Adherent muscle connective tissue fibroblasts are phenotypically and biochemically equivalent to stromal fibro/adipogenic progenitors.
Adherent muscle connective tissue fibroblasts are phenotypically and biochemically equivalent to stromal fibro/adipogenic progenitors.
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DOI：
10.1016/j.mbplus.2019.04.003
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发表时间：
2019-05
影响因子：
--
通讯作者：
Brandan E
中科院分区：
文献类型：
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作者：
Contreras O;Rossi FM;Brandan E
细胞外基质（ECM）提供结构，支持，是骨骼肌中发现的几种细胞的生态位。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。间质成纤维脂肪祖细胞（FAP）是CT成纤维细胞样的间充质祖细胞（MP），在再生和退化中起重要作用。慢性损伤抑制了肌肉成纤维细胞/FAP的正常再生行为。因此，这些间充质祖细胞的分离和研究对于了解它们的行为和生物学至关重要。我们研究了通过预铺板策略培养的成人肌肉CT成纤维细胞（下文称为粘附成纤维细胞[aFbs]）是否属于FAP的异质群体。通过结合显微镜、蛋白质印迹分析、流式细胞术和FACS，我们确定从骨骼肌分离的aFbs与FAP在很大程度上重叠。此外，我们使用PDGFRαEGFP小鼠以证实我们使用EGFP+ FAP的结果。此外，我们的策略允许从鼠DMD模型PDGFRαEGFP; mdx和PDGFRαEGFP去神经小鼠中分离活化的EGFP+ FAP。在这里，我们报告，1小时30分钟的预铺板策略允许分离和培养的高度富集的aFbs群体。这些细胞在表型和生物化学上是贴壁细胞的FAP样群体。此外，aFbs以与FAP相同的方式响应于尼洛替尼，尼洛替尼是FAP凋亡的诱导剂。此外，这些aFbs的流式细胞术表征表明，它们中的85%表达MP标记物PDGFRα，并且从PDGFRαEGFP小鼠中分离的aFbs表明，它们中的75%显示出高EGFP表达。此外，TGF-β1诱导aFbs增殖、肌成纤维细胞分化和ECM产生。我们还能够在去神经支配后2天从DMD小鼠的骨骼肌和PDGFRαEGFP小鼠中分离活化的aFbs。我们的研究结果表明，体外预接种策略允许分离和培养相对纯的aFbs群体，其类似于体外FAP。我们主要研究骨骼肌间充质祖细胞。我们研究肌肉CT成纤维细胞是否类似于成纤维细胞/成脂祖细胞。在这里，我们报告了一个预铺板的策略，用于分离和培养FAP样贴壁细胞的富集群体。贴壁成纤维细胞以与FAP相同的方式对TGF-β信号传导和酪氨酸激酶抑制剂尼洛替尼作出反应。体外预铺板策略允许分离和培养CT成纤维细胞，其在体外类似于FAP。
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 PDGFRαEGFP 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 PDGFRαEGFP; mdx and PDGFRαEGFP 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 PDGFRαEGFP 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 PDGFRαEGFP 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. We are focused in the study of the mesenchymal progenitors in skeletal muscle. We study whether muscle CT fibroblasts are similar to fibro/adipogenic progenitors. Here we report a pre-plating strategy for the isolation and culture of an enriched population of FAPs-like adherent cells. Adherent fibroblasts respond in the same fashion to TGF-β signaling and the tyrosine kinase inhibitor Nilotinib as FAPs. In vitro pre-plating strategy allows the isolation and culture of CT fibroblasts, which resembles FAPs in vitro.