Identification of proliferation and differentiation capability of hippocampal neural stem/progenitor cells by adherent culture

doi:10.3969/j.issn.2095-4344.1592

Abstract

Abstract:

BACKGROUND: In vitro isolation and culture of neural stem/progenitor cells will provide a good cell model for the study of neurodevelopment, neurological diseases, and neural transplantation.
OBJECTIVE: To study the highly effective method for isolation and expansion of hippocampal neural stem/progenitor cells from newborn mice, and to identify the proliferation and differentiation of hippocampal neural stem/progenitor cells using improved adherent culture method.
METHODS: Neural stem/progenitor cells were isolated from the hippocampus of newborn C57BL/6 mice and were expanded for several passages. Combination of polylysine and laminin were used for adherent culture to promote cell attachment. Morphological observation and immunofluorescence cytochemical staining were conducted to detect the expression of neural progenitor-specific marker protein Nestin and proliferation index Ki-67. After 7 days of induction and differentiation, the expression of GFAP, DCX, Tuj1 and S100β was detected by immunofluorescence.
RESULTS AND CONCLUSION: About 82% of the cultured neural stem/progenitor cells expressed Nestin, and about 49% expressed Ki-67. A small number of cells were DCX-positive neurons after induction and differentiation, while most of the cells were positive for GFAP. The ratio of neurons to astrocytes was 1:1.7 identified by Tuj1 and S100β double staining. The neural stem/progenitor cells derived from the hippocampus were efficiently isolated and cultured. The cell proliferation and differentiation abilities were effectively identified after adherent culture, which can provide sufficient cell sources for further experimental research.

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: Neural Stem Cells, Hippocampus, Tissue Engineering

CLC Number:

Hu Yuting, Liu Fei, Liu Jing, Tao Xinrong. Identification of proliferation and differentiation capability of hippocampal neural stem/progenitor cells by adherent culture[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(9): 1330-1335.

Figures/Tables 1

2.1 神经干/祖细胞原代和传代培养形态变化从PBS悬液中收集原代细胞增加了提取细胞的数量，小鼠海马分离提取原代神经干/祖细胞当天，显微镜下可见细胞悬液中有单个细胞和细小组织碎片，单个细胞大部分呈现的是圆形，边缘清晰，透光性好，见图1A；继续培养3 d，悬液中依然有明显的细小组织碎片，漂浮着没有光泽的死细胞，培养皿底部有突起的贴壁细胞，部分单细胞存活聚集增殖形成小球，聚集的神经球稀疏且较松散，见图1B；培养至第5天，组织碎片仍然存在但所占比例较少，存活的神经干/祖细胞继续增殖形成大小不一的神经球，类似于桑葚状，细胞边缘有单个细胞凸起，边界清晰但不规则，透光性好，见图1C。原代细胞培养5 d传代，消化后继续培养3 d，细胞增殖形成直径为50-80 μm大小不等的神经球，见图1D；培养3 d后形成的神经球，细胞消化时能够轻易地将细胞消化下来，见图1E；然而传代后的细胞培养至第5天，形成的神经球偏大，多数细胞直径大于100 μm，神经球中央部分细胞密集，见图1F；细胞消化时不易成功消化，仍然存在未完全消化的神经球状态，见图1G。 2.2 神经干/祖细胞贴壁培养形态变化当只用100 mg/L多聚赖氨酸包被时，单细胞接种，培养4 d，神经干/祖细胞贴壁增殖形成边界清晰的克隆，见图2A，并且有部分神经球贴壁不牢固悬浮于培养液中；当用33 mg/L多聚赖氨酸和2 mg/L层粘连蛋白包被时，培养4 d，神经干/祖细胞增殖形成的克隆有一定程度的铺展，同时存在单个细胞贴壁且形态清晰，见图2B；当用33 mg/L多聚赖氨酸和5 mg/L层粘连蛋白包被时，培养4 d，神经干/祖细胞增殖形成的克隆完全铺展，细胞与细胞之间连接形成网状，见图2C。 2.3 神经干/祖细胞贴壁培养后Nestin的表达用于鉴定的细胞为第3代细胞贴壁培养至第4天的神经干/祖细胞，免疫荧光法检测特异性标志蛋白Nestin的表达。结果显示，贴壁培养后细胞染色清楚，单个细胞的胞浆和细胞核清晰辨别，部分克隆球中央也成功被染色；Nestin阳性即胞浆绿色细胞所占比例约为82%，细胞核被DAPI衬染为蓝色，Nestin阳性表达率高，说明成功培养得到神经干/祖细胞，见图3A。 2.4 神经干/祖细胞贴壁培养后增殖指标Ki-67的表达免疫细胞化学染色结果显示，部分神经干/祖细胞呈Ki-67阳性，经过ImageJ软件统计分析Ki-67阳性细胞占所有细胞比例约49%，见图3B。 2.5 神经干/祖细胞贴壁培养诱导分化及鉴定结果第2代培养第3天的神经干/祖细胞消化成单细胞悬液，贴壁培养诱导分化7 d，免疫荧光染色结果显示，少部分细胞DCX表达阳性，呈红色，说明少部分神经干/祖细胞分化为神经元，见图4A；大部分细胞经过诱导分化后GFAP反应阳性，呈绿色，见图4B；同时早期神经元Tuj1和S100β双染，神经元和星形胶质细胞比例为1∶1.7，见图4C，进一步说明了培养的细胞为神经干/祖细胞，并且具有向不同神经细胞方向分化的能力。"

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