Mesothelial Progenitor Cells and Their Potential

The International Journal of Biochemistry & Cell Biology 36 (2004) 621642
Review
Mesothelial progenitor cells and their potential in tissue engineering

Sarah E. Herrick a, , Steven E. Mutsaers b
a b
School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK 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 The mesothelium consists of a single layer of attened 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 identied, 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-oating population of cells present in the serosal uid, the origin of which is currently unknown. Recent ndings have shown that mesothelial cells can undergo an epithelial to mesenchymal transition, and transform into myobroblasts 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 bres. These ndings 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. 2. 3. 4. 5. 6. 7. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Embryology and morphology of mesothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of the mesothelial cell layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesothelial healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adhesion formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for a multipotential subserosal mesenchymal cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epithelial-mesenchymal transition of mesothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622 622 623 625 627 628 629
Corresponding author. Tel.: +44-161-275-6765; fax: +44-161-275-5945. E-mail address: sarah.herrick@man.ac.uk (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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S.E. Herrick, S.E. Mutsaers / The International Journal of Biochemistry & Cell Biology 36 (2004) 621642
Tissue engineering potential of mesothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Vascular grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Omental grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Nerve grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Does a mesothelial stem cell exist? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.
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1. Introduction The mesothelium lines the peritoneal, pleural and pericardial cavities with visceral and parietal surfaces covering the internal organs and body wall, respectively. 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 inammatory cells and broblast-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 uid and solute transport, immune surveillance and the production of extracellular matrix (ECM) molecules, proteases, cytokines and growth factors. The mesothelium is bathed in serosal uid that resembles an ultraltrate of plasma and contains blood proteins, resident inammatory cells, sugars and various enzymes including amylase and lactate dehydrogenase (Dondelinger, Boverie, & Cornet, 1982). The composition and volume of the serosal uid is indicative of certain pathological states, such as peritonitis, tumorgenesis and endometriosis (Haney, 1993), and it is likely that the mesothelial layer responds as a single unit to changes in serosal uid composition. Indeed, repair of serosal tissue involves increased mesothelial cell proliferation at sites distant to the wound, suggesting diffuse activation of the mesothelium in response to mediators or cells released into the serosal uid, or via cell to cell communication (Mutsaers, McAnulty et al., 1997; Mutsaers, Whitaker, & Papadimitriou, 2002). Although local proliferation of resident cells surrounding 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 multipotential 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 specic niches 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 denition that characterises cells as stem cells. However, a general denition 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 identied, many lines of evidence suggest that a mesothelial progenitor cell does exist. This review will describe the mesothelial cell in terms of its embryological origin, morphological characteristics and diverse functions. Subsequent sections present evidence to support the concept of a free-oating mesothelial progenitor cell present in serosal uid and also discuss mesothelial cell differentiation, novel tissue engineering applications for these cells and possible future research directions in this rapidly developing eld.
2. Embryology and morphology of mesothelial cells Bichart, in 1827 (reviewed by Whitaker, Papadimitriou, & Walters, 1982a) rst observed that serous cavities were lined by a layer of attened 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 mesodermic 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 mesoderm is continuous with the extraembryonic mesoderm 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 intraembryonic 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 continuous 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 cavity, two pleural cavities and a peritoneal cavity. In this phase of development, the mesothelial and submesothelial layers of the coelom are referred to as the pericardium, pleura, and peritoneum respectively, and together as serous membranes (reviewed by Thors 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 considered generally similar at different serosal sites and between different mammalian species (Whitaker et al., 1982a,b; Baradi & Rao, 1976; Whitaker, Papadimitriou, & Walters, 1980). In their fully differentiated state, they form a monolayer of predominantly squamous-like cells approximately 25 m in diameter, with characteristic surface microvilli and occasional 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 characteristics including a polygonal cell shape, cytokeratin intermediate laments (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 stimulation, alpha smooth muscle actin (Afy, Al-Khafaji, Paulino, & Davila, 2002). Ultrastructural analysis of polarised mesothelial cells demonstrates well developed cellcell junctional complexes including tight
Fig. 1. Monolayer imprint of normal rat peritoneal mesothelial cells 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, cuboidal mesothelial cells also exist at various locations including septal folds of the mediastinal pleura, parenchymal 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 differences ultrastructurally. In particular, cuboidal cells have abundant mitochondria and rough endoplasmic reticulum (RER), a well developed Golgi apparatus, microtubules and a greater number of microlaments 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 epithelial surface, the mesothelial layer performs many
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diverse functions which are important in the maintenance of serosal homeostasis. These include transport and movement of uid and particulate material across serosal cavities, regulation of leucocyte migration in response to inammatory mediators, synthesis of pro-inammatory cytokines, growth factors and ECM molecules, control of coagulation and brinolysis, and antigen presentation. These functions are often linked to the phenotypic state of the cell. For instance, the enzymatic prole of squamous and cuboidal mesothelial cells suggests that the former fully differentiated state is involved with membrane transport whereas cuboidal mesothelial cells have a wider spectrum of activity (Whitaker et al., 1980; Ramsey, Tweeddale, Bryant, & Braunstein, 1970; Raftery, 1976; Whitaker et al., 1982a,b). Mesothelial cells secrete glycosaminoglycans (GAGs), in particular hyaluronan, proteoglycans including syndecans and biglycan, and surfactant lubricants, to provide a slippery non-adhesive surface which also protects the serosal surface from abrasion, infection and possibly tumour dissemination. Furthermore, they participate in serosal inammation by secreting various pro-, anti- and immunomodulatory mediators, and by changing microvilli density and adhesion molecule expression, in particular intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), they can regulate trafcking of leucocytes into and out of serosal cavities (Liang & Sasaki, 2000; Bellingan et al., 2002). Mesothelial cells also produce a multitude of cytokines and growth factors which can regulate inammatory responses and stimulate tissue repair. Stimuli such as bacterial products, asbestos, instilled agents and tissue injury induces release of pro-inammatory cytokines and chemokines including interleukin (IL)-1, IL-8, monocyte chemoattractant protein-1 (MCP-1), RANTES, growth-related oncogene- (GRO-), interferon- (IFN-)-inducible protein 10 (IP-10), stromal cell-derived factor-1 (SCD-1) and eotaxin, which recruit neutrophils, monocytes, lymphocytes and eosinophils to the site of challenge (Mutsaers, 2002). Further stimulation by hyaluronan secreted by mesothelial cells, and products released from activated macrophages such as IL-1, tumour necrosis factor- (TNF-) and IFN-, increase the production of chemokines by mesothelial cells so potentiates the inammatory response (Betjes et al.,
1993; Visser et al., 1998; Haslinger, Mandl-Weber, Sellmayer, & Sitter, 2001). However, to regulate the inammatory response, mesothelial cells also secrete anti-inammatory mediators including prostaglandins, prostacyclin and IL-6 (Topley et al., 1993, 1994). Endogenous IL-6 plays a crucial role in local and systemic acute inammatory responses by controlling the levels of pro-inammatory, but not anti-inammatory cytokines (Xing et al., 1998). Mesothelial cells also release growth factors which initiate cell proliferation, differentiation and migration of mesothelial and submesothelial cells surrounding a lesion. Transforming growth factor- (TGF-), platelet-derived growth factor (PDGF), broblast growth factor (FGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and members of the epidermal growth factor (EGF) family are some of the factors likely to regulate these processes (Martin, Hopkinson-Woolley, & McCluskey, 1992; Mutsaers, Bishop, McGrouther, & Laurent, 1997; Jayne, Perry, Morrison, Farmery, & Guillou, 2000; Offner et al., 1996; Langerak et al., 1996; Adamson, Bakowska, & Prieditis, 2000; Warn et al., 2001). Interestingly, KGF and HGF are generally considered as epithelial cell-derived growth factors that stimulate mesenchymal cell proliferation and migration. The nding that mesothelial cells also express the receptors for these factors and can respond to them conrms the dual epithelial/mesenchymal properties characteristic of this cell type (Adamson et al., 2000; Warn et al., 2001). Mesothelial cells also have the capacity to synthesise a variety of ECM macromolecules in vitro, such as collagen types I, III, and IV, elastin, bronectin and laminin and regulate turnover through the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). It has been proposed that the phenotypic state of the cells has a marked inuence on MMP and TIMP expression so that cells with a squamous epithelioid morphology adopt an ECM-degradative phenotype whilst cuboidal cells deposit more matrix components (Marshall et al., 1993). A similar variability in phenotypic differentiation is observed in human and rodent malignant mesothelioma (Dobra, Andang, Syrokou, Karamanos, & Hjerpe, 2000). This tumour, predominantly of the pleura, is particularly aggressive and largely unresponsive to conventional chemotherapy or radiotherapy. In particular, the sarcomatous growth pattern
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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 both the spread and inhibition of tumour growth within serosal cavities. It has been clearly shown that traumatised mesothelial surfaces are privileged sites for tumour cell adhesion (Cunliffe & Sugarbaker, 1989). It has been suggested that this occurs due to upregulation of adhesion molecules on mesothelial cells in response to inammatory mediators, promoting tumour cell adhesion (van der Wal et al., 1997). However, binding via integrins to exposed submesothelial connective tissue is likely to be the main mechanism of attachment (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 demonstrated that tumour growth was increased following exposure to surgical wound uid 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 tumour growth (Hofer et al., 1998). This may occur by stimulating tumour cell proliferation but also through upregulation of cell adhesion molecules on mesothelial 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 mesothelial cells into the serosal uid may inhibit tumour cell adhesion (Casey & Skubitz, 2000; Jones, Gardner, Catterall, & Turner, 1995). Conditioned medium from conuent mesothelial cell cultures containing large amounts of hyaluronan prevented tumour cell attachment, but this inhibition was overcome following 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 factor and brin stabilisers plasminogen activator inhibitor (PAI)-1 and -2, as well as brinolytic mediators including the plasminogen activators (PA) urokinase PA (uPA) and tissue PA (tPA) by the mesothelium, demonstrates an importance in regulating haemostasis and brin clearance (Sitter et al., 1995). Following serosal injury, there is a ne balance between these processes, which if disrupted may result in the formation of adhesions, bands of brous tissue that occur in up to 95% of patients following surgery. Adhesions initially form as brin-rich deposits between damaged, closely opposed serosal surfaces. If there is insufcient serosal brinolytic activity, these brin-rich adhesions persist, become organised by invading broblasts and endothelial cells and with subsequent collagen deposition form permanent brous 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 regeneration of the mesothelium. For a more extensive review of mesothelial cell function see Mutsaers (2002).
4. Mesothelial healing Hertzler (1919) was the rst to observe that small and large peritoneal wounds healed in the same amount of time. He concluded that the mesothelium could not regenerate solely by proliferation and centripetal migration of cells at the wound edge as occurs 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 begins within 24 h of injury with the appearance of a population of rounded cells, predominantly neutrophils 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 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 conditions, 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 stimulated 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-
jury. Later, kinetic studies using [3 H]-thymidine incorporation in rodent models conrmed 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 studies showed that uninjured murine testicular mesothelium has a 0.25% basal mitotic activity, which upon stimulation by the exogenous addition of peritoneal inammatory lavage cells and activated macrophages, increased to values greater than 12% (Mutsaers et al., 2002). As inammatory cells collect on the wound surface within the rst 24 h of injury, it is likely that they play a signicant 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
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 dened by thick arrows, is identied by a high density of cells, many of which are inammatory 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 from Mutsaers et al. (2000).
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complete within 710 days of injury when the wound area is covered by cells displaying all the characteristics 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 additional sources for the regenerating mesothelial cells. These include: macrophage transformation (Eskeland & Kjaerheim, 1966; Ryan, Grobety, & Majno, 1973), exfoliation of mature or proliferating mesothelial cells from adjacent or opposing serosal surfaces (Whitaker & Papadimitriou, 1985; Johnson & Whitting, 1962; Mutsaers et al., 2000; Fotev et al., 1987; Cameron, Hassan, & De, 1957; Watters & Buck, 1972), pre-existing free-oating serosal progenitor cells that implant on the wound and differentiate into mesothelial cells (Ryan et al., 1973), subserosal mesenchymal precursors that convert into mesothelial 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 experimental evidence suggests that a free-oating serosal progenitor 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-oating serosal cells. Further evidence for a free-oating progenitor arises from peritoneal uid studies where a signicantly higher number of viable free-oating mesothelial cells were recovered from experimental animals 2 days following injury compared with the control uninjured animals (Whitaker & Papadimitriou, 1985; Fotev et al., 1987). Our own group has performed cell-tracking and labelling studies in rodent models and conclusively shown that serosal healing involves the incorporation and proliferation of free-oating 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 regenerating mesothelium as demonstrated by cell junction formation. Peritoneal macrophages also attached to injured areas but failed to incorporate whereas peritoneal broblasts failed to attach, as did mesothelial cells to uninjured areas. This suggests that free-oating mesothelial cells are able to adhere to exposed and deposited ECM substrates such as collagen, bronectin, vitronectin and possibly brin following injury, undergo cell division and integrate into the mesothelial layer. It is not known whether these free-oating cells are desquamated mesothelial cells from the serosal lining, a resident peritoneal uid sub-population or a dedicated 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 nding still needs to be conrmed.
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 adhesions demonstrated that they were all well vascularised and innervated and contained clusters of smooth muscle cells, the origin of which was unclear (Herrick et al., 2000; Sulaiman et al., 2001). It has been proposed that adhesions form as a consequence of reduced brinolytic activity in serosal tissues. This has been shown both in human studies and genetically modied 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 brinolytic pathway (Sitter et al., 1995). If mesothelial healing is impaired, there is a reduction in local PA secretion which reduces brinolytic activity. Two major therapeutic approaches have been investigated to prevent adhesion formation: brinolytic
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agents and barrier devices such as membranes and gels. However, due to complications associated with bleeding, systemic brinolysis, injury to the internal organs and vessels, impaired wound healing and difculty 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 brinolytic capacity. Another approach to increase the rate of serosal repair is through the exogenous addition of mesothelial cells. Several groups have demonstrated that instillation of autologous mesothelial cells at the time of injury prevents adhesion formation (Di Paolo, Vanni, & Sacchi, 1990; Bertram et al., 1999). Di Paolo et al. (1990) found that intraperitoneal (i.p.) injection of cultured autologous omental mesothelial cells in rabbits with staphylococcal-induced peritonitis signicantly reduced the formation of adhesions. In a clinical study by the same group, four uremic peritoneal dialysis patients recovering from severe peritonitis were injected i.p. with 3 108 of their own mesothelial cells, previously 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 the prevention 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 mesothelial cells increased serosal repair so prevented adhesion formation, although this has not been conrmed. These ndings again support the concept that a free-oating progenitor mesothelial cell is involved in mesothelial repair however, they also raise a number of important questions. For example, it is not clear whether the free-oating injected cells are different from the resident mesothelial cells of the serosal lining, 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-oating mesothelial cells obtained from peritoneal lavage and peritoneal wall into injured serosa (Foley-Comer et al., 2002), suggesting that omental mesothelial cells alone may not be the only cells involved in mesothelial regeneration. The concept that these free-oating mesothelial progenitor cells may have stem cell-like qualities is supported by the ndings of Lucas, Warejcka, Zhang, Newman, and Young (1996). They isolated and cultured 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. Adhesion number was signicantly reduced in the animals receiving living MSCs at the time of surgery in a concentration dependent manner, whereas adhesion had increased in the animals receiving MSCs 46 h after 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 brin-rich adhesions, such as brinolytic proteases or growth factors that stimulate mesothelial healing. Cells injected 46 h after injury are likely to have been trapped within deposited brin and may have differentiated into broblasts 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 adherent and free-oating serosal cells following injury to determine the exact roles they play in normal and abnormal 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 derived 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 characteristics 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 ndings have customarily been explained by the theory that there exists a population of subserosal multipotential cells with the ability to differentiate along both mesenchymal and mesothelial pathways; a concept originally suggested by Klemperer and Rabin (1931). Indeed Raftery (1973) described the involvement of a subserosal precursor cell in the repair of the mesothelium, that appeared intermediate in form between primitive mesenchymal cells on one hand and proliferating broblasts or endothelial cells on the other. Bolen et al. (1986, 1987) provided the best support for a multipotential subserosal cell using light, ultrastructural and immunohistochemical techniques to examine intermediate lament expression in reactive and non-reactive human serosal tissue. The group demonstrated that normal surface mesothelial cells express low and high molecular weight cytokeratins whereas submesothelial cells express only vimentin. However, in biopsies from injured serosa, submesothelial cells lost vimentin immunoreactivity and progressively acquired high and low molecular weight cytokeratins. It was suggested that these cells were differentiating towards a mesothelial cell phenotype and were responsible for the re-establishment of surface mesothelium. However, Whitaker et al. (1992) in a similar study were unable to reproduce these ndings and suggested that the staining pattern seen by Bolen and colleagues may be a result of mature mesothelial cells migrating into the subserosal connective tissue. In another study, Amari, Taguchi, Iwahara, Shibuya, and Naoe (2002) reported that cultured spheroids composed of free-oating multicellular clusters of rat pleural 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-broblast growth factor receptor
antibody, suggesting FGF plays a key role in the phenotypic conversion of broblasts into regenerated mesothelial cells. Further support for a multipotential submesothelial cell comes from experimental ndings 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 transient expression of cytokeratin 18 in subserosal mesenchymal cells. At a later stage, new muscle expressing smooth muscle myosin and desmin, was detected in the subserosal layer in the absence of mitotic activity in the original smooth muscle layer (Pampinella et al., 1996). In agreement with their previous ndings (Buoro et al., 1993), the authors concluded that resident keratin expressing subserosal mesenchymal cells transformed into myobroblasts and subsequently into fetal-type smooth muscle cells a well as regenerating mesothelial cells (Buoro et al., 1993; Pampinella et al., 1996). Taken together these ndings would seem to support the view that a multipotential subserosal mesenchymal cell exists which can differentiate into myobroblasts and possibly smooth muscle cells as well as mesothelial cells. However, new evidence suggests that the mesothelial cells themselves may be multipotential and have the ability to differentiate into various different cell types. Indeed, irradiation and kinetic studies have also questioned the role of subserosal 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 uid demonstrate cobblestone epithelioid morphology in culture. However, it has long been known that these cells can change to a broblastic phenotype with repeated passage, reducing cytokeratin and increasing vimentin expression (Mackay, Tracy, & Craighead, 1990). Various growth factors can also induce mesothelial cells to change phenotype and express many of the characteristics associated with broblasts such as increased motility and enhanced ECM production (Fig. 3). For example, EGF induces the reversible change to a broblastic phenotype 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) broblastic phenotypes, at different passage numbers of the same cell preparation. Micrographs courtesy of Jason Tee.
of 1 integrins, in particular 21, facilitating an enhanced 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 mesothelial cells (Harvey & Amlot, 1983; Owens & Milligan, 1994; Yang, Kim, Lee, Park, & Kim, 1999). Various benign disorders, including liver cirrhosis, endometriosis or serosal inammation, produce effusions that often contain increased numbers of mesothelial cells thought to be derived from the reactive serosa. In culture, these cells demonstrate both broblastic 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 different stages of differentiation, and it is likely that in disease, inammatory factors and other mediators direct cells down various phenotypic pathways. The expression 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 described as a mesothelial lineage marker. Whitaker et al. (1992) rst suggested that mature mesothelial cells could transform into broblast-like cells in vivo and invade the underlying subserosal connective tissue. Indeed, they suggested that this could account for the intermediate lament staining pattern observed by Bolen et al. (1986). This seems an unusual concept because in contrast to mesenchymal stromal cells, epithelial-like cells infrequently convert into broblasts in mature tissue, apart from during wound healing or tumour progression (Hay, 1995). However, two recent reports investigating the pathological effects of continuous ambulatory peritoneal dialysis (CAPD) have provided strong evidence to support this concept. CAPD is known to cause peritoneal brosis leading to a failure of ultraltration however, the mechanisms involved in this process are not clear. In the rst study, Yang, Chen, and Lin (2003) demonstrated that transforming growth factor-1 (TGF-1) induced human omental mesothelial cells to transdifferentiate into myobroblasts in vitro with the characteristic appearance of prominent RER, conspicuous smooth muscle actin myolaments, intermediate and gap junctions and active deposition of ECM. Gene expression analysis revealed a complex modulation of gene expression involving cytoskeletal organisation, cell adhesion, ECM production, cell proliferation, innate immunity, stress responses and many other essential metabolic processes as the mesothelial cells underwent transformation. The authors proposed that the differentiated epithelial cells of the mesothelium convert into myobroblasts and that the pathological features observed following CAPD may be due to the recruitment of brogenic cells from the mesothelium during serosal inammation and wound healing. The second study by Ynez-Mo et al. (2003) also demonstrated that human mesothelial cells undergo a conversion from an epithelial to mesenchymal phenotype which occurred in patients following serosal injury. Peritoneal mesothelial cells isolated from dialysis uid efuents displayed a mesenchymal phenotype 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 transcriptional repressor snail. They also acquired a migratory phenotype with up-regulation of 2 integrins. Major probrotic and inammatory 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 cytokeratins, on broblast-like cells embedded in the subserosal layer, suggesting that these cells were derived 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 broblast-like cells with pseudopodial protrusions and increased migratory, invasive and brogenic features (Hay, 1995). However, it is currently unknown whether the mesothelial cells remain as myobroblasts, continue to differentiate into smooth muscle cells or revert back to surface mesothelial cells. Furthermore, it is unclear whether the mesothelial cells that undergo trandifferentiation are a resident population in the mesothelial layer, originate from a serosal uid subpopulation or are from a circulating blood-derived source. Nevertheless, the authors do suggest that in light of these recent ndings, the earlier concept of a multipotential subserosal cell being able to convert to both epithelial mesothelial cells and myobroblasts (Raftery, 1973; Bolen et al., 1986) should be questioned. Furthermore, it raises the interesting possibility that mesothelial transdifferentiation may be wholly or partly responsible for the pathological changes that occur in the serosal layer following trauma caused by, for example, CAPD, irradiation, malignancy or surgery.
8.1. Vascular grafts Despite considerable clinical research, no biological or synthetic grafts have been developed as an ideal substitute for small diameter arteries (Nerem & Seliktar, 2001). When acellular articial prostheses are used in the reconstruction of small diameter vessels, failure frequently occurs because the luminal surface is thrombogenic resulting in thrombus formation and re-occlusion following implantation. Cell seeding should decrease thrombogenicity of implanted vascular 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, rabbit or mouse, initiate an inammatory 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 rst to demonstrate that a mesothelial membrane could be grown on the outer surface of a free-oating diffusion chamber placed in the peritoneal cavity of rats. Later ultrastructural studies showed that mesothelial cells deposited and organised ECM; including thick collagen bres, the amorphous components of elastic bres and basement 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 brinolytic and antithrombotic properties of mesothelial cells (Louagie et al., 1986), Clarke, Pittilo, Machin, and Woolf (1984) proposed that autologous mesothelial cells may represent a practical alternative to endothelial cells in vascular grafts. Subsequently, many groups have investigated the efcacy of using mesothelial cells, mainly derived from the omentum, as endothelial replacements (Sparks et al., 2002; Bull et 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 mesothelial cells promoted luminal cell cover, displayed anti-thrombogenic activity, inhibited platelet aggregation and released more prostacyclin than unseeded grafts in canine abdominal aorta replacements. However, later studies using digested omental extract seeded onto knitted Dacron scaffolds and implanted
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.
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as bilateral femoral artery replacements, suggested that the mesothelial cells were not retained on the graft 24 h later (Bearn et al., 1992). Furthermore, bronectin coated small diameter polytetrauoroethylene (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 replaced 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 improvement in short term patency compared to PTFE alone (Theuer et al., 1996). Despite these disappointing ndings, Campbell, Efendy, and Campbell (1999) using an alternative seeding method, have produced more favourable results. Free-oating silastic tubing was implanted into the peritoneal cavity of rats and rabbits and after two weeks, the ones that remained free-oating were removed and processed. When the tubes were everted and histologically assessed they consisted of an intima of non-thrombotic mesothelial cells, a media of smooth muscle-like cells or myobroblasts embedded in a collagen and elastic matrix, and an outer collagenous 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 myolament expression (evidence of smooth muscle phenotype) in the grafts over time, which was also observed by cyclically 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 biocompatible with the host tissue, need no articial mesh as part of the wall, have a nonthrombogenic surface and develop elastic lamellae. Moreover, they have demonstrated patency for at least 4 months with 1020%
contractile responses compared with the control artery after transplantation. However, many questions remain unanswered such as; how similar the inner surface lining of mesothelial cells are to true endothelial cells, and are mesothelial cells in the intimal layer subsequently replaced by local ingrowth of endothelial cells following transplantation to high pressure arterial sites. Indeed, if the free-oating 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 bioarticial vascular grafts as reviewed by Moldovan 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 denaturation 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 uid (Tiwari et al., 2003) is a better method for generating tissue engineered grafts, awaits further investigation. Until then, the use of mesothelial cells as endothelial cell replacements still remains a possibility and may prove important in, for example, the development of autologous coronary artery bypass grafts or arteriovenous access stulae for hemodialysis patients.
8.2. Omental grafts The scientic community has neglected the omentum 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 embedded 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 oats passively within the peritoneal cavity but rapidly adheres to inamed or damaged tissues. In a post-mortem study, Weibel and Manjo (1973) found that the omentum was the organ most frequently involved in adhesion formation and many workers have suggested that omental adhesions offer protection against more severe complications such as peritonitis and ischaemic bowel disease (Williams & White, 1986; Hasgood, 1990). Part of the omentums ability to rescue injured tissue is likely to be due to its angiogenic (Goldsmith, Grifth, Kupferman, & Catsimpoolas, 1984; Goldsmith, Grifth, & Catsimpoolas, 1986) and neurotrophic (Chamorro et al., 1993) properties; hence, its use as a pedicle graft tissue for clinical conditions 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 neurodegenerative 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 became revascularised 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 involved. In another study, Chamorro et al. (1993) used free omental grafts to facilitate nerve graft regeneration in rats by surrounding the nerve graft with omentum. Early revascularisation and directional growth of sprouting axons was encouraged, thus increasing the efciency 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 often incomplete due to loss or misdirection of nerve bres and neuroma formation. The use of nerve replacements composed of articial tubes seeded with isolated mesothelial cells as an alternative to primary nerve suture has been introduced as a biological approach to nerve injuries. Initial studies in a rat model by Lundborg et al. (1982) investigated the regeneration 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 axonal 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 uid, 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 studies demonstrated that when rabbit hypoglossal nerves were repaired using mesothelial chambers, a signicantly 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 found around the tube lacked primary inammatory signs at follow-up after 1 year and showed no signs of compression (Dahlin & Lundborg, 2001). Similar to the studies of Chamorro 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 functional 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 mesothelial cells was not described in these studies, articial tubes lined by mesothelial cells appear to be important 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 remarkably poorly understood and in general, there is a lack of a unifying denition as well as specic markers to dene them. A rich reservoir of adult stem cells resides in specic niches within the bone marrow microenvironment as well as in a variety of connective tissues, where they are maintained in an undifferentiated 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 example, when appropriately induced, mesenchymal stem cells (MSCs) have the potential to differentiate along specic mesenchymal lineages (multipotency) and form tissues that include endothelium, muscle, bone, cartilage and fat (reviewed by Tuan, Boland & Tuli, 2003). Although a mesothelial stem cell has not been identied, growing evidence based on its primitive embryological origin and ability to transdifferentiate 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 origin of associated mesothelial tumours. Since then, as previously described, tissue culture and animal experimental studies have convincingly demonstrated that adult mesothelial cells are capable of transdifferentiating from an epithelial to mesenchymal phenotype and this seems to depend on the presence of certain 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 specic mesenchymal cell lineages is still lacking. Munoz-Chapuli et al. (1999) recently hypothesised that 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 progenitor. 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 mesothelium differentiates into endothelium or smooth muscle through an epithelial-mesenchymal transition (EMT) process. This nding raised the interesting question of whether the coelomic mesothelium retains its ability 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 specic growth factors. The authors suggest that a cell line derived from rat epicardial mesothelial cells acts in a similar manner to the bipotential vascular progenitor cells, a stem cell population originally described by Yamashita et al. (2000). As well as the intriguing possibility that adult mesothelial progenitor cells are able to produce endothelium and smooth muscle, ndings from several experimental models suggest that these cells may also form skeletal muscle and cartilage. For instance, during the healing phase of a chemical-induced peritonitis, skeletal muscle bres were found to develop de novo in the peritoneal lining of the adult rat diaphragm. The location and orientation of the bres suggested an origin from mesothelial or submesothelial cells in granulation tissue rather than intrinsic diaphragmatic muscle satellite cells (Levine & Saltzman, 1994; Drakontides, Danon, & Levine, 1999). However, more extensive studies are required to conrm these ndings. If the mesothelium is the source of new skeletal muscle bres, as the authors state, it will be important to determine if the diaphragmatic mesothelium is different from mesothelium in other locations. Indeed, it would be desirable to imitate the environment of the inamed diaphragmatic peritoneum in
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other areas of skeletal muscle damage where regeneration is needed. Although rare, it is of no surprise that biopsies taken from human malignant mesothelioma express markers of osseous and cartilaginous differentiation (Donna & Betta, 1986; Yousem & Hochholzer, 1987; Kiyozuka et al., 1999; Andrion, Mazzucco, Bernardi, & Mollo, 1989). Furthermore, in experimental models, bone and cartilage were found in peritoneal malignant mesotheliomas that were induced by i.p. injection of asbestos bres (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 ossication (or osseous 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 described earlier. However, in light of recent ndings, 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-oating in the serosal uid or alternatively, may be derived from a circulating multipotential cell population which enters serosal cavities via the vasculature. This matter is further complicated by the observation that cells of a haemopoietic origin, identied through bone marrow transplant and Ly5 antigen expression, are able to differentiate into myobroblasts and smooth muscle cells in response to a foreign body implanted into the peritoneal cavity (Campbell, Efendy, Han, Girjes, & Campbell, 2000). Depending on the local environment these progenitor cells may be able to progress down various differentiation pathways. Growth factors 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-oating cells are in continuous communication with peritoneal uid 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 myobroblasts, smooth muscle cells and endothelial cells. In addition, they may detach from the basement membrane and become free-oating progenitor cells in the serosal uid 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 specic lineages upon stimulation, other key features of stem cells are to remain in a quiescent undifferentiated state until provided with the signal to divide asymmetrically and undergo many more replicative cycles than normal. Future studies have yet to determine if these are 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 broblasts and in vivo senesce following exposure to dialysis uids (Thomas et al., 1997; Gotloib, Wajsbrot, & Shostak, 2003). Furthermore, whether serosal uid contains survival factors that allow mesothelial progenitors to remain viable and proliferative or if it contains chemoattractants that cause circulating progenitor cells to home to
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areas of serosal injury are all intriguing questions still to be answered.
10. Future directions To begin to address the question of whether a mesothelial stem cell exists, immediate studies should focus on isolating reservoirs of mesothelial progenitor cells harboured in the three serosal cavities, either resident within various sites of the mesothelial lining or free-oating in serosal uid, both pre- and post-injury. At present, there is only limited empirical information on how to select, propagate, differentiate and characterise mesothelial cells (Simsir et al., 1999; Stylianou, Jenner, Davies, Coles, & Williams, 1990; Faris et al., 1994; Ross et al., 1998). In particular, the cell markers that could be used are not well dened but those that are currently considered include cytokeratins 8 and 18, vimentin (LaRocca & Rheinwald, 1984), calretinin (Fetsch, Simsir, & Abati, 2001), desmin (Hurlimann, 1994), HBME-1 (Donna et al., 1997), WT-1 (Muir, Cheville, & Lager, 2001) mesothelin (Chang & Pastan, 1996) and CD31 (Chung-Welch, Patton, Shepro, & Cambria, 1997). Future studies that address this issue of identication are crucial, although the point along a differentiation pathway (stem, progenitor or mature) at which the cells are isolated may alter the expression of some of these markers. Furthermore, whether traditional mesothelial cell culture conditions select particular subsets of cells and stimulate them to progress down a certain differentiation pathway needs to be considered. Even though this eld is still in its infancy, the limited ndings presented to date are encouraging and suggest that an accessible and abundant supply of mesothelial progenitor cells is available for tissue repair, regenerative medicine and tissue engineering applications. Exploitation of these progenitor cells may offer the opportunity to effectively repair a variety of tissues and expected long-term clinical applications are numerous. For example, in the prevention of adhesions autologous mesothelial progenitor cells may be injected into the patient or immobilising factors administered that recruit the progenitor cells to the serosal cavity of the patient before surgery. In addition, the development of biocompatible peritoneal grafts to repair anastomosis and hernia defects is an
interesting possibility. Mesothelial cells still remain a viable alternative to endothelial cells in tissue engineered small diameter vascular grafts and as a inner lining for nerve replacements. These therapeutic approaches may involve incorporating mesothelial progenitor cells induced to a particular differentiation state in either culture using specic growth factors, or genetic manipulation, or in situ in combination with appropriate biomaterials and mediators, prior to implantation in the patient. In vivo or ex vivo genetic manipulation of mesothelial cells has already been investigated for the delivery of therapeutic proteins to protect the peritoneal membranes of peritoneal dialysis patients (Hoff et al., 1998; Hekking et al., 2003). In addition, Nagy et al. (1995) used this approach to demonstrate sustained systemic delivery of recombinant proteins, which may have applications for the treatment of inherited or acquired disorders requiring delivery of therapeutic proteins to the circulation. However, to fully appreciate the potential of mesothelial progenitor cells, it is essential that future studies are directed at elucidating their cellular and molecular characteristics to optimise identication, isolation and expansion and to fully understand their differentiation capacity as well as the role they play in normal and abnormal repair events.
Acknowledgements We thank Dr. Grenham Ireland and Dr. Denis Headon for useful comments. We acknowledge research funding from the Medical Research Council, UK (SEH) and Heart Foundation and Raine Medical Research Foundation, Australia (SEM).
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Mesothelial Progenitor Cells and Their Potential

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The International Journal of Biochemistry & Cell Biology 36 (2004) 621642

Mesothelial progenitor cells and their potential in tissue engineering

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areas of serosal injury are all intriguing questions still to be answered.

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