Objective: To investigate whether there is mitochondrial transfer in dental mesenchymal stem cells (MSCs) and its significance for the odontogenic differentiation. Methods: Flow cytometry and immunohistochemical staining were used to isolate dental mesenchymal stem cells. Immunofluorescence staining and live cell imaging were applied to determine whether there is mitochondrial transfer in dental MSCs. Transcriptome sequencing data re-analysis of human dental pulp stem cells (DPSCs) and bone marrow mesenchymal stem cells (BMSCs) from gene expression omnibus (GEO) data base demonstrated the importance of mitochondrial transfer in dental MSCs. Cells were managed with mitochondrial transfer inhibitor ML141 with dimethyl sulfoxide as the control. Immunofluorescence staining, senescence-associated β-galactosidase (SA-β-gal) staining, reactive oxygen species (ROS) assay, 5-ethynyl-2'-deoxyuridine(Edu) labelling, cell counting kit-8 (CCK-8) assay, Western blotting, live cell imaging and transmission electron microscope were used to investigate cell morphology, ROS level, cellular senescence, cell proliferation, MSCs marker paired related homeobox 1 (Prrx1) and Sp7 transcription factor (Sp7) expression, mitochondrial transfer and mitochondrial morphology, respectively. Further, after using ML141 during the induction of odontogenic differentiation, alkaline phosphatase (ALP) chromogenic kit was used to detect ALP activity and real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of odontogenic differentiation-related genes Alp, Sp7, dentin matrix protein 1 (Dmp1), and dentin salivary phosphoprotein (Dspp), which were applied to investigate the effect of mitochondrial transfer on odontogenic differentiation. Results: An ultrafine tunneling nanotubes (TNTs) structure labelled with F-actin existed between dental MSCs, and the presence of transferring mitochondria in this structure was also confirmed. Transcriptome sequencing data suggested that the gene expression profiles were significantly different between DPSCs and BMSCs. Genes related to mitochondrial transfer and mitochondrial dynamic were significantly increased in DPSCs compared to BMSCs. Compared with the control group, treatment with 1, 5, 10 μmol/L ML141, the mitochondrial transfer inhibitor, had little significant effects on the cell morphology, cytoskeleton and ROS level. SA-β-gal activity and the proportion of SA-β-gal positive cells in the ML141-treated groups [(3.93±0.21)%, (3.23±0.42)%, (4.06±0.84)%] had no significant differences with the control group [(3.83±0.28)%] (all P>0.05). In the cell proliferation assay, the proportion of EdU positive cells in the ML141-treated groups [(20.00±3.82)%, (19.48±1.96)%, (12.55±2.86)%] had no significant differences (all P>0.05) with the control group [(18.57±0.87)%], whereas the CCK-8 assay showed similar results in ML141-treated group of 1, 5 μmol/L all P>0.05. Western blotting results showed that the protein expression levels of PRRX1 and SP7 in the ML141-treated group had no significant differences with the control group. Live cell imaging showed that compared with the control group [(31.42±4.01)%], the proportion of TNTs and mitochondrial transfer in the ML141-treated groups [(13.45±1.46)%, (10.36±3.47)%, (9.32±1.11)%] were significantly decreased in dental MSCs (all P<0.001). Scanning electron microscope showed that the mitochondrial morphology of dental MSCs in the ML141-treated group was similar to the control group, with globular and short-rod shape. After 7 days of odontogenic differentiation, the ALP staining intensity of the ML141-treated group was significantly lower than the control group. After 21 days of induction, RT-qPCR results showed that compared with control group, the relative mRNA expressions of Alp, Sp7, Dmp1 and Dspp were significantly decreased in the ML141-treated group (all P<0.05), indicating that the suppression of mitochondrial transfer in dental MSCs inhibited the odontogenic differentiation. Conclusions: Mitochondrial transfer exists between dental MSCs, and inhibition of mitochondrial transfer impairs the odontogenic differentiation.
目的： 探究牙源性间充质干细胞间是否存在线粒体转移以及线粒体转移对其成牙向分化的影响。 方法： 流式细胞术分选胚胎13~14 d小鼠下颌第一磨牙牙胚间充质细胞，得到牙源性间充质干细胞，通过免疫组织化学染色明确牙源性间充质干细胞属性。通过免疫荧光染色及活细胞成像检测牙源性间充质干细胞间是否存在线粒体转移；分析基因表达数据库中的人牙源性间充质干细胞与骨髓间充质干细胞转录组测序数据，探究线粒体相关基因在两组样本间是否存在差异。以二甲基亚砜作为对照组，使用不同浓度（1、5、10 μmol/L）线粒体转移抑制剂ML141对原代细胞进行处理，利用鬼笔环肽免疫荧光染色、细胞活性氧检测、衰老相关β-半乳糖苷酶（SA-β-gal）染色、5-乙炔-2′-脱氧尿苷（EdU）、细胞计数试剂盒（CCK-8）实验、蛋白质印迹法、活细胞成像以及透射电子显微镜观察间充质干细胞间线粒体转移被抑制后对细胞形态、细胞衰老、细胞活性氧、细胞增殖、间充质干细胞标志物配对相关同源基因1（Prrx1）和成牙分化相关基因Sp7转录因子（Sp7）蛋白水平、线粒体转移水平以及线粒体形态的影响。进一步在成牙向分化诱导过程中使用线粒体转移抑制剂，通过碱性磷酸酶（ALP）显色试剂盒检测ALP活性，实时荧光定量PCR（RT-qPCR）检测成牙分化相关基因Alp、Sp7、牙本质基质蛋白1（Dmp1）和牙本质涎磷蛋白（Dspp）表达，观察抑制牙源性间充质干细胞间线粒体转移对其成牙向分化能力的影响。 结果： 牙源性间充质干细胞间存在细长形态、被F-肌动蛋白（F-actin）标记的隧道纳米管（TNTs）结构，同时观察到该结构中存在正在转移的线粒体。转录组测序数据显示，牙源性间充质干细胞和骨髓间充质干细胞基因表达谱特征存在明显区别，牙源性间充质干细胞特异性上调线粒体转移及线粒体动力学相关基因。相较于对照组，1、5、10 μmol/L ML141处理组牙源性间充质干细胞的细胞形态、细胞骨架无明显变化，活性氧水平无明显变化，SA-β-gal活性、SA-β-gal阳性细胞比例［分别为（3.93±0.21）%、（3.23±0.42）%、（4.06±0.84）%］相较对照组［（3.83±0.28）%］差异均无统计学意义（均P>0.05）。细胞增殖实验中，1、5、10 μmol/L ML141处理组EdU阳性细胞比例［分别为（20.00±3.82）%、（19.48±1.96）%、（12.55±2.86）%］相较对照组［（18.57±0.87）%］差异均无统计学意义（均P>0.05），同时CCK-8结果也显示，与对照组相比，1、5 μmol/L ML141处理对细胞增殖活性无显著影响均P>0.05。蛋白质印迹法结果显示，ML141处理组PRRX1和SP7的表达与对照组无明显差异。活细胞成像结果显示，牙源性间充质干细胞间TNTs的形成比例和线粒体转移ML141处理组［分别为（13.45±1.46）%、（10.36±3.47）%、（9.32±1.11）%］相较对照组［（31.42±4.01）%］均显著降低（均P<0.001）。电镜结果显示，ML141处理组牙源性间充质干细胞线粒体形态与对照组相似，均呈球状、短棒状。成牙向分化7 d，ML141处理组ALP染色强度明显低于对照组；21 d RT-qPCR结果显示，与对照组相比，ML141处理组成牙分化相关基因Alp、Sp7、Dmp1和Dspp的mRNA相对表达水平均显著降低（均P<0.05），表明抑制牙源性间充质干细胞间线粒体转移使其成牙向分化能力降低。 结论： 牙源性间充质干细胞间存在线粒体转移，抑制线粒体转移影响其成牙向分化。.