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微环境诱导iPSCs和BMSCs向神经元样细胞分化——Reelin对细胞分化和极性化的调节作用

付苏, 石贞玉, 范文娟, 符星, 邓锦波^*, 王强^*

河南大学神经生物研究所，开封 475004

摘要

本文旨在研究小鼠诱导性多能干细胞(induced pluripotent stem cells, iPSCs)和骨髓间充质干细胞 (bone marrow mesenchymal stem cells, BMSCs)在大脑微环境的诱导下向神经元样细胞分化的过程，探讨与细胞极性化相关的关键分子，以及Reelin蛋白对干细胞的分化的影响。用细胞脑片共培养以及细胞脑匀浆上清共培养的方法，将iPSCs和BMSCs分别与野生型(WT)和reelin基因缺失小鼠(reeler小鼠)的海马脑片以及脑匀浆上清共培养，观察二者在脑片微环境下的细胞分化和极性化改变。结果显示，与WT和reeler小鼠脑片共培养的iPSCs和BMSCs均出现神经元样细胞的分化；与WT小鼠海马脑片共培养的这两种细胞呈双C字型海马片层化分布，同时表现出很强的方位性，而与reeler小鼠海马脑片共培养的细胞分布则无明显规律性，呈均匀分布。脑匀浆上清培养基共培养的 iPSCs和BMSCs在共培养72 h后，部分类神经元样细胞可以被神经元特异标记物(NeuN)所标记，显示出分化成为功能性神经元。而在共培养初期(2~4天)，reeler微环境培养基中神经元分化和突起生长相对滞后。以上结果提示，在大脑微环境的诱导下，iPSCs和BMSCs可以向神经元样细胞，甚至向功能性神经元分化，Reelin蛋白参与了细胞极性化过程，而Reelin缺乏可造成神经细胞极性紊乱，延迟神经元分化及突起生长。

关键词： 诱导多能干细胞; 骨髓间充质干细胞; Reelin蛋白; 大脑微环境; 细胞分化; 细胞极性化

[Microenvironments induce iPSCs and BMSCs into neuron-like cells--Reelin’s regulative role in cell differentiation and polarization.] [Article in Chinese]

FU Su, SHI Zhen-Yu, FAN Wen-Juan, FU Xing, DENG Jin-Bo^*, WANG Qiang^*

Institute of Neurobiology, Henan University, Kaifeng 475004, China

Abstract

The present study was aimed to investigate how the induced pluripotent stem cells (iPSCs) and bone marrow mesenchymal stem cells (BMSCs) differentiate into neuron-like cells under the induction of hippocampal microenvironments and Reelin’s regulation. iPSCs or BMSCs were co-cultured with WT (wild type) or genotypic hippocampal slice and cerebral homogenate supernatant, then the stem cells’ differentiation under the induction of hippocampal environment was observed by using immunofluorescence technique. In the meantime, stem cells were co-cultured with hippocampal slice and cerebral conditioned medium of reeler (Reelin deletion) mouse respectively. The results showed that both adhesive iPSCs and BMSCs on WT hippocampal slice exhibited lamination of double “C” shape with high density on granular and pyramidal layers. The stem cells could differentiate into neuron-like cells with obvious polarization on WT hippocampal slice. In pyramidal cell layer, the differentiated neuron-like cells were oriented vertically with similar shapes of pyramidal cell in vivo, and the cells within molecule layer were arranged horizontally. In addition, adhesive iPSCs and BMSCs could differentiate into Nestin positive neural stem cells and NeuN positive neurons, respectively, under WT hippocampal microenvironment. On the other hand, under induction of hippocampal microenvironment of reeler mouse, iPSCs and BMSCs differentiation could also be seen, but their lamination was in disorder, and cell polarization was irregular. Moreover, differentiation and polarization of the iPSCs and BMSCs were delayed. These results suggest both iPSCs and BMSCs can differentiate into neuron-like cells under the induction of hippocampal microenvironments. Reelin is involved in the regulation of neuronal differentiation and cell polarization. Without Reelin, the cellular lamination and polarization appear irregular, and the stem cells’ differentiation is delayed.

Key words: iPSCs; BMSCs; Reelin; cerebral microenvironment; cell differentiation; cell polarization

收稿日期：2015-02-04　　录用日期：2015-05-04

通讯作者：邓锦波,王强　　E-mail: jinbo_deng@henu.edu.cn, kfwq@henu.edu.cn

引用本文：

付苏, 石贞玉, 范文娟, 符星, 邓锦波, 王强. 微环境诱导iPSCs和BMSCs向神经元样细胞分化——Reelin对细胞分化和极性化的调节作用[J]. 生理学报 2015; 67 (4): 357-369.

FU Su, SHI Zhen-Yu, FAN Wen-Juan, FU Xing, DENG Jin-Bo, WANG Qiang. [Microenvironments induce iPSCs and BMSCs into neuron-like cells--Reelin’s regulative role in cell differentiation and polarization.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (4): 357-369 (in Chinese with English abstract).