Peltzer Juliette, Lund Kyle, Goriot Marie-Emmanuelle, Grosbot Marion, Lataillade Jean-Jacques, Mauduit Philippe, Banzet Sébastien
Institut de Recherche Biomédicale des Armées, Clamart, France.
UMR-MD-1197, INSERM, Université Paris 11, Ministère de la défense, Villejuif, France.
Front Cell Dev Biol. 2020 Dec 15;8:581436. doi: 10.3389/fcell.2020.581436. eCollection 2020.
Mesenchymal stromal cell (MSC)-based cell therapy has received great interest in regenerative medicine. Priming the cells during the culture phase can improve their efficacy and/or survival after injection. The literature suggests that MSC extracellular vesicles (EV) can recapitulate a substantial part of the beneficial effects of the cells they originate from, and that micro-RNAs (miRNAs) are important players in EV biological action. Here, our aim was to determine if two classical priming methods of MSC, interferon-gamma (IFNγ) and hypoxia (HYP), could modify their EV miRNA content. Human bone marrow MSCs (BM-MSCs) from five healthy donors were cultured with IFNγ or in HYP or in control (CONT) conditions. The conditioned media were collected after 48 h in serum-free condition and EV were isolated by ultracentrifugation. Total RNA was isolated, pools of CONT, IFN, and HYP cDNA were prepared, and a miRNA profiling was performed using RT-qPCR. Then, miRNAs were selected based on their detectability and measured on each individual EV sample. Priming had no effect on EV amount or size distribution. A set of 81 miRNAs was detected in at least one of the pools of EVs. They were measured on each individual sample; 41 miRNAs were detected in all samples. The principal component analysis (PCA) failed to discriminate the groups. HYP induced a significant decrease in EV hsa-miR-34a-3p content and IFN induced a significant increase in five miRNAs (hsa-miR-25-3p, hsa-miR-106a-5p, hsa-miR-126-3p, hsa-miR-451a, and hsa-miR-665). Taken together, we found only limited alterations in the miRNA landscape of MSC EV with a high inter-individual variability.
基于间充质基质细胞（MSC）的细胞疗法在再生医学领域备受关注。在培养阶段对细胞进行预处理可以提高其注射后的疗效和/或存活率。文献表明，MSC细胞外囊泡（EV）可以概括其来源细胞的大部分有益作用，并且微小RNA（miRNA）是EV生物学作用的重要参与者。在此，我们的目的是确定两种经典的MSC预处理方法，即干扰素-γ（IFNγ）和缺氧（HYP），是否可以改变其EV miRNA含量。将来自五名健康供体的人骨髓MSC（BM-MSC）在IFNγ条件下、缺氧条件下或对照（CONT）条件下培养。在无血清条件下培养48小时后收集条件培养基，并通过超速离心分离EV。分离总RNA，制备CONT、IFN和HYP cDNA文库，并使用RT-qPCR进行miRNA谱分析。然后，根据miRNA的可检测性进行选择，并在每个单独的EV样品上进行测量。预处理对EV的数量或大小分布没有影响。在至少一个EV文库中检测到一组81种miRNA。在每个单独的样品上对它们进行测量；在所有样品中检测到41种miRNA。主成分分析（PCA）未能区分各组。HYP导致EV hsa-miR-34a-3p含量显著降低，IFN导致五种miRNA（hsa-miR-25-3p、hsa-miR-106a-5p、hsa-miR-126-3p、hsa-miR-451a和hsa-miR-665）显著增加。综上所述，我们发现MSC EV的miRNA图谱仅有有限的变化，且个体间差异很大。
