Use of Ferritin Expression, Regulated by Neural Cell-Specific Promoters in Human Adipose Tissue-Derived Mesenchymal Stem Cells, to Monitor Differentiation with Magnetic Resonance Imaging In Vitro

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.

本研究旨在建立一种基于铁蛋白转基因表达、受神经分化诱导型启动子调控，并结合磁共振成像（magnetic resonance imaging，MRI）的干细胞神经分化监测方法。将携带人铁蛋白重链1（human ferritin heavy chain 1，FTH1）基因的慢病毒，分别与三种神经细胞特异性启动子组合：人突触素1启动子（human synapsin 1 promoter，SYN1p，适用于神经元标记）、人胶质纤维酸性蛋白启动子（human glial fibrillary acidic protein promoter，GFAPp，适用于星形胶质细胞标记）、人髓鞘碱性蛋白启动子（human myelin basic protein promoter，MBPp，适用于少突胶质细胞标记），转导人脂肪来源间充质干细胞（human adipose tissue-derived mesenchymal stem cells，hADMSCs），成功构建三组神经分化诱导型铁蛋白表达（neural-differentiation-inducible ferritin-expressing，NDIFE）hADMSCs：SYN1p-FTH1、GFAPp-FTH1及MBPp-FTH1。采用细胞计数试剂盒8（Cell Counting Kit-8，CCK-8）法评估NDIFE hADMSCs的增殖速率；在分化诱导前后，通过蛋白质印迹法（western blotting）与免疫荧光染色检测NDIFE hADMSCs的铁蛋白表达水平；采用普鲁士蓝铁染色与电感耦合等离子体质谱法检测细胞内铁含量；体外通过MRI测量R2弛豫率。结果显示，对照组与NDIFE hADMSCs的增殖速率无显著统计学差异（P > 0.05）。SYN1p-FTH1、GFAPp-FTH1及MBPp-FTH1 hADMSCs在神经分化后，分别特异性表达神经元、星形胶质细胞与少突胶质细胞的标志物。神经分化可使NDIFE hADMSCs的铁蛋白表达上调2倍、细胞内铁含量上调3倍、R2弛豫率上调2~3倍，进而使T2加权图像出现显著低信号（P < 0.05），上述结果经交叉验证。综上，本研究在hADMSCs中建立了神经分化与MRI信号（R2弛豫率）之间的关联，采用MRI结合神经分化诱导型铁蛋白表达的方法，可实现对人脂肪来源间充质干细胞神经分化过程的有效监测。