miR-146b facilitates the differentiation of induced pluripotent stem cells into neuron-like cells

doi:10.3969/j.issn.2095-4344.0517

Abstract

Abstract:

BACKGROUND: To facilitate the neuronal differentiation of induced pluripotent stem cells (iPSCs) is of great benefit for the repair of nerve injury and nerve regeneration.
OBJECTIVE: To investigate the expression of miR-146b in iPSCs differentiation, and its effects on the neural differentiation of iPSCs.
METHODS: After 7-day neural induction of mouse iPSCs, the expressions of miR-146b and pluripotent stem cell markers were detected by real-time PCR. Then, we generated miR-146b overexpressing iPSCs followed by the neural induction. The expression of stem cell and neuroectoderm markers were examined by cell immunofluorescence and real-time PCR respectively. Meanwhile, the expression of Tuj-1, a nerve cell marker, was also detected.
RESULTS AND CONCLUSION: Compared with the normal iPSCs, the expressions of pluripotent stem cell markers Nanog, Oct4 and Sox2 were significantly down-regulated in iPSCs undergoing 7-day neural induction, while the level of miR-146b was increased obviously. In miR-146b-overexpressing iPSCs, we found that the expression of Oct4 was substantially decreased, while there were no statistical changes in the expression of Nanog and Sox2. Meanwhile, during the neural differentiation, miR-146b overexpression significantly down-regulated the expression of Nanog, Oct4 and Sox2, and up-regulated the expression of neuroectoderm markers, Nestin, Sox1 and Pax6. After 18 days of neural induction, the miR-146b overexpression significantly increased the number of Tuj-1-positive cells. Taken together, miR-146b plays a vital role in facilitating the differentiation of iPSCs into neuron-like cells.

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, MicroRNAs, Neurons, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Li Qiang, Zhang Ming-wei, Li Jian-ming, Hu Jun-lin. miR-146b facilitates the differentiation of induced pluripotent stem cells into neuron-like cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2711-2716.

Figures/Tables 1

2.1 小鼠诱导多能干细胞向神经元样细胞诱导分化后干性标记分子的表达诱导多能干细胞向神经元样细胞诱导分化7 d时，反映细胞干性标记分子Oct4，Sox2和Nanog的表达水平较诱导前显著下调(P < 0.01，图1A)，提示大多数诱导多能干细胞干性消失并开始出现细胞分化。诱导7 d后细胞中miR-146b的水平是诱导前的2.08倍，二者间差异有显著性意义(P < 0.01，图1B)。 2.2 利用慢病毒构建过表达miR-146b的诱导多能干细胞小鼠诱导多能干细胞过表达miR-146b 48 h时，采用RT- qPCR检测miR-146b的表达水平，结果如图2所示，在感染之后过表达组的miR-146b水平较对照组显著升高，两组间差异有显著性意义(P < 0.01)，提示lenti miR-146b病毒过表达效果确切。 2.3 miR-146b过表达对诱导多能干细胞干性的影响 lenti miR-146b过表达效果经验证之后，实验探讨过表达miR-146b对诱导多能干细胞干性的影响。过表达miR-146b之后诱导多能干细胞干性标记分子Sox2和Nanog的表达水平与对照组细胞无明显差异，而过表达组的Oct4蛋白水平却较对照组显著降低，两组间差异有显著性意义(P < 0.01，图3A)。细胞免疫化学结果显示，过表达lenti-miR-146b后细胞干性标记分子Sox2和Nanog的表达水平与对照组细胞无明显差异，Oct4的表达水平则显著低于对照组(P < 0.01)，而细胞形态等未见明显变化(图3B-I)。 2.4 miR-146b过表达对诱导多能干细胞神经分化的影响 miR-146b过表达后诱导多能干细胞中Oct4表达水平显著降低，鉴于Oct4蛋白与神经外胚层的发育密切相关[22-23]，在进一步的研究中首先观察了miR-146b过表达对诱导多能干细胞向神经元样细胞分化过程的影响。成功转染lenti- miR-146b的诱导多能干细胞经神经分化7 d后，细胞多能性的标记分子Oct4，Sox2和Nanog的表达水平较诱导前显著降低(图4A)，提示大多数诱导多能干细胞干性消失并开始出现细胞分化，该结果与前面所得结果一致，以上结果说明诱导多能干细胞中miR-146b的过表达并不影响诱导多能干细胞的分化特性。随后，分别检测了经神经分化诱导后过表达组和对照组细胞神经干细胞标记分子的表达，结果显示两组细胞经相同的诱导过程之后，过表达组的神经干细胞标记分子Nestin、Sox1和Pax6的表达均显著高于对照组(P < 0.01，图4B)，提示miR-146b可促进诱导多能干细胞向神经方向分化。 2.5 miR-146b过表达可促进诱导多能干细胞的神经分化为进一步证实miR-146b过表达对诱导多能干细胞神经分化是否具有促进作用，行细胞免疫化学实验检测细胞中神经细胞标记分子Tuj-1的表达。如图5所示，诱导分化18 d后，对照组和过表达组细胞均可见Tuj-1的表达，但过表达组的阳性率和信号强度明显高于对照组，提示miR-146b可促进诱导多能干细胞向神经方向分化。"

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