Adherent muscle connective tissue fibroblasts are phenotypically and biochemically equivalent to stromal fibro/adipogenic progenitors

Abstract

Extracellular matrix (ECM) gives structure, support, and is the niche for several cells found in skeletal muscle. ECM is mainly produced by muscle connective tissue (CT) fibroblasts during development and regeneration. Stromal fibroadipogenic progenitors (FAPs) are CT fibroblasts-like mesenchymal progenitors (MPs) with important roles in regeneration and degeneration. Chronic damage restrains the normal regenerative behavior of muscle fibroblasts/FAPs. Thus, the isolation and study of these mesenchymal progenitors are of crucial importance for understanding their behavior and biology. We investigated whether adult muscle CT fibroblasts (hereafter referred to as adherent fibroblasts [aFbs]) cultured via pre-plating strategy belong to a heterogeneous population of FAPs. By combining microscopy, western blot analyses, flow cytometry, and FACS we determined that aFbs isolated from skeletal muscle largely overlap with FAPs. In addition, we used the PDGFRα EGFP mice in order to corroborate our results with EGFP + FAPs. Moreover, our strategy allows the isolation of activated EGFP + FAPs from the murine DMD model PDGFRα EGFP ; mdx and PDGFRα EGFP denervated mice. Here we report that 1 h 30 min of pre-plating strategy allows the isolation and culture of a highly enriched population of aFbs. These cells are phenotypically and biochemically a FAPs-like population of adherent cells. In addition, aFbs respond in the same fashion as FAPs to Nilotinib, an inducer of FAPs apoptosis. Moreover, flow cytometry characterization of these aFbs suggests that 85% of them express the MP marker PDGFRα, and isolation of aFbs from the PDGFRα EGFP mice suggests that 75% of them show high EGFP expression. Furthermore, TGF-β1 induces aFbs proliferation, myofibroblast differentiation, and ECM production. We were also able to isolate activated aFbs from skeletal muscle of the DMD mice and from the PDGFRα EGFP mice 2-days after denervation. Our findings suggest that the in vitro pre-plating strategy allows the isolation and culture of a relatively pure aFbs population, which resembles FAPs in vitro .

TGF-β-driven downregulation of the Wnt/β-Catenin transcription factor TCF7L2/TCF4 in PDGFRα+ fibroblasts

“…Others and we have demonstrated that sub-populations of stromal MSCs co-express both TCF7L2 and the tyrosine kinase receptor PDGFRα in development and adulthood (Contreras et al, 2016; Contreras et al, 2019a,b; Murphy et al, 2011; Vallecillo-García et al, 2017). As a consequence of the stromal expansion caused by injury both TCF7L2 and PDGFRα protein levels are increased in dystrophic tissue, denervated muscle, and after chronic barium chloride-induced damage (Contreras et al, 2016).…”

“…Chronic upregulation of TGF-β is found in several models of organ damage, where it is known to regulate tissue fibrosis severity (Kim et al, 2018; Pessina et al, 2015). Activated extracellular TGF-β not only promotes MSCs survival and proliferation but also primes these progenitor cells to become myofibroblasts (Contreras et al, 2019b, Contreras et al, 2019c; Cho et al, 2018; Kim et al, 2018; Lemos et al, 2015). TCF7L2 + MSCs are closely associated with CD68 + macrophages (one of the major cell sources of TGF-β in DMD) (Bentzinger et al, 2013; Contreras et al, 2016; Juban et al, 2018; Tidball and Villalta, 2010).…”

“…Even though TCF7L2 was initially described as a marker for CT fibroblasts (Kardon et al, 2003; Mathew et al, 2011; Merrell et al, 2015), increasing evidence suggests that CT fibroblasts shared many cell properties with FAPs (Contreras et al, 2019b; Malecova et al, 2018; Wosczyna and Rando, 2018). A recent report demonstrates that TCF7L2 protein but not mRNA levels are increased during adipocyte differentiation, thereby TCF7L2 plays a regulatory role in fibroblast-adipocyte fate decisions in vitro and in vivo (Chen et al, 2018).…”

“…Cells were isolated from undamaged muscles. For the TGF-β1 treatment experiment, after 72 h to 96 h in culture and 70–80% confluence, FAPs were stimulated with 5 ng/ml TGF-β1 (Contreras et al, 2019b). Cells were then collected for further analyses.…”

“…Interestingly, TGF-β inhibition improves the pathophysiology of MD (Accornero et al, 2014; Acuña et al, 2014; Ceco and McNally, 2013; Cohn et al, 2007; Danna et al, 2014). Mechanistically, TGF-β signaling stimulates FAPs and MSCs proliferation, extracellular matrix (ECM) production and myofibroblast differentiation (Contreras et al, 2019b; Hinz et al, 2007; Lemos et al, 2015; Uezumi et al, 2011). Hence, understanding the role of TGF-β signaling in MSCs may offer novel opportunities for the development of effective anti-fibrotic strategies and to improve tissue function.…”