Cardiac Nestin+ mesenchymal stromal cells enhance healing of ischemic heart through Periostin-mediated M2 macrophage polarization. Mus musculus

Background—Mesenchymal stem cells (MSCs) have shown therapeutic potency for treating cardiovascular diseases, but their therapeutic efficacy shows significant heterogeneity depending on their tissue of origin. We herein sought to identify the optimal source of MSCs for cardiovascular disease therapy. Methods—Heart- and bone marrow (BM)-derived GFP+ cells positive for the MSCs marker, Nestin, were flow cytometrically sorted from 7-day-postnatal Nestin-GFP transgenic mice. To study their biological characteristics in vitro, we characterized their self-renewal capacity, multi-lineage differentiation ability, and surface markers. To investigate their therapeutic potential in vivo, we intramyocardially injected Nestin+ cells (3×105) into the infarct border zone of mice subjected to acute myocardial infarction (MI), and performed echocardiography and Masson’s-Trichrome staining at 1 and 3 weeks post-MI. We characterized gene profiles with RNA-sequencing analysis, and analyzed the putative reparative mechanism, that of Periostin-mediated M2 macrophages polarization, by immunofluorescence staining, qPCR, ELISA, and flow cytometry in vivo and in vitro. Results—In vitro, the heart- and BM-derived Nestin+ cells (Nes+cMSCs and Nes+bmMSCs, respectively) were found to possess self-renewal ability, show similar tri-lineage differentiation potentials, and express some MSCs-related surface markers. Importantly, Nes+cMSCs significantly improved cardiac function, attenuated left ventricular (LV) remodeling, and decreased infarct size in the mouse MI model, compared with Nes+bmMSCs- or saline-treated MI controls. Nes+cMSC treatment notably reduced the total number of CD68+ pan-macrophages while inducing the polarization of macrophages toward an anti-inflammatory M2 phenotype in ischemic myocardium, compared with the saline-treated MI control. Periostin, which was highly expressed in Nes+cMSCs, could promote the polarization of M2-subtype macrophages in vitro and in vivo. Finally, Periostin knockdown remarkably reduced the therapeutic effects of Nes+cMSCs, inhibiting the survival and LV remodeling of MI model mice and significantly decreasing the number of M2 macrophages at lesion sites. Conclusions—Nestin+ cMSCs have greater efficacy than Nestin+ bmMSCs for cardiac repair following acute MI, using a reparative mechanism that acts at least partly through Periostin-mediated M2 macrophage polarization. Overall design: Examination of 2 different Nestin+ cells from Bone marrow and heart of 7-day old Nestin-GFP transgenic mice.

背景——间充质干细胞（Mesenchymal Stem Cells, MSCs）在心血管疾病治疗中已展现出治疗潜力，但其治疗效果因组织来源不同呈现显著异质性。本研究旨在确定适用于心血管疾病治疗的最佳间充质干细胞来源。方法——我们从出生7天的Nestin-GFP转基因小鼠体内，通过流式细胞术分选出表达间充质干细胞标志物巢蛋白（Nestin）的绿色荧光蛋白（GFP）阳性细胞，分别来源于心脏与骨髓（BM）。为体外探究其生物学特性，我们对其自我更新能力、多向分化潜能及表面标志物进行了系统鉴定。为体内研究其治疗潜能，我们向构建了急性心肌梗死（MI）模型的小鼠的梗死周边区域心肌内注射3×10^5个Nestin+细胞，并于心肌梗死后1周和3周分别开展超声心动图检测与马松三色染色。我们通过RNA测序（RNA-sequencing）分析基因表达谱，并借助体内外免疫荧光染色、qPCR、酶联免疫吸附实验（ELISA）及流式细胞术，解析了骨膜蛋白（Periostin）介导的M2巨噬细胞极化这一潜在修复机制。结果——体外实验表明，心脏来源与骨髓来源的Nestin+细胞（分别记为Nes+cMSCs与Nes+bmMSCs）均具备自我更新能力，拥有相似的三系分化潜能，并表达部分间充质干细胞相关表面标志物。值得注意的是，相较于Nes+bmMSCs治疗组与生理盐水处理的心肌梗死对照组，Nes+cMSCs可显著改善小鼠心肌梗死模型的心脏功能，减轻左心室（LV）重构，缩小梗死面积。与生理盐水处理的心肌梗死对照组相比，Nes+cMSCs治疗可显著减少缺血心肌中CD68+泛巨噬细胞的总数，同时诱导巨噬细胞向抗炎性M2表型极化。在Nes+cMSCs中高表达的骨膜蛋白，可在体内外促进M2型巨噬细胞极化。最后，敲低骨膜蛋白可显著削弱Nes+cMSCs的治疗效果，抑制心肌梗死模型小鼠的存活与左心室重构，并显著减少病变部位的M2巨噬细胞数量。结论——相较于Nes+bmMSCs，Nes+cMSCs在急性心肌梗死后的心脏修复中具有更优的治疗效果，其修复机制至少部分通过骨膜蛋白介导的M2巨噬细胞极化实现。整体实验设计：对取自出生7天的Nestin-GFP转基因小鼠骨髓与心脏组织的2种不同来源的Nestin+细胞进行检测分析。