Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)
Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)
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DOI：
10.21769/bioprotoc.2255
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发表时间：
2017-05-05
期刊：
影响因子：
0.8
通讯作者：
Krasnodembskaya, Anna D.
中科院分区：
文献类型：
--
作者：
Jackson, Megan, V;Krasnodembskaya, Anna D.
间充质干细胞/基质细胞（MSC）是成体干细胞，其已显示在急性呼吸窘迫综合征（ARDS）和脓毒症的临床前模型中改善存活、增强细菌清除和减轻炎症。这些疾病的特征是不受控制的炎症，通常由细菌感染支持。MSC免疫调节作用的机制尚不完全清楚。我们试图研究MSC细胞与肺泡巨噬细胞（AM）的接触依赖性通讯，肺泡巨噬细胞是在肺部炎症反应和抗菌防御中发挥重要作用的专职吞噬细胞。通过使用基本的直接共培养系统，共聚焦显微镜和流式细胞术，我们可视化并有效地量化了MSC线粒体通过隧道纳米管（TNT）转移到AM。为了模拟人AM，从人供体血液中分离原代单核细胞，并在粒细胞巨噬细胞集落刺激因子（GM-CSF）的存在下分化成巨噬细胞（单核细胞衍生的巨噬细胞，MDM），从而允许适应AM样表型（de Almeida et al. 2000; Guilliams等人，2013年）。取人骨髓来源的MSC，用荧光标记后，充分洗涤，以1：20（MSC/MDM）的比例接种于含MDM的组织培养板中，共培养24 h。TNT形成和线粒体转移通过共聚焦显微镜观察，并通过流式细胞术进行半定量。通过使用我们在这里描述的方法，我们确定MSC使用TNT作为将线粒体转移到巨噬细胞的手段。进一步的研究表明，线粒体转移增强了巨噬细胞的氧化磷酸化和吞噬作用。当TNT的形成被细胞松弛素B阻断时，MSC对巨噬细胞吞噬的作用被完全消除。这是第一项证明TNT介导的线粒体从MSC转移到先天免疫细胞的研究。
Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence. With the use of a basic direct co-culture system, confocal microscopy and flow cytometry we visualised and effectively quantified MSC mitochondrial transfer to AM through tunnelling nanotubes (TNT). To model the human AM, primary monocytes were isolated from human donor blood and differentiated into macrophages (monocyte derived macrophages, MDM) in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), thus allowing adaptation of an AM-like phenotype (de Almeida et al., 2000; Guilliams et al., 2013). Human bone-marrow derived MSC, were labelled with mitochondria-specific fluorescent stain, washed extensively, seeded into the tissue culture plate with MDMs at the ratio of 1: 20 (MSC/MDM) and co-cultured for 24 h. TNT formation and mitochondrial transfer were visualised by confocal microscopy and semi-quantified by flow cytometry. By using the method we described here we established that MSC use TNTs as the means to transfer mitochondria to macrophages. Further studies demonstrated that mitochondrial transfer enhances macrophage oxidative phosphorylation and phagocytosis. When TNT formation was blocked by cytochalasin B, MSC effect on macrophage phagocytosis was completely abrogated. This is the first study to demonstrate TNT-mediated mitochondrial transfer from MSC to innate immune cells.