Direct cell contact influences bone marrow mesenchymal stem cell fate

Abstract

Adult bone marrow-derived mesenchymal stem cells (MSC) can differentiate into various cell types of mesenchymal origin, but mechanisms regulating such cellular changes are unclear. We have conducted co-culture experiments to examine whether mesenchymal stem cell differentiation is influenced by indirect or direct contact with differentiated cells. Cultured adult mesenchymal stem cells showed some characteristics of synthetic state vascular smooth muscle cells (SMC). When co-cultured with vascular endothelial cells (EC) without cell contact, they exhibited abundant well-organised smooth muscle alpha-actin (α-actin) filaments. Direct co-culture with endothelial cells resulted in increased smooth muscle alpha-actin mRNA and protein, yet also comprehensive disruption of smooth muscle alpha-actin filament organisation. In order to assess whether these cell contact effects on mesenchymal stem cells were cell type specific, we also analysed direct co-cultures of mesenchymal stem cells with dermal fibroblasts. However, these experiments were characterised by the appearance of abundant spindle-shaped myofibroblast-like cells containing organised smooth muscle alpha-actin filaments. Thus, direct contact with distinct differentiated cells may be a critical determinant of mesenchymal stem cell fate in blood vessels and other connective tissues.

Introduction

Mature blood vessel walls have a complex three-dimensional structural organisation that reflects compartments of highly differentiated cells and their associated extracellular matrix (Drake, Hungerford, & Little, 1998; Hungerford & Little, 1999). The endothelial cells (EC) that line the lumen are separated from the smooth muscle cell (SMC) containing medial layer by a subendothelial extracellular matrix and the internal elastic lamina. Medial SMC are arranged in concentric lamellar layers that are separated by an elastic fibre-rich matrix. Fibroblasts of the outer collagen-rich adventitial layer are, in turn, separated from medial SMC by the outer elastic lamina. Recent studies have demonstrated that adult bone marrow stromal cell populations include spindle-shaped mesenchymal stem cells (MSC) that have the potential to differentiate into cells characteristic of blood vessels (Galmiche, Koteliansky, Briere, Herve, & Charbord, 1993; Kashiwakura et al., 2003; Kinner, Zaleskas, & Spector, 2002) in addition to bone, cartilage, tendon, muscle, fat and neural tissues (Pittenger et al., 1999). An MSC population fated to become SMC with self-expansion abilities has been identified using a tissue-specific promoter sorting approach (Kashiwakura et al., 2003).

Understanding the factors controlling the recruitment and differentiation of medial SMC and adventitial fibroblasts during vascular development are of importance in the treatment and prevention of vascular disease. EC tubes form during early vasculogenesis (Jain, 2003) then recruit mesenchymal cells which differentiate into the SMC that are responsible for deposition of vascular matrix and contractility. EC recruitment of mesenchymal cells, and their influence on SMC differentiation are poorly understood but probably involve growth factors such as PDGF-BB and TGF-β1 (Antonelli-Orlidge, Saunders, Smith, & D’Amore, 1989; Hellstrom, Kalen, Lindahl, Abramsson, & Betsholtz, 1999; Sato, Tsuboi, Lyons, Moses, & Rifkin, 1990). Newly recruited SMC are proliferative and deposit abundant extracellular matrix (Gerrity, Adams, & Cliff, 1975), but in maturing vessels SMC synthesise less matrix and become more quiescent and contractile (Kocher & Gabbiani, 1986). In culture, mature SMC characteristically exhibit a continuum of synthetic to contractile phenotypes (Owens, 1995). Fibroblasts recruited to the outer vessel wall deposit a collagen-rich adventitial matrix which constrains vessel wall elasticity. The origins of these adventitial fibroblasts are not well defined, but they may be derived from more than one embryological origin including mesenchymal cells (Dettman, Denetclaw, Ordahl, & Bristow, 1998). A cell continuum may exist from fibroblast to myofibroblast to SMC, all of which may derive from a common stem-like cell (Sartore et al., 2001). In developing coronary arteries, epicardial cells undergo an epithelial–mesenchymal transformation to form mesenchymal cells which are progenitors of both SMC and adventitial fibroblasts (Dettman et al., 1998).

During atherogenesis and vascular repair processes, contact and communication between EC and uncommitted MSC are likely to influence MSC differentiation into medial SMC and adventitial fibroblasts. Neointimal SMC, which play a key role in atherogenesis, originate at least in part from bone marrow (Sata et al., 2002, Shimizu et al., 2001). Bone marrow-derived cells in contact with EC within intimal lesions exhibit a SMC-like synthetic phenotype (Ross, 1993). Circulating SMC progenitors (Simper, Stalboerger, Panetta, Wang, & Caplice, 2002) originating in bone marrow may home to specific repair sites in the vasculature where the local vascular cells they contact (Kiger, Jones, Schulz, Rogers, & Fuller, 2001) may dictate their phenotypic behaviour (Blau, Brazelton, & Weimann, 2001). In vitro porcine cell studies indicated a key role for EC in regulating SMC differentiation (Chamley-Campbell & Campbell, 1981). However using rat SMC, bovine EC conditioned medium dramatically downregulated SM alpha-actin (α-actin) expression (Vernon, Thompson, & Owens, 1992). Direct co-culture of EC with mouse embryonic fibroblast 10T1/2 cells, or with transforming growth factor-beta (TGF-β), significantly upregulated SM α-actin expression and induced differentiation into contractile SMC-like cells (Hirschi, Rohovsky, & D’Amore, 1998). However, another group was unable to detect smooth muscle myosin expression in these cells after TGF-β treatment (Kumar & Owens, 2003). Direct co-culture of EC with SMC is known to locally activate TGF-β (Flaumenhaft et al., 1993). All these studies have monitored SMC phenotype by expression of cytoskeletal markers, but not direct examination of cytoskeletal organisation.

In this study we have investigated, at both expression and cytoskeletal organisation levels, how co-culture with EC and dermal fibroblasts influences MSC determination. We show that direct co-cultures of MSC with EC leads not only to increased SM α-actin expression but also comprehensively disrupts SM α-actin filament organisation. Interestingly, direct co-culture of MSC with fibroblasts leads to differentiation into myofibroblast-like cells. Thus, resident tissue cells are key determinants of the fate of recruited MSC.