br Materials and methods br Results
Materials and methods
Discussion In this study we provide evidence that lung stromal methylergometrine manufacturer and their differentiated progeny have different capacities for the support of lung EpiSPC. We demonstrate that the expression of CD166 on primary isolated lung stromal cells correlates with a marked reduction in proliferative capacity and a commitment to myofibroblastic differentiation. This is consistent with previous findings in the bone marrow suggesting that CD166 marks a population of lung stromal cells with an increased propensity for myofibroblastic differentiation (Battula et al., 2009; Buhring et al., 2009). When injuries occur, tissue integrity is restored by the contraction of myofibroblasts around the wound and the deposition of new ECM forms the foundation for tissue re-epithelialization (Duffield et al., 2012; Scotton and Chambers, 2007). By virtue of these properties, myofibroblasts are also considered to be the driving force behind pulmonary fibrosis where their accumulation in the interstitial space results in excess deposition of ECM that distorts the architecture and function of the lung (Duffield et al., 2013). However, despite the immense interest in their role in pulmonary fibrosis, the cellular origin of myofibroblasts is still a contentious issue. Historically, it has been widely touted that myofibroblasts were derived from the transdifferentiation of epithelial cells through a process known as epithelial–mesenchymal transition (Willis and Borok, 2007). However, the recent identification of tissue resident multipotent MSC has led us to reconsider the genesis of myofibroblasts in solid organs (McQualter et al., 2009; Summer et al., 2007; Lama et al., 2007; Mailleux et al., 2005; Rock et al., 2011; Shan et al., 2008; Walker et al., 2011). Lineage-tracing studies now suggest that myofibroblasts are primarily derived from tissue resident MSC, with little evidence of a direct lineage relationship between epithelial cells and myofibroblasts in the adult lung (Rock et al., 2011). Our data supports the concept that myofibroblasts are derived from resident lung stromal cells that differentiate into myofibroblasts in response to TGF-β. Although the etiology of fibrosis is not well understood, it is known that TGF-β is a key driver of myofibroblast differentiation (Desmouliere et al., 1993; Sime et al., 1997; Thannickal et al., 2003; Gu et al., 2007) and has been shown to stimulate ECM secretion, increase contractility and activate pro-survival signaling pathways (Horowitz et al., 2007; Gauldie et al., 2006), all of which contribute to the perpetuation of fibrosis. What is not known is how the classical TGF-β mediated activation of lung stromal cells to a contractile, matrix-secreting phenotype affects epithelial repair. In this study we demonstrate that primary undifferentiated stromal cell progenitors act as EpiSPC support cells, whereas their more differentiated (α-SMApos) progeny do not. We also demonstrate that ex vivo expansion of lung stromal cells results in the selective expansion or induced differentiation of non-supportive stromal cells. However, the epithelial-supportive capacity could be restored by administration of a TGF-β receptor inhibitor (SB431542). There was no change in the expression of the myofibroblast differentiation marker αSMA between the epithelial-supportive and non-supportive stromal cells in vitro. This suggests that the epithelial-supportive capacity of lung stromal cells is regulated by the stimulation or inhibition of TGF-β signaling rather than differentiation alone. We also show that FGF-10 expression in stromal cells was downregulated after classical activation with TGF-β1 and upregulated after the inhibition of TGF-β with SB431542. Thus, the epithelial-supportive capacity of lung stromal cells is directly correlated with the level of FGF-10 expression. In the developing lung, FGF-10 is elaborated by lung stromal cells in the distal tip mesenchyme and is critical for the process of branching morphogenesis during lung development (Mailleux et al., 2005; Sekine et al., 1999; Min et al., 1998; Park et al., 1998; Bellusci et al., 1997). We have also previously shown that FGF-10 can partly replace the requirement for lung stromal cell support in our CFU-Epi assay (McQualter et al., 2010). More recently, it has also been shown that FGF-10pos lung stromal cells are recruited to the subepithelial space of naphthalene-injured airways as part of the primary wound healing response (Volckaert et al., 2011). The findings in this study support the idea that FGF-10-expressing stromal cells are important mediators of EpiSPC-mediated repair in the adult lung.