Superparamagnetic iron oxide labeling influences in vitro differentiation of induced pluripotent stem cells

doi:10.3969/j.issn.2095-4344.0487

Abstract

Abstract:

BACKGROUND: Superparamagnetic iron oxide (SPIO) labeling technology is a classic noninvasive tracing method, which has been widely used in the stem cell transplantation. Induced pluripotent stem cells (iPSCs) are currently one of the most promising seed cells for cell transplantation. Whether SPIO labeling can also be used to noninvasively trace induced pluripotent stem cells is rarely reported, and concern has been raised about whether SPIO markedly impacts the differentiation of iPSCs.
OBJECTIVE: To investigate the effects of SPIO labeling on the differentiation of iPSCs in vitro.
METHODS: Rat fibroblasts were isolated and cultured. Efficient recombinant vector and plasmids that were packaged by virus and contained target genes (Oct4, Sox2, Klf4 and c-Myc) were transfected into 293T cells for virus packaging and production. The packaging lentiviral vectors that contained target genes infected rat fibroblasts to obtain iPSCs. SPIO-labeled (experimental) or unlabeled (control) iPSCs were subjected to neural induction and differentiation. Prussian blue staining and transmission electron microscope observation were performed for SPIO-labeled iPSCs. Immunohistochemical method was used to detect neuron-specific enolase expression after induced differentiation. Flow cytometry was used to detect the proportion of neurons and glial cells differentiated from iPSCs.
RESULTS AND CONCLUSION: There were dense iron particles in the cytoplasm of SPIO-labeled iPSCs shown by Prussian staining and under transmission electron microscope. Differentiated iPSCs were positive for neuron-specific enolase. In addition, the proportion of neurons and glial cells showed no difference between the experimental and control groups. To conclude, SPIO labeling has no obvious effect on the capacity of iPSCs differentiating into neurons. Reasonable application of this new cell labeling technique will promote the development of seed cells in regenerative medicine.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Induced Pluripotent Stem Cells, Craniocerebral Trauma, Superoxides, Tissue Engineering

CLC Number:

R349.2

Xie Qing-song, Shan Yu-dong, Fu Xiao-jun, Xu Xin-long, Hua Jie, Wen Lu-tong. Superparamagnetic iron oxide labeling influences in vitro differentiation of induced pluripotent stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2114-2119.

Figures/Tables 1

2.1 荧光显微镜观察分离培养获得的成纤维细胞取传代后48 h的大鼠成纤维细胞，荧光显微镜观察可见细胞贴壁生长，呈梭形且生长速度快(图1)，胞浆中可见波形蛋白染色，获得的成纤维细胞纯度约为96%。 2.2 慢病毒基因重组载体系统的构建分别用EcoRⅠ酶切慢病毒载体(图2)和含有目的基因(Oct4，Sox2，Klf4和c-Myc)的质粒，割胶回收线性化的载体及含有目的基因的质粒片段。将包含有目的基因的质粒片段与该慢病毒载体连接，得到慢病毒基因重组载体(图3)。将含有目的基因的慢病毒基因重组载体转染到293T细胞中获得大量能够表达该重组载体的高质量病毒。 2.3 标记前后iPSCs的形态取培养的iPSCs，吸掉细胞培养基，用PBS冲洗，胰酶消化，在荧光显微镜下观察，此时的iPSCs细胞质中未见明显的有色成分(图4A)；在光学显微镜下观察SPIO转染标记的iPSCs，此时的iPSCs细胞质中局部染色，呈浅褐色(图4B)。SPIO标记细胞行普鲁士蓝染色后，光镜观察可见细胞质内大量蓝色致密颗粒(图4C)，iPSCs的磁化标记有效率达95%以上(SPIO标记阳性率=阳性细胞数/细胞总数×100%)。透射电镜观察可见iPSCs胞浆内含大量黑色超顺磁氧化铁(图4D)。 2.4 免疫组化检测神经元细胞标记物神经元特异性烯醇化酶的阳性表达诱导分化后大部分细胞呈神经元特异性烯醇化酶阳性表达，iPSCs诱导分化率为(82.28±17.72)%，见图5A，B，而使用一抗为PBS的对照组则无阳性表达，见图5C，D。 2.5 流式细胞仪检测分化为神经元样细胞及胶质样细胞的比例实验组的神经元样细胞占总细胞的比例为74.4%，对照组的神经元样细胞占总细胞的比例为76.6%，两组差异无显著性意义(P > 0.05)。实验组的胶质样细胞和对照组的胶质样细胞占总细胞的比例分别为25.6%和23.4%，两组差异无显著性意义(P > 0.05)。见图6。"

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