Effect of cell passage on differentiation of bone marrow mesenchymal stem cells into neural stem cells

doi:10.3969/j.issn.2095-4344.2016.41.002

Abstract

Abstract:

BACKGROUND: It is unclear whether serial cell passage in vitro influences the differentiation of bone marrow mesenchymal stem cells into neural stem cells.
OBJECTIVE: To investigate the effect of cell passage on the differentiation of bone marrow mesenchymal stem cells into neural stem cells.
METHODS: Rat bone marrow mesenchymal stem cells were isolated and cultured by the whole bone marrow adherence method. Bone marrow mesenchymal stem cells at passages 3, 6, 9, 12 were incubated in serum-free medium. After culture for 7 and 14 days, cell biological characterization was observed and differenitaiton ability into neural stem cells was observed by detecting Nestin expression in cells using flow cytometry. Then, the cells were further induced to differentiate and cell multipotential differentiation capacity was detected by measurement of nerve enolase and glial acidic protein expression.
RESULTS AND CONCLUSION: Under induction, bone marrow mesenchymal stem cells at different passages were all differentiated into Nestin-positive neural stem cells. However, there was a significant difference in differentiation proportion of cells at different passages (P < 0.05). Strongest differentiation ability was found in the passage 6 cells, with the Nestin expression up to (93.7±2.3)% at 7 days of induction and (96.2±1.8)% at 14 days of induction. The proportion of differentiated cells at passages 6 and 9 was signfiicantly higher than that at passages 3 and 12. Moreover, adherent cells were positive for nerve enolase and glial acidic protein. All these findings indicate that the differentiation of bone marrow mesenchymal stem cells into neural stem cells is correlated with cell passage. Cells at lower or higher passages are both detrimental to cell differentiation.

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: Bone Marrow, Mesenchymal Stem Cells, Serial Passage, Cell Differentiation, Neural Stem Cells, Tissue Engineering

CLC Number:

R394.2

Liang Wei, Liu Zhou, Xu Zhi-en, Lin Li-feng, Fang Hong-ming. Effect of cell passage on differentiation of bone marrow mesenchymal stem cells into neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(41): 6092-6097.

Figures/Tables 2

2.1 骨髓间充质干细胞的细胞表型和生长曲线流式细胞仪检测第3代骨髓间充质干细胞的细胞表型，其中CD45阳性细胞比例为(1.4±0.5)%，CD34阳性细胞比例为(1.2±0.3)%，而CD90阳性细胞比例为(93.2±2.1)%，CD29阳性细胞比例为(89.6±7.9)%，提示经3代以上的传代培养可获得高纯度的大量骨髓间充质干细胞。镜下可见骨髓间充质干细胞的早期传代细胞多呈长梭形或纺锤形(图1A)，至第12代时呈多角型且细胞体积变大，胞浆明显增加(图1B)。生长曲线分析可见，体外培养的骨髓间充质干细胞生长有潜伏期、对数生长期和平台期(图2)。第3代骨髓间充质干细胞培养第2天就进入对数生长期，倍增时间平均为3.5 d，第6天进入平台期；第6代骨髓间充质干细胞潜伏期只有一两天，培养到第4天达到对数生长期，五六天进入平台期；第9代骨髓间充质干细胞倍增时间平均为3.7 d，第6天进入平台期；第12代骨髓间充质干细胞倍增时间为3 d，第4天进入平台期。以上说明体外培养的骨髓间充质干细胞生长增殖活跃，传代后第3-5天为较好的后续实验处理时间。 2.2 神经干细胞特异性标记物免疫表型鉴定结果分别取第3，6，9，12 代骨髓间充质干细胞，用胰酶消化成单细胞悬液后，按3×108 L-1细胞浓度接种于6孔板，悬浮于培养液中，逐渐聚集成大量大小不等的细胞团(图3)，分别诱导培养14 d后进行Nestin荧光染色，结果显示骨髓来源的神经干细胞球均呈Nestin阳性(图4)。流式细胞仪检测出各代细胞Nestin阳性率不同，各组间差异有显著性意义(P < 0.05)，其中第6代骨髓间充质干细胞向神经干细胞分化比例最高，不同代细胞的分化比例为第6代>第9代>第12代>第3代(表1)。 2.3 骨髓源性神经干细胞的多向分化能力鉴定将细胞球消化成单细胞悬液，贴壁培养7 d后行细胞免疫荧光染色，结果显示细胞球贴壁分化后可表达神经细胞标志物NSE、GFAP。NSE阳性细胞呈多极神经元形态，胞体发出一两个长短不一的突起，部分细胞突起相互连接成网状(图5A)。GFAP阳性细胞呈梭形，扁平状，胞体相对较大，胞体向四周发出放射状的突起，突起末端部分展成薄片状(图5B)，蓝色荧光为细胞核(DAPI)。以上证明诱导形成的细胞球为神经干细胞球，且能够分化成为神经元和神经胶质细胞。"

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