Contreras Osvaldo, Rossi Fabio M, Brandan Enrique
Departamento de Biología Celular y Molecular and Center for Aging and Regeneration (CARE-ChileUC), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
Biomedical Research Centre, Department of Medical Genetics, 2222 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada.
Matrix Biol Plus. 2019 Apr 17;2:100006. doi: 10.1016/j.mbplus.2019.04.003. eCollection 2019 May.
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 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 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; and PDGFRα 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 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 mice 2-days after denervation. Our findings suggest that the pre-plating strategy allows the isolation and culture of a relatively pure aFbs population, which resembles FAPs .
细胞外基质（ECM）赋予骨骼肌结构和支撑，并为其中发现的多种细胞提供微环境。ECM主要由肌肉结缔组织（CT）成纤维细胞在发育和再生过程中产生。基质纤维脂肪生成祖细胞（FAPs）是CT成纤维细胞样间充质祖细胞（MPs），在再生和退变中起重要作用。慢性损伤会抑制肌肉成纤维细胞/FAPs的正常再生行为。因此，分离和研究这些间充质祖细胞对于了解它们的行为和生物学特性至关重要。我们研究了采用预铺板策略培养的成年肌肉CT成纤维细胞（以下简称贴壁成纤维细胞[aFbs]）是否属于FAPs的异质群体。通过结合显微镜检查、蛋白质印迹分析、流式细胞术和荧光激活细胞分选，我们确定从骨骼肌分离的aFbs与FAPs在很大程度上重叠。此外，我们使用PDGFRα小鼠，以便用EGFP FAPs来证实我们的结果。此外，我们的策略允许从鼠DMD模型PDGFRα和PDGFRα去神经支配小鼠中分离活化的EGFP FAPs。在此我们报告，1小时30分钟的预铺板策略允许分离和培养高度富集的aFbs群体。这些细胞在表型和生化方面是贴壁细胞的FAPs样群体。此外，aFbs对FAPs凋亡诱导剂尼洛替尼的反应方式与FAPs相同。此外，这些aFbs的流式细胞术表征表明，其中85%表达MP标志物PDGFRα，从PDGFRα小鼠中分离aFbs表明，其中75%显示高EGFP表达。此外，转化生长因子-β1诱导aFbs增殖、肌成纤维细胞分化和ECM产生。我们还能够从DMD小鼠的骨骼肌和去神经支配2天后的PDGFRα小鼠中分离活化的aFbs。我们的研究结果表明，预铺板策略允许分离和培养相对纯的aFbs群体，其类似于FAPs。
