to generate vascular replacements and grafts to bridge
transected nerve fibres. These findings suggest that mesothelial cell progenitors are able to switch between different cell
phenotypes depending on the local environment. However, only by performing detailed investigations involving selective cell
isolation, clonal analysis together with cell labelling and tracking studies, will we begin to determine the true existence of a
mesothelial stem cell.
2003 Elsevier Ltd. All rights reserved.
Keywords: Peritoneum; Stem cells; Epithelial-mesenchymal transitions; Adhesions; Serosa
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
2. Embryology and morphology of mesothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
3. Functions of the mesothelial cell layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
4. Mesothelial healing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
5. Adhesion formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
6. Evidence for a multipotential subserosal mesenchymal cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
7. Epithelial-mesenchymal transition of mesothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629
Corresponding author. Tel.: +44-161-275-6765; fax: +44-161-275-5945.
E-mail address: [email protected] (S.E. Herrick).
1357-2725/$ see front matter 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2003.11.002
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8. Tissue engineering potential of mesothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
8.1. Vascular grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
8.2. Omental grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
8.3. Nerve grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
9. Does a mesothelial stem cell exist? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
10. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
1. Introduction
The mesothelium lines the peritoneal, pleural and
pericardial cavities with visceral and parietal surfaces
covering the internal organs and body wall, respec-
tively. It comprises a monolayer of epithelial-like
cells resting on a thin basement membrane supported
by sub-serosal connective tissue containing blood
vessels, lymphatics, resident inflammatory cells and
fibroblast-like cells (Wang, 1974; Ishihara et al., 1980;
Albertine, Wiener-Kronish, Roos, & Staub, 1982). The
sole function of the mesothelial layer was traditionally
thought to provide a protective, non-adhesive surface
to facilitate intracoelomic movement. However, it is
now recognised as a dynamic cellular membrane with
many physiological functions including the control
of fluid and solute transport, immune surveillanceand the production of extracellular matrix (ECM)
molecules, proteases, cytokines and growth factors.
The mesothelium is bathed in serosal fluid that re-
sembles an ultrafiltrate of plasma and contains blood
proteins, resident inflammatory cells, sugars and var-
ious enzymes including amylase and lactate dehydro-
genase (Dondelinger, Boverie, & Cornet, 1982). The
composition and volume of the serosal fluid is indica-
tive of certain pathological states, such as peritonitis,
tumorgenesis and endometriosis (Haney, 1993),and it
is likely that the mesothelial layer responds as a singleunit to changes in serosal fluid composition. Indeed,
repair of serosal tissue involves increased mesothelial
cell proliferation at sites distant to the wound, suggest-
ing diffuse activation of the mesothelium in response
to mediators or cells released into the serosal fluid, or
via cell to cell communication (Mutsaers, McAnulty
et al., 1997; Mutsaers, Whitaker, & Papadimitriou,
2002).
Although local proliferation of resident cells sur-
rounding a lesion is one source of healing cells, recent
reports suggest that the repair of many organs in the
adult organism also involves incorporation of multipo-
tential stem cells and as such, has generated exciting
prospects in cell and tissue engineering (Bianco &
Robey, 2001; Goodell, 2001; Tuan, Boland, & Tuli,
2003).A rich reservoir of these cells resides in specificniches within the bone marrow microenvironment as
well as in a variety of connective tissues where they are
maintained in an undifferentiated and quiescent state.
At present, there is a lack of a unifying definition that
characterises cells as stem cells. However, a general
definition is a cell capable of extensive self-renewal
that can give rise to successively more differentiated
progeny cells (Wagers, Christensen, & Weissman,
2002). Although a classic mesothelial stem cell has
not been identified, many lines of evidence suggest that
a mesothelial progenitor cell does exist. This reviewwill describe the mesothelial cell in terms of its embry-
ological origin, morphological characteristics and di-
verse functions. Subsequent sections present evidence
to support the concept of a free-floating mesothelial
progenitor cell present in serosal fluid and also dis-
cuss mesothelial cell differentiation, novel tissue engi-
neering applications for these cells and possible future
research directions in this rapidly developing field.
2. Embryology and morphology of mesothelial
cells
Bichart, in 1827 (reviewed by Whitaker, Papad-
imitriou, & Walters, 1982a)first observed that serous
cavities were lined by a layer of flattened cells similar
to those of the lymphatics.Minot (1890)subsequently
proposed the term mesothelium following a detailed
study of its embryological origin that showed this layer
to be the epithelial lining of mammalian mesoder-
mic cavities. It is now understood that during human
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development, the intraembryonic mesoderm on each
side of the neural groove differentiates into paraxial,
intermediate and lateral mesoderm. The lateral meso-
derm is continuous with the extraembryonic meso-derm covering the yolk sac and amnion. At the end of
week 3, small spaces appear in the lateral mesoderm
that fuse, dividing the mesoderm into two layers: the
intraembryonic somatic or parietal layer and the in-
traembryonic splanchnic or visceral layer. The somatic
mesoderm and overlying embryonic ectoderm form
the embryonic body wall (somatopleure), whereas the
splanchnic mesoderm and embryonic endoderm form
the embryonic gut wall (splanchnopleure). A contin-
uous mesothelial membrane lines the margin of these
two layers and so borders the intraembryonic coelom.
Between 5 and 7 weeks, the coelom is sub-divided by
a process of septation into a future pericardial cav-
ity, two pleural cavities and a peritoneal cavity. In
this phase of development, the mesothelial and sub-
mesothelial layers of the coelom are referred to as the
pericardium, pleura, and peritoneum respectively, and
together as serous membranes (reviewed byThors and
Drukker, 1997). Mesothelial cells are therefore of a
primitive mesodermal origin, but share characteristics
of both epithelial and mesenchymal cells (Whitaker,
Manning, Robinson, & Shilkin, 1992).
Morphologically, mesothelial cells are consideredgenerally similar at different serosal sites and be-
tween different mammalian species (Whitaker et al.,
1982a,b; Baradi & Rao, 1976; Whitaker, Papadi-
mitriou, & Walters, 1980). In their fully differenti-
ated state, they form a monolayer of predominantly
squamous-like cells approximately 25 m in diame-
ter, with characteristic surface microvilli and occa-
sional cilia. The microvilli vary in shape, length and
density between adjacent cells and between different
organs (Mutsaers, Whitaker, & Papadimitriou, 1996).
Mesothelial cells display many epithelial character-istics including a polygonal cell shape, cytokeratin
intermediate filaments (cytokeratins 6, 8, 18 and 19)
(Czernobilsky, Moll, Levy, & Franke, 1985), and the
ability to secrete a basement membrane. However,
they also show features of mesenchymal cells such
as the presence of vimentin, desmin and upon stimu-
lation, alpha smooth muscle actin (Afify, Al-Khafaji,
Paulino, & Davila, 2002). Ultrastructural analysis of
polarised mesothelial cells demonstrates well devel-
oped cellcell junctional complexes including tight
Fig. 1. Monolayer imprint of normal rat peritoneal mesothelialcells showing immunoreactivity for zonula occludens-1 expression,
a plaque protein associated with tight junctions, localised to the
plasma membrane. Bar, 10 m. Reproduced with permission from
Foley-Comer et al. (2002).
junctions (zonula occludens) located towards their
luminal aspect, adherens junctions, gap junctions and
desmosomes (Pelin, Hirvonen, & Linnainmaa, 1994)
(Fig. 1). They also express E-, N- and P-cadherins,
but unlike true epithelia, N-cadherin predominates
(Simsir, Fetsch, Mehta, Zakowski, & Abati, 1999).
Although mainly squamous in appearance, cuboidalmesothelial cells also exist at various locations includ-
ing septal folds of the mediastinal pleura, parenchy-
mal organs (liver, spleen), the milky spots of the
omentum, and the peritoneal side of the diaphragm
overlying the lymphatic lacunae (Wang, 1998). They
also predominate following injury or stimulation of
the serosal surface (Mutsaers et al., 2002; Whitaker &
Papadimitriou, 1985). The two forms of mesothelial
cell, squamous-like and cuboidal, also show differ-
ences ultrastructurally. In particular, cuboidal cells
have abundant mitochondria and rough endoplasmic
reticulum (RER), a well developed Golgi apparatus,microtubules and a greater number of microfilaments
compared with squamous cells, suggesting a more
metabolically active state (Kluge & Hovig, 1967;
Fukata, 1963; Baradi & Campbell, 1974).
3. Functions of the mesothelial cell layer
As well as providing a slippery, non-adhesive ep-
ithelial surface, the mesothelial layer performs many
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has been associated with a worse prognosis and may
represent a less differentiated tumour (Fusco et al.,
1993).
Mesothelial cells have also been implicated in boththe spread and inhibition of tumour growth within
serosal cavities. It has been clearly shown that trau-
matised mesothelial surfaces are privileged sites for
tumour cell adhesion (Cunliffe & Sugarbaker, 1989).
It has been suggested that this occurs due to upregula-
tion of adhesion molecules on mesothelial cells in re-
sponse to inflammatory mediators, promoting tumour
cell adhesion (van der Wal et al., 1997). However,
binding via integrins to exposed submesothelial con-
nective tissue is likely to be the main mechanism of at-
tachment (Sugarbaker, 1991).Tumour growth is then
potentiated by growth factors released from activated
mesothelial cells.
Several studies have also shown that following
surgical trauma, tumour growth is also increased at
sites distal to the injury (Hofer, Shrayer, Reichner,
Hoekstra, & Wanebo, 1998). Animal studies demon-
strated that tumour growth was increased following
exposure to surgical wound fluid or a combination of
the growth factors, TGF- and FGF, suggesting that
mediators produced after surgical trauma or by the
tumour cells themselves, enhance local and distal tu-
mour growth (Hofer et al., 1998). This may occur bystimulating tumour cell proliferation but also through
upregulation of cell adhesion molecules on mesothe-
lial cells promoting their attachment and invasion into
serosal tissues.
Many studies have demonstrated that adhesion of
tumour cells to hyaluronan bound to mesothelial cells
is important for the spread of ovarian and colorectal
tumours (Casey & Skubitz, 2000; Harada et al., 2001;
Lessan, Aguiar, Oegema, Siebenson, & Skubitz, 1999;
Catterall, Jones, & Turner, 1999).However, evidence
also suggests that secretion of hyaluronan by mesothe-lial cells into the serosal fluid may inhibit tumour cell
adhesion (Casey & Skubitz, 2000; Jones, Gardner,
Catterall, & Turner, 1995). Conditioned medium
from confluent mesothelial cell cultures containing
large amounts of hyaluronan prevented tumour cell
attachment, but this inhibition was overcome follow-
ing hyaluronidase treatment (Jones et al., 1995).It is
likely that free hyaluronan in the conditioned medium
bound to CD44 on the tumour cells and prevented
them from binding to hyaluronan on the mesothelial
cell surface. Removal of free hyaluronan may explain
why tumour cells adhered to mesothelial cells in other
studies.
The secretion of pro-coagulants such as tissue factorand fibrin stabilisers plasminogen activator inhibitor
(PAI)-1 and -2, as well as fibrinolytic mediators in-
cluding the plasminogen activators (PA) urokinase
PA (uPA) and tissue PA (tPA) by the mesothelium,
demonstrates an importance in regulating haemostasis
and fibrin clearance (Sitter et al., 1995). Following
serosal injury, there is a fine balance between these
processes, which if disrupted may result in the for-
mation of adhesions, bands of fibrous tissue that
occur in up to 95% of patients following surgery.
Adhesions initially form as fibrin-rich deposits be-
tween damaged, closely opposed serosal surfaces. If
there is insufficient serosal fibrinolytic activity, these
fibrin-rich adhesions persist, become organised by in-
vading fibroblasts and endothelial cells and with sub-
sequent collagen deposition form permanent fibrous
adhesions within a week of injury (Sulaiman et al.,
2000). Although the pathophysiology of adhesion
formation is poorly understood, it is proposed that
adhesions develop if regeneration of the mesothelial
layer is impaired. However, there is much controversy
regarding the mechanisms involved in normal serosal
repair, in particular the cells involved in the regenera-tion of the mesothelium. For a more extensive review
of mesothelial cell function seeMutsaers (2002).
4. Mesothelial healing
Hertzler (1919)was the first to observe that small
and large peritoneal wounds healed in the same
amount of time. He concluded that the mesothe-
lium could not regenerate solely by proliferation and
centripetal migration of cells at the wound edge asoccurs for the healing of epithelium. Since then,
many studies involving a wide range of experimental
model systems have been performed to elucidate the
mechanisms regulating the regeneration process.
It is generally agreed that the healing process be-
gins within 24 h of injury with the appearance of
a population of rounded cells, predominantly neu-
trophils and macrophages, on the wound surface
(Mutsaers et al., 2002). Mesothelial cells at the
wound edge undergo cell division and the epithelial
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sheet temporally transforms into spindle-shaped fi-
broblastic cells that migrate onto the denuded wound
area (Whitaker & Papadimitriou, 1985; Johnson &
Whitting, 1962; Bridges & Whitting, 1964;Mutsaers,Whitaker, & Papadimitriou, 2000). We have shown
that proliferative factors (Mutsaers, McAnulty et al.,
1997) and chemotactic factors, such as HGF, are
likely to play a major role in stimulating this repair
process (Warn et al., 2001). Under normal condi-
tions, the mesothelium is a slowly renewing tissue
with 0.160.5% of cells undergoing mitosis at any
one time (Mutsaers et al., 2000; Fotev, Whitaker, &
Papadimitriou, 1987). However, they can be stimu-
lated to divide by a variety of agents as well as by
direct physical damage. Watters and Buck (1973)
showed that mesothelial cells on opposing serosal
surfaces undergo maximal division 2 days after in-
Fig. 2. Monolayer imprint of tritiated thymidine treated murine serosal lesions at (A) 24 h, (B) 2 days and (C) 4 days after injury. Dark
nuclei are labelled with silver grains (small arrows) and represent cells undergoing division. The centre of the lesion (c), the margin between
the centre and edge of the lesion defined by thick arrows, is identified by a high density of cells, many of which are inflammatory cells.
At 24 h, few mesothelial cells surrounding the wound are undergoing division. By 2 days approximately 28% of these cells are dividing.
At 4 days, the majority of dividing cells are at the wound centre and are characterised as mesothelial cells. Bar, 125 m. Reproduced with
permission fromMutsaers et al. (2000).
jury. Later, kinetic studies using [3H]-thymidine in-
corporation in rodent models confirmed that 2860%
of mesothelial cells at the wound edge and on the
opposing surface were dividing 2448 h after injury(Fig. 2) (Whitaker & Papadimitriou, 1985; Mutsaers
et al., 2000; Fotev et al., 1987). Our subsequent stud-
ies showed that uninjured murine testicular mesothe-
lium has a 0.25% basal mitotic activity, which upon
stimulation by the exogenous addition of peritoneal
inflammatory lavage cells and activated macrophages,
increased to values greater than 12% (Mutsaers et al.,
2002). As inflammatory cells collect on the wound
surface within the first 24 h of injury, it is likely that
they play a significant role in inducing mesothelial
cell proliferation and stimulating serosal repair.
Irrespective of the size of the damaged wound area,
type of trauma or animal species, serosal healing is
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complete within 710 days of injury when the wound
area is covered by cells displaying all the charac-
teristics of mesothelial cells (Mutsaers et al., 2002;
Whitaker & Papadimitriou, 1985; Raftery, 1973;Teranishi, Sakaguchi, & Itaya, 1977). It is unlikely
that the processes of cell division and migration alone
account for these similar healing times. Based on this
evidence, a number of groups have proposed addi-
tional sources for the regenerating mesothelial cells.
These include: macrophage transformation (Eskeland
& Kjaerheim, 1966; Ryan, Grobety, & Majno,
1973), exfoliation of mature or proliferating mesothe-
lial cells from adjacent or opposing serosal sur-
faces (Whitaker & Papadimitriou, 1985; Johnson &
Whitting, 1962; Mutsaers et al., 2000; Fotev et al.,
1987; Cameron, Hassan, & De, 1957; Watters &
Buck, 1972),pre-existing free-floating serosal progen-
itor cells that implant on the wound and differentiate
into mesothelial cells (Ryan et al., 1973), subserosal
mesenchymal precursors that convert into mesothe-
lial cells and migrate to the wound surface (Raftery,
1973; Ellis, Harrison, & Tugh, 1965; Bolen,
Hammar, & McNutt, 1986;Davila & Crouch, 1993),
and bone marrow-derived circulating precursors
(Wagner, Johnson, Brown, & Wagner, 1982).
The origin of these regenerating cells is highly
controversial. Nevertheless, extensive experimentalevidence suggests that a free-floating serosal pro-
genitor is probably involved. For instance, studies
have demonstrated that mesothelial regeneration
is impaired following selective irradiation at the
site of injury but recoverable after the addition of
peritoneal lavage cells (Whitaker & Papadimitriou,
1985). Moreover,Cleaver, Hopkins, Ng Nee Kwong,
& Raftery (1974) showed that the healing rate of
mesothelium was retarded following post-operative
peritoneal lavages, possibly due to the removal of
the free-floating serosal cells. Further evidence for afree-floating progenitor arises from peritoneal fluid
studies where a significantly higher number of viable
free-floating mesothelial cells were recovered from
experimental animals 2 days following injury com-
pared with the control uninjured animals (Whitaker &
Papadimitriou, 1985; Fotev et al., 1987). Our own
group has performed cell-tracking and labelling stud-
ies in rodent models and conclusively shown that
serosal healing involves the incorporation and prolifer-
ation of free-floating mesothelial cells (Foley-Comer
et al., 2002). We found that both cultured and
lavage-derived mesothelial cells implanted onto a
peritoneal wound surface and underwent cell division
with subsequent incorporation into the regeneratingmesothelium as demonstrated by cell junction forma-
tion. Peritoneal macrophages also attached to injured
areas but failed to incorporate whereas peritoneal fi-
broblasts failed to attach, as did mesothelial cells to un-
injured areas. This suggests that free-floating mesothe-
lial cells are able to adhere to exposed and deposited
ECM substrates such as collagen, fibronectin, vit-
ronectin and possibly fibrin following injury, undergo
cell division and integrate into the mesothelial layer.
It is not known whether these free-floating cells are
desquamated mesothelial cells from the serosal lining,
a resident peritoneal fluid sub-population or a dedi-
cated circulating precursor cell population. However,
cell depletion studies using whole body X-irradiation
(Whitaker & Papadimitriou, 1985; Venables, Ellis,
& Burns, 1967) do not appear to support the claim
that a bone marrow-derived precursor is involved in
mesothelial healing, but this finding still needs to be
confirmed.
5. Adhesion formation
Adhesions are a common consequence of serosal
injury in all three serosal cavities leading to serious
complications such as intestinal obstruction, chronic
pain and infertility in women. A detailed histological
and ultrastructural study of human peritoneal adhe-
sions demonstrated that they were all well vascularised
and innervated and contained clusters of smooth mus-
cle cells, the origin of which was unclear (Herrick
et al., 2000; Sulaiman et al., 2001).
It has been proposed that adhesions form as a con-
sequence of reduced fibrinolytic activity in serosal tis-sues. This has been shown both in human studies and
genetically modified mouse models (Holmdahl et al.,
1997; Sulaiman, Dawson, Laurent, Bellingan, &
Herrick, 2002).In serosal tissue, mesothelial cells are
the major source of PA, which are proteases essential
to the fibrinolytic pathway (Sitter et al., 1995). If
mesothelial healing is impaired, there is a reduction in
local PA secretion which reduces fibrinolytic activity.
Two major therapeutic approaches have been inves-
tigated to prevent adhesion formation: fibrinolytic
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agents and barrier devices such as membranes and
gels. However, due to complications associated with
bleeding, systemic fibrinolysis, injury to the internal
organs and vessels, impaired wound healing and dif-ficulty of application, these approaches have shown
limited success. The future direction in preventing
adhesions is likely to be the application of growth
factors and mediators designed to increase the rate of
serosal repair and so re-establish the tissues normal
fibrinolytic capacity.
Another approach to increase the rate of serosal re-
pair is through the exogenous addition of mesothelial
cells. Several groups have demonstrated that instilla-
tion of autologous mesothelial cells at the time of in-
jury prevents adhesion formation (Di Paolo, Vanni, &
Sacchi, 1990; Bertram et al., 1999). Di Paolo et al.
(1990)found that intraperitoneal (i.p.) injection of cul-
tured autologous omental mesothelial cells in rabbits
with staphylococcal-induced peritonitis significantly
reduced the formation of adhesions. In a clinical study
by the same group, four uremic peritoneal dialysis pa-
tients recovering from severe peritonitis were injected
i.p. with 3108 of their own mesothelial cells, previ-
ously cultured and frozen. At laparoscopy 3 and 6 days
post-implantation, there were morphological signs of
cell incorporation in peritoneal biopsies suggesting
this technique may have important applications for theprevention of adhesions in humans (Di Paolo et al.,
1991). In a rat surgical model, Bertram et al. (1999)
also found that i.p. injection of cultured autologous rat
omental mesothelial cells immediately after abrasion
of the peritoneum reduced the number of adhesions
compared to the control group. It is assumed from
these studies that the addition of exogenous mesothe-
lial cells increased serosal repair so prevented adhe-
sion formation, although this has not been confirmed.
These findings again support the concept that a
free-floating progenitor mesothelial cell is involved inmesothelial repair however, they also raise a number
of important questions. For example, it is not clear
whether the free-floating injected cells are different
from the resident mesothelial cells of the serosal lin-
ing, or if their differentiation state changes during
culture or when introduced back into the peritoneal
cavity. Furthermore, omental mesothelial cells may
display phenotypic characteristics that are different
from mesothelial cell populations present in other
locations. Our studies demonstrated incorporation of
free-floating mesothelial cells obtained from peri-
toneal lavage and peritoneal wall into injured serosa
(Foley-Comer et al., 2002), suggesting that omental
mesothelial cells alone may not be the only cellsinvolved in mesothelial regeneration.
The concept that these free-floating mesothelial pro-
genitor cells may have stem cell-like qualities is sup-
ported by the findings of Lucas, Warejcka, Zhang,
Newman, and Young (1996). They isolated and cul-
tured mesenchymal stem cells (MSCs) from skeletal
muscle of neonatal rats and assessed their effect on
the formation of peritoneal adhesions. They compared
the implantation of different concentrations of MSCs
with dead MSCs or smooth muscle cells isolated from
adult animals. Cells were injected i.p. immediately
following surgical injury or at 46 h post-surgery. Ad-
hesion number was significantly reduced in the ani-
mals receiving living MSCs at the time of surgery in
a concentration dependent manner, whereas adhesion
had increased in the animals receiving MSCs 46 h af-
ter surgery. Dead MSCs and smooth muscle cells had
no effect on adhesion formation compared with saline
controls. The authors proposed that MSCs have the
capacity to differentiate into mesothelial cells capable
of repopulating injured serosa and so prevent adhesion
formation. Alternatively, the MSCs produce factors
that inhibit the formation of the initial fibrin-rich adhe-sions, such as fibrinolytic proteases or growth factors
that stimulate mesothelial healing. Cells injected 46 h
after injury are likely to have been trapped within de-
posited fibrin and may have differentiated into fibrob-
lasts rather than mesothelial cells, produced collagen
and formed stronger and more extensive adhesions.
Cell tracking studies were not performed in this study
so the fate of the injected cells remains unknown. It
is crucial that future studies elucidate the origin, state
of differentiation and ultimate fate of resident adher-
ent and free-floating serosal cells following injury todetermine the exact roles they play in normal and ab-
normal mesothelial repair.
6. Evidence for a multipotential subserosal
mesenchymal cell
Another popular theory as to the origin of the
regenerating mesothelial cells is that they are de-
rived from multipotential subserosal mesenchymal
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cells, which when appropriately stimulated, begin to
differentiate into mesothelial cells while migrating
to the injured surface. Many groups have described
the presence of cells with epithelial-like characteris-tics in the subserosal layer of biopsies from various
pathological conditions (Bolen et al., 1986; Davila
& Crouch, 1993;Bolen, Hammar, & McNutt, 1987;
Dobbie, 1990) and from experimental animal models
(Johnson & Whitting, 1962; Yen et al., 1996; Buoro
et al., 1993; Pampinella et al., 1996). These findings
have customarily been explained by the theory that
there exists a population of subserosal multipoten-
tial cells with the ability to differentiate along both
mesenchymal and mesothelial pathways; a concept
originally suggested byKlemperer and Rabin (1931).
IndeedRaftery (1973)described the involvement of a
subserosal precursor cell in the repair of the mesothe-
lium, that appeared intermediate in form between
primitive mesenchymal cells on one hand and prolif-
erating fibroblasts or endothelial cells on the other.
Bolen et al. (1986, 1987)provided the best support for
a multipotential subserosal cell using light, ultrastruc-
tural and immunohistochemical techniques to exam-
ine intermediate filament expression in reactive and
non-reactive human serosal tissue. The group demon-
strated that normal surface mesothelial cells express
low and high molecular weight cytokeratins whereassubmesothelial cells express only vimentin. However,
in biopsies from injured serosa, submesothelial cells
lost vimentin immunoreactivity and progressively ac-
quired high and low molecular weight cytokeratins. It
was suggested that these cells were differentiating to-
wards a mesothelial cell phenotype and were respon-
sible for the re-establishment of surface mesothelium.
However, Whitaker et al. (1992) in a similar study
were unable to reproduce these findings and suggested
that the staining pattern seen by Bolen and colleagues
may be a result of mature mesothelial cells migrat-ing into the subserosal connective tissue. In another
study, Amari, Taguchi, Iwahara, Shibuya, and Naoe
(2002) reported that cultured spheroids composed of
free-floating multicellular clusters of rat pleural fi-
broblasts, demonstrated differentiation of surface cells
into that consistent with mesothelial cells. These cells
expressed microvilli, formed adherens junctions and
were immunoreactive for cytokeratin. This change
in phenotype was inhibited following incubation of
spheroids with anti-fibroblast growth factor receptor
antibody, suggesting FGF plays a key role in the
phenotypic conversion of fibroblasts into regenerated
mesothelial cells.
Further support for a multipotential submesothe-lial cell comes from experimental findings following
short-term bladder obstruction in a rabbit model. This
form of injury induced thickening of the subserosal
layer with smooth muscle hypertrophy, and a tran-
sient expression of cytokeratin 18 in subserosal mes-
enchymal cells. At a later stage, new muscle express-
ing smooth muscle myosin and desmin, was detected
in the subserosal layer in the absence of mitotic ac-
tivity in the original smooth muscle layer (Pampinella
et al., 1996).In agreement with their previous findings
(Buoro et al., 1993),the authors concluded that resi-
dent keratin expressing subserosal mesenchymal cells
transformed into myofibroblasts and subsequently into
fetal-type smooth muscle cells a well as regenerat-
ing mesothelial cells (Buoro et al., 1993; Pampinella
et al., 1996). Taken together these findings would seem
to support the view that a multipotential subserosal
mesenchymal cell exists which can differentiate into
myofibroblasts and possibly smooth muscle cells as
well as mesothelial cells. However, new evidence sug-
gests that the mesothelial cells themselves may be
multipotential and have the ability to differentiate into
various different cell types. Indeed, irradiation and ki-netic studies have also questioned the role of sub-
serosal cells for mesothelial regeneration (Whitaker &
Papadimitriou, 1985; Mutsaers et al., 2000).
7. Epithelial-mesenchymal transition of
mesothelial cells
Classically, isolated mesothelial cells from normal
serosal tissue or fluid demonstrate cobblestone ep-
ithelioid morphology in culture. However, it has longbeen known that these cells can change to a fibroblas-
tic phenotype with repeated passage, reducing cytok-
eratin and increasing vimentin expression (Mackay,
Tracy, & Craighead, 1990). Various growth factors
can also induce mesothelial cells to change pheno-
type and express many of the characteristics associated
with fibroblasts such as increased motility and en-
hanced ECM production (Fig. 3). For example, EGF
induces the reversible change to a fibroblastic pheno-
type that is accompanied by an increased expression
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Fig. 3. Primary cultures of human pericardial mesothelial cells
representing (A) epithelioid and (B) fibroblastic phenotypes, at dif-
ferent passage numbers of the same cell preparation. Micrographs
courtesy of Jason Tee.
of1 integrins, in particular21, facilitating an en-
hanced adhesion to and migration on collagen type
I (Leavesley, Stanley, & Faull, 1999). Furthermore,
EGF, PDGF and IL-1 beta have also been shown to
stimulate increased collagen production in mesothe-
lial cells (Harvey & Amlot, 1983; Owens & Milligan,
1994;Yang, Kim, Lee, Park, & Kim, 1999).
Various benign disorders, including liver cirrhosis,
endometriosis or serosal inflammation, produce effu-
sions that often contain increased numbers of mesothe-lial cells thought to be derived from the reactive serosa.
In culture, these cells demonstrate both fibroblastic
and epithelioid morphologies, a pattern which is stable
throughout early passages (Gulyas, Dobra, & Hjerpe,
1999).It has been suggested that these two different
cell morphologies represent mesothelial cells at differ-
ent stages of differentiation, and it is likely that in dis-
ease, inflammatory factors and other mediators direct
cells down various phenotypic pathways. The expres-
sion of Wilms tumor susceptibility gene (WT1) and
certain proteoglycans, syndecan-4 and glypican, are
proposed to be associated with progression through the
differentiation process (Dobra et al., 2000;Gulyas &
Hjerpe, 1999, 2003) with WT1 often being describedas a mesothelial lineage marker.
Whitaker et al. (1992) first suggested that mature
mesothelial cells could transform into fibroblast-like
cells in vivo and invade the underlying subserosal con-
nective tissue. Indeed, they suggested that this could
account for the intermediate filament staining pattern
observed by Bolen et al. (1986). This seems an un-
usual concept because in contrast to mesenchymal
stromal cells, epithelial-like cells infrequently convert
into fibroblasts in mature tissue, apart from during
wound healing or tumour progression (Hay, 1995).
However, two recent reports investigating the patho-
logical effects of continuous ambulatory peritoneal
dialysis (CAPD) have provided strong evidence to sup-
port this concept. CAPD is known to cause peritoneal
fibrosis leading to a failure of ultrafiltration however,
the mechanisms involved in this process are not clear.
In the first study,Yang, Chen, and Lin (2003)demon-
strated that transforming growth factor-1 (TGF-1)
induced human omental mesothelial cells to transdif-
ferentiate into myofibroblasts in vitro with the char-
acteristic appearance of prominent RER, conspicuous
smooth muscle actin myofilaments, intermediate andgap junctions and active deposition of ECM. Gene ex-
pression analysis revealed a complex modulation of
gene expression involving cytoskeletal organisation,
cell adhesion, ECM production, cell proliferation, in-
nate immunity, stress responses and many other es-
sential metabolic processes as the mesothelial cells
underwent transformation. The authors proposed that
the differentiated epithelial cells of the mesothelium
convert into myofibroblasts and that the pathological
features observed following CAPD may be due to the
recruitment of fibrogenic cells from the mesotheliumduring serosal inflammation and wound healing.
The second study byYnez-Mo et al. (2003)also
demonstrated that human mesothelial cells undergo a
conversion from an epithelial to mesenchymal phe-
notype which occurred in patients following serosal
injury. Peritoneal mesothelial cells isolated from dial-
ysis fluid effluents displayed a mesenchymal pheno-
type that appeared to be related to both the duration
of CAPD and to whether peritonitis had occurred.
Mesothelial cells lost their epithelial morphology and
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showed a decrease in the expression of cytokeratins
and E-cadherin through induction of the transcrip-
tional repressorsnail. They also acquired a migratory
phenotype with up-regulation of 2 integrins. Ma-jor profibrotic and inflammatory cytokines, such as
TGF-1 and IL-1B, appeared to be involved in this
process. In addition, assessment of peritoneal biopsy
specimens from patients undergoing CAPD showed
the presence of mesothelial markers, ICAM-1 and cy-
tokeratins, on fibroblast-like cells embedded in the
subserosal layer, suggesting that these cells were de-
rived from a local conversion of mesothelial cells.
The authors described this phenotypic conversion
as transdifferentiation, a complex and generally
reversible process that starts with the disruption of
intercellular junctions and loss of apical-basolateral
polarity typical of epithelial cells. With time, the cells
transform into fibroblast-like cells with pseudopodial
protrusions and increased migratory, invasive and fi-
brogenic features (Hay, 1995).However, it is currently
unknown whether the mesothelial cells remain as
myofibroblasts, continue to differentiate into smooth
muscle cells or revert back to surface mesothelial
cells. Furthermore, it is unclear whether the mesothe-
lial cells that undergo trandifferentiation are a resident
population in the mesothelial layer, originate from a
serosal fluid subpopulation or are from a circulatingblood-derived source. Nevertheless, the authors do
suggest that in light of these recent findings, the ear-
lier concept of a multipotential subserosal cell being
able to convert to both epithelial mesothelial cells and
myofibroblasts (Raftery, 1973; Bolen et al., 1986)
should be questioned. Furthermore, it raises the inter-
esting possibility that mesothelial transdifferentiation
may be wholly or partly responsible for the patholog-
ical changes that occur in the serosal layer following
trauma caused by, for example, CAPD, irradiation,
malignancy or surgery.
8. Tissue engineering potential of mesothelial cells
Although there is a lack of information regarding the
differentiation potential of mesothelial cells, for over
a century these cells have been used to repair damaged
tissues and organs, as well as being employed in a
number of new tissue engineering applications.
8.1. Vascular grafts
Despite considerable clinical research, no biolog-
ical or synthetic grafts have been developed as anideal substitute for small diameter arteries (Nerem &
Seliktar, 2001). When acellular artificial prostheses
are used in the reconstruction of small diameter ves-
sels, failure frequently occurs because the luminal sur-
face is thrombogenic resulting in thrombus formation
and re-occlusion following implantation. Cell seeding
should decrease thrombogenicity of implanted vas-
cular grafts but this application is hampered by the
limited availability of autologous vascular endothelial
cells, and so alternative cell types have been sought.
It has long been recognised that foreign objects
introduced into the peritoneal cavity of the rat, rab-
bit or mouse, initiate an inflammatory response with
the resultant granulation tissue covered by a layer of
mesothelium (Ryan et al., 1973; Campbell & Ryan,
1983; Mosse, Campbell, & Ryan, 1985). Eskeland
and Kjaerheim (1966) were first to demonstrate that
a mesothelial membrane could be grown on the outer
surface of a free-floating diffusion chamber placed
in the peritoneal cavity of rats. Later ultrastructural
studies showed that mesothelial cells deposited and
organised ECM; including thick collagen fibres, the
amorphous components of elastic fibres and base-ment membrane-like structures restricted to the basal
region of the cell layer (Rennard et al., 1984). Based
on these observations, in addition to the known fibri-
nolytic and antithrombotic properties of mesothelial
cells (Louagie et al., 1986), Clarke, Pittilo, Machin,
and Woolf (1984)proposed that autologous mesothe-
lial cells may represent a practical alternative to
endothelial cells in vascular grafts. Subsequently,
many groups have investigated the efficacy of using
mesothelial cells, mainly derived from the omentum,
as endothelial replacements (Sparks et al., 2002; Bullet al., 1988; Bearn et al., 1992; Verhagen et al., 1998;
Theuer et al., 1996).
Studies by Bull et al. (1988) showed that Dacron
arterial grafts seeded with autologous mesothe-
lial cells promoted luminal cell cover, displayed
anti-thrombogenic activity, inhibited platelet aggre-
gation and released more prostacyclin than unseeded
grafts in canine abdominal aorta replacements. How-
ever, later studies using digested omental extract
seeded onto knitted Dacron scaffolds and implanted
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as bilateral femoral artery replacements, suggested
that the mesothelial cells were not retained on the
graft 24h later (Bearn et al., 1992). Furthermore,
fibronectin coated small diameter polytetrafluoroethy-lene (PTFE) scaffolds seeded with cultured omental
mesothelial cells showed poor patency and increased
neointimal thickening compared with non-seeded
grafts following implantation into the carotid artery
of the same dog (Verhagen et al., 1998). Other studies
in which the infrarenal inferior vena cava was re-
placed with interposition grafts of either a peritoneal
tube, PTFE or PTFE lined peritoneum, demonstrated
that peritoneal lined grafts maintained a continuous
circumferential cellular lining but showed no im-
provement in short term patency compared to PTFE
alone (Theuer et al., 1996).
Despite these disappointing findings, Campbell,
Efendy, and Campbell (1999) using an alternative
seeding method, have produced more favourable re-
sults. Free-floating silastic tubing was implanted into
the peritoneal cavity of rats and rabbits and after two
weeks, the ones that remained free-floating were re-
moved and processed. When the tubes were everted
and histologically assessed they consisted of an in-
tima of non-thrombotic mesothelial cells, a media of
smooth muscle-like cells or myofibroblasts embedded
in a collagen and elastic matrix, and an outer collage-nous adventitia. The grafts remained patent, showed
reasonable tensile strength and were responsive to
contractile agonists for at least 4 months. The role
of haemodynamic stress, active stretch and neuronal
imput on the differentiation of the cells within the
mesothelial tubes was investigated in a subsequent
study. Following end-to-end anastomosis with the
aorta, there was a progressive increase in myofilament
expression (evidence of smooth muscle phenotype) in
the grafts over time, which was also observed by cycli-
cally stretching the tubes in vitro (Efendy, Campbell,& Campbell, 2000). In contrast, innervation of the
tubes following transplantation into the rat anterior
eye chamber appeared to have little effect on the
differentiation of cells towards a smooth muscle cell
phenotype. The authors state that these grafts have
several advantages over others in that they are biocom-
patible with the host tissue, need no artificial mesh as
part of the wall, have a nonthrombogenic surface and
develop elastic lamellae. Moreover, they have demon-
strated patency for at least 4 months with 1020%
contractile responses compared with the control artery
after transplantation. However, many questions re-
main unanswered such as; how similar the inner sur-
face lining of mesothelial cells are to true endothelialcells, and are mesothelial cells in the intimal layer
subsequently replaced by local ingrowth of endothe-
lial cells following transplantation to high pressure
arterial sites. Indeed, if the free-floating mesothelial
cells of the peritoneal cavity are able to provide all
the cell types found in the transplanted graft, is this
through a transdifferentiation process as described
previously?
Many authors remain to be convinced of the use
of the peritoneal cavity as a feasible environment for
growing functional bioartificial vascular grafts as re-
viewed byMoldovan and Havemann (2002).Cebotari,
Walles, Sorrentino, Haverich, and Mertsching (2002)
repeated the work of Campbell et al. (1999) using
decellularised allogenic scaffolds and, although they
found repopulation of the implanted grafts in the
given time period, they also showed extensive denat-
uration of collagen and graft degeneration. Whether
prior seeding vascular scaffolds with mesothelial cells
isolated from the omentum (Pearce et al., 1987; Pasic
et al., 1994; Salacinski, Punshon, Krijgsman,
Hamilton, & Seifalian, 2001) or peritoneal fluid
(Tiwari et al., 2003) is a better method for generatingtissue engineered grafts, awaits further investigation.
Until then, the use of mesothelial cells as endothe-
lial cell replacements still remains a possibility and
may prove important in, for example, the develop-
ment of autologous coronary artery bypass grafts
or arteriovenous access fistulae for hemodialysis
patients.
8.2. Omental grafts
The scientific community has neglected the omen-
tum for many years, although recent interest has
stemmed from its multiple uses in reconstructive
surgery (Liebermann-Meffert, 2000). The omentum
is essentially composed of two mesothelial sheets
which enclose predominately adipocytes embed-
ded in a highly vascularised connective tissue. The
greater part of the omentum is associated with the
stomach, small intestines and transverse colon and
forms an apron-like structure covering abdominal
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organs. The omentum is particularly susceptible to
forming adhesions as it floats passively within the
peritoneal cavity but rapidly adheres to inflamed or
damaged tissues. In a post-mortem study, Weibel andManjo (1973) found that the omentum was the or-
gan most frequently involved in adhesion formation
and many workers have suggested that omental ad-
hesions offer protection against more severe com-
plications such as peritonitis and ischaemic bowel
disease (Williams & White, 1986; Hasgood, 1990).
Part of the omentums ability to rescue injured tis-
sue is likely to be due to its angiogenic (Goldsmith,
Griffith, Kupferman, & Catsimpoolas, 1984; Gold-
smith, Griffith, & Catsimpoolas, 1986) and neu-
rotrophic (Chamorro et al., 1993) properties; hence,
its use as a pedicle graft tissue for clinical condi-
tions involving revascularisation of ischaemic parts
of the brain, kidney, spleen, heart and spinal cord
(Goldsmith, Chen, & Duckett, 1973; Goldsmith,
Duckett, & Chen, 1975).
Free omental grafts have been used in the treatment
of numerous human disorders including neurodegen-
erative diseases such as Alzheimers disease, chronic
leg ulcers and gastric ulcers (Weinzweig, Schlechter,
Baraniewski, & Schuler, 1997). Piano et al. (1998)
used free omental grafts to treat severe necrotising
fasciitis and observed that necrotic tissue becamerevascularised resulting in acceptance of the graft
and healing of the defect. The exact mechanism of
this early revascularisation is unknown, however, it
has been suggested that various growth factors such
as FGF (Chamorro et al., 1993) and VEGF (Zhang
et al., 1997; Mandl-Weber, Cohen, Haslinger,
Kretzler, & Sitter, 2002), which are present in high
levels and can be isolated from the omentum, are in-
volved. In another study,Chamorro et al. (1993)used
free omental grafts to facilitate nerve graft regenera-
tion in rats by surrounding the nerve graft with omen-tum. Early revascularisation and directional growth
of sprouting axons was encouraged, thus increasing
the efficiency of nerve regeneration. It is worth noting
that the fate and role of the mesothelial cells was not
determined in any of these studies and therefore it is
not clear whether they were in part responsible for
the success of these grafts, through either the release
of growth factors or themselves being incorporated
into the repairing tissue.
8.3. Nerve grafts
Regeneration of severed peripheral nerves is of-
ten incomplete due to loss or misdirection of nervefibres and neuroma formation. The use of nerve re-
placements composed of artificial tubes seeded with
isolated mesothelial cells as an alternative to primary
nerve suture has been introduced as a biological ap-
proach to nerve injuries. Initial studies in a rat model
by Lundborg et al. (1982) investigated the regen-
eration of a transected sciatic nerve through either
preformed mesothelial chambers or autologous nerve
grafts bridging a 10 mm gap. Within the mesothelial
chambers, an organised multifascicular nerve trunk
formed between proximal and distal stumps. After 3
months there was no difference with respect to ax-
onal density or distribution of axons between the two
grafts. Furthermore, the conduction velocities across
the gaps were similar. In the mesothelial chambers,
the regenerating nerve was surrounded by a loose
cellular stroma and a small amount of interstitial
fluid, which was found to contain trophic activity for
cultured rodent sensory neurons.
In a subsequent study, nerve regrowth occurred
when a preformed mesothelial tube bridged the gap
between left and right sciatic nerves that had been
transferred to the backs of rats (Danielsen, Dahlin,Lee, & Lundborg, 1983). When the gap was 10 mm or
less, a well developed nerve structure was generated
in the chamber between the nerve ends, and axons
from the left sciatic nerve reinnervated muscles in the
right limb via the right sciatic nerve. Additional stud-
ies demonstrated that when rabbit hypoglossal nerves
were repaired using mesothelial chambers, a sig-
nificantly faster migration of radio-labelled proteins
in the distal nerve segment was observed compared
to sutured nerves (Danielsen, Lundborg, & Frizell,
1986).Remarkably, the thin mesothelial lining foundaround the tube lacked primary inflammatory signs
at follow-up after 1 year and showed no signs of
compression (Dahlin & Lundborg, 2001). Similar to
the studies ofChamorro et al. (1993),Castaneda and
Kinne (2002) performed siatic nerve transections in
rats and found that 2530 mm defects bridged by an
omental graft were fully healed with increased func-
tional recovery and less scarring than end to end repair.
It was suggested that grafts incorporating mesothelial
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cells may have an advantage as they allow sliding of
the repair site against surrounding tissues due to the
secretion of surfactants (Dahlin & Lundborg, 2001).
Although the origin, fate or function of the mesothe-lial cells was not described in these studies, artificial
tubes lined by mesothelial cells appear to be impor-
tant alternatives to conventional repair techniques for
primary nerve repair and reconstruction of segmental
defects.
9. Does a mesothelial stem cell exist?
The biology of adult stem cells remains remark-
ably poorly understood and in general, there is a lack
of a unifying definition as well as specific markers
to define them. A rich reservoir of adult stem cells
resides in specific niches within the bone marrow mi-
croenvironment as well as in a variety of connective
tissues, where they are maintained in an undiffer-
entiated and quiescent state. The ability to produce
cells that can progress down a variety of distinct
cell lineages, even as clonally isolated cells, is one
of the main characteristics of stem cells. For exam-
ple, when appropriately induced, mesenchymal stem
cells (MSCs) have the potential to differentiate along
specific mesenchymal lineages (multipotency) andform tissues that include endothelium, muscle, bone,
cartilage and fat (reviewed by Tuan, Boland & Tuli,
2003). Although a mesothelial stem cell has not been
identified, growing evidence based on its primitive
embryological origin and ability to transdifferenti-
ate strongly supports the idea that a population of
mesothelial progenitor cells exist. Indeed,Donna and
Betta (1986)proposed that the mesothelial cell was
not only totipotent but represented real mesoderm that
retained the potential to differentiate along embryonic
developmental lines including to cartilage and bone.Thus, they suggested the term mesoderma instead
of mesothelioma to recognise the mesodermal ori-
gin of associated mesothelial tumours. Since then, as
previously described, tissue culture and animal exper-
imental studies have convincingly demonstrated that
adult mesothelial cells are capable of transdifferenti-
ating from an epithelial to mesenchymal phenotype
and this seems to depend on the presence of cer-
tain growth factors or cytokines (Yang et al., 2003;
Yanez-Mo et al., 2003). However, conclusive evidence
demonstrating that adult human mesothelial cells are
capable of differentiating along specific mesenchymal
cell lineages is still lacking.
Munoz-Chapuli et al. (1999)recently hypothesisedthat hemangioblasts, the common progenitor of the
endothelial and hematopoietic cell lineages, originated
from embryonic splanchic mesothelium, and that the
differentiation of endothelial and blood cells was
therefore from a common mesothelial-derived progen-
itor. A later study provided evidence to support this
theory. Using cell-labelling techniques and quail-chick
chimeras,Perez-Pomares and Munoz-Chapuli (2002)
showed that during development, epicardial mesothe-
lium differentiates into endothelium or smooth muscle
through an epithelial-mesenchymal transition (EMT)
process. This finding raised the interesting question
of whether the coelomic mesothelium retains its abil-
ity to transform into multipotent mesenchymal cells
in the adult. Based on this assumption, Wada, Osler,
Reese, and Bader (2003) recently showed that in
culture, explants of adult rat epicardial mesothelium
retain the ability to produce mesenchyme including
smooth muscle cells in response to specific growth
factors. The authors suggest that a cell line derived
from rat epicardial mesothelial cells acts in a sim-
ilar manner to the bipotential vascular progenitor
cells, a stem cell population originally described byYamashita et al. (2000).
As well as the intriguing possibility that adult
mesothelial progenitor cells are able to produce en-
dothelium and smooth muscle, findings from several
experimental models suggest that these cells may also
form skeletal muscle and cartilage. For instance, dur-
ing the healing phase of a chemical-induced peritoni-
tis, skeletal muscle fibres were found to develop de
novo in the peritoneal lining of the adult rat diaphragm.
The location and orientation of the fibres suggested
an origin from mesothelial or submesothelial cellsin granulation tissue rather than intrinsic diaphrag-
matic muscle satellite cells (Levine & Saltzman,
1994; Drakontides, Danon, & Levine, 1999). How-
ever, more extensive studies are required to confirm
these findings. If the mesothelium is the source of new
skeletal muscle fibres, as the authors state, it will be
important to determine if the diaphragmatic mesothe-
lium is different from mesothelium in other locations.
Indeed, it would be desirable to imitate the environ-
ment of the inflamed diaphragmatic peritoneum in
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other areas of skeletal muscle damage where regener-
ation is needed.
Although rare, it is of no surprise that biopsies taken
from human malignant mesothelioma express markersof osseous and cartilaginous differentiation (Donna
& Betta, 1986; Yousem & Hochholzer, 1987; Kiyo-
zuka et al., 1999; Andrion, Mazzucco, Bernardi, &
Mollo, 1989). Furthermore, in experimental models,
bone and cartilage were found in peritoneal malig-
nant mesotheliomas that were induced by i.p. injec-
tion of asbestos fibres (Rittinghausen, Ernst, Muhle,
& Mohr, 1992). Surprisingly, however, Fadare,
Bifulco, Carter and Parkash (2002) found evidence
of cartilaginous differentiation in human peritoneal
tissue biopsies which did not appear to be associated
with an intra-abdominal malignancy. Indeed, in the
human peritoneum, several other well-documented
cases of mesenteric heterotopic ossification (or os-
seous metaplasia) and/or cartilaginous differentiation
have been reported (Lemeshev, Lahr, Denton, Kent, &
Diethelm, 1983; Wilson, Montague, Salcuni, Bordi, &
Rosai, 1999; Yannopoulos, Katz, Flesher, Geller,
& Berroya, 1992).The source of the cells that undergo
this differentiation process remains controversial but
the traditional view is that they are derived from a
population of subserosal multipotential cells as de-
scribed earlier. However, in light of recent findings,it is also possible that a population of mesothelial
cells may have the ability to form cells of different
mesenchymal lineages. These progenitor cells may
be resident in the mesothelial layer, free-floating in
the serosal fluid or alternatively, may be derived from
a circulating multipotential cell population which en-
ters serosal cavities via the vasculature. This matter is
further complicated by the observation that cells of a
haemopoietic origin, identified through bone marrow
transplant and Ly5 antigen expression, are able to
differentiate into myofibroblasts and smooth musclecells in response to a foreign body implanted into the
peritoneal cavity (Campbell, Efendy, Han, Girjes, &
Campbell, 2000). Depending on the local environ-
ment these progenitor cells may be able to progress
down various differentiation pathways. Growth fac-
tors levels, cellcell interactions, cell density and
physical and mechanical stimuli may all contribute
to the end product of differentiation. In addition, the
mesothelial layer and free-floating cells are in con-
tinuous communication with peritoneal fluid and so
Fig. 4. Hypothetical representation of a mesothelial progenitor
cell residing in the serosal monolayer. Following injury, these
cells may transdifferentiate into subserosal progenitor cells with
the capacity to further differentiate into myofibroblasts, smooth
muscle cells and endothelial cells. In addition, they may detach
from the basement membrane and become free-floating progenitor
cells in the serosal fluid before repopulating serosal lesions.
any changes in, for example, levels of cytokines and
growth factors, proteases, oxygen, and pH, may also
affect ultimate progenitor cell fate (Fig. 4).
As well as an ability to differentiate along specific
lineages upon stimulation, other key features of stem
cells are to remain in a quiescent undifferentiated state
until provided with the signal to divide asymmetri-
cally and undergo many more replicative cycles than
normal. Future studies have yet to determine if theseare characteristics of mesothelial progenitor cells. At
present, little is know regarding aspects of ageing or
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viable and proliferative or if it contains chemoattrac-
tants that cause circulating progenitor cells to home to
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