Monosialotetrahexosyl ganglioside at an optimal concentration: inducing neuron-like differentiation of human umbilical cord mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.0490

Abstract

Abstract:

BACKGROUND: Studies have confirmed that monosialotetrahexosyl anglioside can induce human umbilical cord mesenchymal stem cells to differentiate into neuron-like cells, but little is reported on its optimal concentration.
OBJECTIVE: To explore the optimal concentration of monosialotetrahexosyl ganglioside that induces human umbilical cord mesenchymal stem cells to differentiate into neuron-like cells in vitro.
METHODS: Human umbilical cord mesenchymal stem cells were isolated by using collagenase digestion method, and after expansion, passage 3 cells were randomly allocated into five groups. When 70%-80% of cells were confluent, 50, 100, 150 and 200 mg/L monosialotetrahexosyl ganglioside induction solutions were added in corresponding experimental groups, while cells in the blank control group were cultured in the same volume of L-DMEM medium. Cell morphology was observed under inverted phase contrast microscope. Expression levels of microtubule-associated protein 2, neurofilament protein and glial fibrillary acidic protein were measured by using immunohistochemistry at 6 hours after induction.
RESULTS AND CONCLUSION: Human umbilical cord mesenchymal stem cells were isolated successfully and sub-cultured stably. These cells could express surface markers of mesenchymal stem cells. Monosialotetrahexosyl ganglioside at the optimal concentration of 150 mg/L was confirmed to induce the neuron-like differentiation of human umbilical cord mesenchymal stem cells, and differentiated cells could express microtubule-associated protein 2 and neurofilament protein as neuron-specific markers.

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: Umbilical Cord, Mesenchymal Stem Cells, G(M1) Ganglioside, Cell Differentiation, Neurons, Tissue Engineering

CLC Number:

R394.2

Zhang Peng, Zhao Zong-mao, Li Jian-hua, Liu Hui, Liu Yong-jun, Li Min-jie, Chen Ming-wei, Shen Lun,He Lei. Monosialotetrahexosyl ganglioside at an optimal concentration: inducing neuron-like differentiation of human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2039-2044.

Figures/Tables 1

2.1 hUMSCs形态变化以及冻存、复苏、传代对细胞生长的影响多数原代培养的细胞在12 h内能够贴壁，呈多边形、菱形，逐渐变为长梭形，细胞生长快速，传代稳定，至第10代仍能保持旺盛的增殖能力。将第6代、第10代冻存细胞复苏后以0.8×104/孔的密度接种到24孔板中，大多数细胞在接种24 h内贴壁，在第1，2天处于缓慢增长期，第3天增殖速度明显加快，当细胞融合达到80%时，增殖速度开始减慢，细胞培养至第7天时，细胞呈放射状或漩涡状排列，见图1。复苏各代细胞生长速度的比较无显著差异，细胞生长曲线见图2。 2.2 hUMSCs表面标志物的表达流式细胞仪检测显示：人脐带间充质干细胞表面标志物CD73、CD90和CD105表达均为阳性，CD19、CD45、CD11b、CD34、HLA-DR(MHC-Ⅱ)表达均为阴性(图3)。 2.3 各组神经样细胞数量及形态变化各组在诱导后1-3 h均可见神经元样细胞数量增加，50 mg/L单唾液酸四己糖神经节苷脂组最少，200 mg/L单唾液酸四己糖神经节苷脂组最多。诱导后4 h时，200 mg/L单唾液酸四己糖神经节苷脂组神经元样细胞数量不再增加，其他3组神经元样细胞数量继续增加，这4组细胞形态由纺锤样向双极细胞和多极细胞转变，空白对照组形态未见明显变化。诱导后5 h时，50，100 mg/L单唾液酸四己糖神经节苷脂组神经元样细胞数量增加不明显，部分细胞开始脱落，150 mg/L单唾液酸四己糖神经节苷脂组神经元样细胞数量最多，但也伴随少量细胞脱落，200 mg/L单唾液酸四己糖神经节苷脂组神经元样细胞数量明显下降，呈片状脱落。诱导后6 h时，50，100 mg/L单唾液酸四己糖神经节苷脂组神经元样细胞数量不再增加，150 mg/L单唾液酸四己糖神经节苷脂组神经元样细胞数量无明显变化，200 mg/L单唾液酸四己糖神经节苷脂组仅见少量神经元样细胞。由图4可以看出，200 mg/L单唾液酸四己糖神经节苷脂组前3 h阳性细胞率最高，升高速率也最快，其阳性率达最高值37%，然后神经元样细胞迅速脱落，至6 h降至最低。50，100 mg/L单唾液酸四己糖神经节苷脂组神经元样细胞阳性率缓慢上升，至第5小时达最高值，之后逐渐下降。150 mg/L单唾液酸四己糖神经节苷脂组前5 h上升速率较稳定，至第5小时达最高值42%，之后也均逐渐下降。由此可见，150 mg/L单唾液酸四己糖神经节苷脂组为最佳诱导组。 2.4 诱导细胞鉴定结果诱导6 h后，大部分神经元样细胞的神经元特异性标记物MAP-2和NF-H免疫细胞化学染色阳性(图5)，免疫组织化学染色呈棕黄色，免疫荧光染色呈黄绿色，胶质细胞特异性标记物GFAP免疫细胞化学染色阴性。空白对照组GFAP、MAP-2和NF-H免疫细胞化学染色均为阴性。由表1可见，任意单唾液酸四己糖神经节苷脂组组间MAP-2及NF-H表达差异均有显著性意义(P < 0.01)。"

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