mesothelial progenitor cells and their potential

8/13/2019 Mesothelial Progenitor Cells and Their Potential

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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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622 S.E. Herrick, S.E. Mutsaers/ The International Journal of Biochemistry & Cell Biology 36 (2004) 621642

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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S.E. Herrick, S.E. Mutsaers/ The International Journal of Biochemistry & Cell Biology 36 (2004) 621642 623

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


11/22


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


12/22


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


13/22


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


14/22


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


15/22


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

senescence of mesothelial cells, except that in culture

they senesce 2.5-fold faster than fibroblasts and in vivo

senesce following exposure to dialysis fluids (Thomas

et al., 1997; Gotloib, Wajsbrot, & Shostak, 2003).

Furthermore, whether serosal fluid contains survival

factors that allow mesothelial progenitors to remain

viable and proliferative or if it contains chemoattrac-

tants that cause circulating progenitor cells to home to


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