Song Chengang, Wang Jiachuan, Mo Cuiping, Mu Shuhua, Jiang Xiaogang, Li Xiaoyun, Zhong Shizhen, Zhao Zhenfu, Zhou Guangqian
Department of Anatomy, Institute of Clinical Anatomy, Southern Medical University, Guangzhou, China.
School of Medicine, Shenzhen University, Shenzhen, China; Department of Pathology, Shenzhen Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine, Shenzhen, China.
PLoS One. 2015 Jul 15;10(7):e0132480. doi: 10.1371/journal.pone.0132480. eCollection 2015.
The purpose of this study was to establish a method for monitoring the neural differentiation of stem cells using ferritin transgene expression, under the control of a neural-differentiation-inducible promoter, and magnetic resonance imaging (MRI). Human adipose tissue-derived mesenchymal stem cells (hADMSCs) were transduced with a lentivirus containing the human ferritin heavy chain 1 (FTH1) gene coupled to one of three neural cell-specific promoters: human synapsin 1 promoter (SYN1p, for neurons), human glial fibrillary acidic protein promoter (GFAPp, for astrocytes), and human myelin basic protein promoter (MBPp, for oligodendrocytes). Three groups of neural-differentiation-inducible ferritin-expressing (NDIFE) hADMSCs were established: SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1. The proliferation rate of the NDIFE hADMSCs was evaluated using a Cell Counting Kit-8 assay. Ferritin expression was assessed with western blotting and immunofluorescent staining before and after the induction of differentiation in NDIFE hADMSCs. The intracellular iron content was measured with Prussian blue iron staining and inductively coupled plasma mass spectrometry. R2 relaxation rates were measured with MRI in vitro. The proliferation rates of control and NDIFE hADMSCs did not differ significantly (P > 0.05). SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1 hADMSCs expressed specific markers of neurons, astrocytes, and oligodendrocytes, respectively, after neural differentiation. Neural differentiation increased ferritin expression twofold, the intracellular iron content threefold, and the R2 relaxation rate two- to threefold in NDIFE hADMSCs, resulting in notable hypointensity in T2-weighted images (P < 0.05). These results were cross-validated. Thus, a link between neural differentiation and MRI signals (R2 relaxation rate) was established in hADMSCs. The use of MRI and neural-differentiation-inducible ferritin expression is a viable method for monitoring the neural differentiation of hADMSCs.
本研究的目的是建立一种方法，利用在神经分化诱导型启动子控制下的铁蛋白转基因表达以及磁共振成像（MRI）来监测干细胞的神经分化。用携带与人铁蛋白重链1（FTH1）基因偶联的慢病毒转导人脂肪组织来源的间充质干细胞（hADMSCs），该基因与三种神经细胞特异性启动子之一偶联：人突触素1启动子（SYN1p，用于神经元）、人胶质纤维酸性蛋白启动子（GFAPp，用于星形胶质细胞）和人髓鞘碱性蛋白启动子（MBPp，用于少突胶质细胞）。建立了三组神经分化诱导型铁蛋白表达（NDIFE）的hADMSCs：SYN1p - FTH1、GFAPp - FTH1和MBPp - FTH1。使用细胞计数试剂盒 - 8测定法评估NDIFE hADMSCs的增殖率。在NDIFE hADMSCs诱导分化前后，通过蛋白质印迹法和免疫荧光染色评估铁蛋白表达。用普鲁士蓝铁染色和电感耦合等离子体质谱法测量细胞内铁含量。在体外通过MRI测量R2弛豫率。对照和NDIFE hADMSCs的增殖率无显著差异（P>0.05）。神经分化后，SYN1p - FTH1、GFAPp - FTH1和MBPp - FTH1 hADMSCs分别表达神经元、星形胶质细胞和少突胶质细胞的特异性标志物。神经分化使NDIFE hADMSCs中的铁蛋白表达增加两倍，细胞内铁含量增加三倍，R2弛豫率增加两到三倍，导致T2加权图像中出现明显的低信号（P<0.05）。这些结果得到了交叉验证。因此，在hADMSCs中建立了神经分化与MRI信号（R2弛豫率）之间的联系。使用MRI和神经分化诱导型铁蛋白表达是监测hADMSCs神经分化的一种可行方法。
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