Human umbilical cord blood-derived mesenchymal stem cells differentiate into neuron-like cells induced by combination of mouse nerve growth factor and brain-derived neurotrophic factor

doi:10.3969/j.issn.2095-4344.0482

Abstract

Abstract:

BACKGROUND: We have found that mouse nerve growth factor has the ability to induce differentiation of umbilical cord blood mesenchymal stem cells (UCB-MSCs) into neurons in vitro. In order to further explore the method of improving the induction efficiency of nerve cells, we attempt to combine a variety of cell growth factors for cell induction.
OBJECTIVE: To explore the effect of mouse nerve growth factor combined with brain-derived neurotrophic factor on the differentiation of UCB-MSCs into neuron-like cells in vitro.
METHODS: After the donated primary UCB-MSCs were resuscitated and cultured, the passage 5 UCB-MSCs were divided into five groups. The first four groups served as pre-induced groups, and fibroblast growth factor and epidermal growth factor were added to pre-induce cells for 24 hours, and mouse nerve growth factor and brain-derived neurotrophic factor, alone or in combination, were used thereafter to induce UCB-MSCs, while in control group, only the same amount of cell medium was added. The last group was non-pre-induced group, in which the cells were cultured in the cell culture medium for 24 hours, and then mouse nerve growth factor and brain-derived neurotrophic factor were both added to induce UCB-MSCs. The morphological changes of cells were observed under inverted microscope. The expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) was detected by immunocytochemistry technique. Real-time qPCR was used to detect the relative expression of NSE and GFAP at mRNA level.
RESULTS AND CONCLUSION: (1) The cell morphology of UCB-MSCs was in long shuttle shape and spindle shape with unequal size. After induction, the cell bodies gradually retracted and became rounded, and the projections extended to one-side or multi-sides, presenting with the neuron-like changes. (2) Immunocytochemistry and real-time qPCR results showed that NSE and GFAP were positive in each experimental group, and the positive rate and mRNA expression of NSE and GFAP in the combined induction group were higher than those in the other groups. (3) Either mouse nerve growth factor or brain-derived neurotrophic factor could induce UCB-MSCs to differentiate into neuron-like cells. Moreover, there was a cumulative effect between the two cytokines, and their combined use could effectively improve the efficiency of induction.

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: Fetal Blood, Mesenchymal Stem Cells, Nerve Growth Factor, Brain-Derived Neurotrophic Factor, Cell Differentiation, Neurons, Tissue Engineering

CLC Number:

R394.2

Chen Jun, Yang Zi-jin, Li Hong-mei. Human umbilical cord blood-derived mesenchymal stem cells differentiate into neuron-like cells induced by combination of mouse nerve growth factor and brain-derived neurotrophic factor[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2692-2698.

Figures/Tables 2

2.1 脐血间充质干细胞的形态特征倒置显微镜下观察第5代脐血间充质干细胞，其形态为大小不一的长梭形、纺锤形(图1A)。传代培养五六天细胞可达80%融合状态，此时镜下观察可见细胞集落呈漩涡状、放射状生长(图1B)。 2.2 诱导过程中脐血间充质干细胞形态学变化加入成纤维生长因子及表皮生长因子预诱导24 h后，细胞胞体回缩变圆，细胞边缘伸出单个或多个突起，而未经预诱导的脐血间充质干细胞则未见明显形态变化(图2)。正式诱导3 d后，细胞胞体以胞核为中心进一步回缩，细胞突起逐渐变细伸长，形态类似长蝌蚪形、细长条形，呈类神经元样细胞改变；正式诱导5-7 d后，可见局部区域内相邻细胞间通过突触相互连接。各实验组细胞有类似的形态变化，但联合诱导组的细胞形态改变最为显著(图3)。在7 d诱导时间内所有实验组细胞生长状态均良好，未见明显的细胞脱落、死亡现象。 2.3 免疫细胞化学染色鉴定对诱导第1，3，5，7天各实验组细胞行免疫细胞化学染色，发现各实验组细胞NSE、GFAP均阳性表达，表明经诱导后脐血间充质干细胞向神经元样细胞和星形胶质细胞分化。对相同诱导天数下各组细胞染色情况进行观察对比，发现mNGF+BDNF组细胞染色最深，NSE、GFAP阳性细胞多于mNGF组、BDNF组，对照组染色最浅，阳性细胞数较少，部分为非特异性染色(图4)。对比各实验组细胞NSE、GFAP阳性率(表2，3)，发现在7 d诱导时间内，mNGF组、BDNF组、mNGF+BDNF组、非预诱导组NSE、GFAP细胞阳性率均呈逐渐上升趋势，而对照组则逐渐下降；在诱导天数相同的情况下，mNGF+BDNF组NSE、GFAP细胞阳性率均高于mNGF组、BDNF组以及对照组，且高于非预诱导组，差异均有显著性意义(P < 0.05)。 2.4 实时荧光定量PCR检测结果各实验组细胞NSE mRNA和GFAP mRNA在诱导第1，3，5，7天时均有表达，且mNGF组、BDNF组、mNGF+BDNF组、非预诱导组二者表达量均呈上升趋势，在第7天时表达量最高，而对照组表达量则逐渐降低，总体趋势同免疫细胞化学染色结果相一致。相同诱导天数下mNGF+BDNF组NSE mRNA和GFAP mRNA的表达量高于mNGF组、BDNF组以及对照组，差异有显著性意义(P < 0.05)。另外，进行预诱导的mNGF+BDNF组NSE mRNA和GFAP mRNA的表达量明显高于非预诱导组，差异有显著性意义(P < 0.05)，见图5，6。"

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