Human umbilical cord blood mesenchymal stem cells differentiate into neuron-like cells: in vitro induction by mouse nerve growth factor

doi:10.3969/j.issn.2095-4344.0492

Abstract

Abstract:

BACKGROUND: Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) are a kind of adult stem cells in the human umbilical cord blood, which have the potential to differentiate into neuron-like cells and can be used for the treatment of a variety of nervous system diseases. How to effectively induce hUCB-MSCs differentiation into neuron-like cells is always a hotspot in the stem cell research, which is of high scientific research value.
OBJECTIVE: To explore the induction effect of mouse nerve growth factor (mNGF) on the differentiation of human umbilical cord blood-derived mesenchymal stem cells into neuron-like cells in vitro.
METHODS: The donated hUCB-MSCs were resuscitated and the cell morphology after culture was observed to draw a cell growth curve. Passage 5 cells were cultured in the culture medium containing 0 (blank control), 50, 100, 150, 200 μg/L mNGF, and the cell morphology was observed and recorded daily under inverted phase contrast microscope. Immunocytochemistry detection was used to examine the expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) at 7 days of induction, and then the NSE and GFAP positive expression was calculated by Image-Pro Plus 6.0 software.
RESULTS AND CONCLUSION: The viability of resuscitated hUCB-MSCs was up to over 90%. The cell morphology was in long shuttle shape and spindle shape with unequal size, and the cells presented with “S”-shaped growth curve and entered the logarithmic growth phase at 3-6 days. Typical neuron-like changes were observed after induction by mNGF; however, there was no change in the cell morphology in the control group. Immunocytochemical staining showed that the induced cells were positive for both NSE and GFAP, and the highest positive rates of NSE and GFAP were observed after induction by 100 μg/L mNGF (P < 0.05). In the control group, there was no positive expression of NSE and GFAP. To conclude, mNGF can induce the in vitro differentiation of hUCB-MSCs into neuron-like cells, and 100 μg/L mNGF can achieve the best induction effect.

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: Nerve Growth Factor, Fetal Blood, Mesenchymal Stem Cells, Neurons, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Chen Jun, Yang Zi-jin. Human umbilical cord blood mesenchymal stem cells differentiate into neuron-like cells: in vitro induction by mouse nerve growth factor[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2027-2032.

Figures/Tables 1

2.1 脐血间充质干细胞复苏后的细胞活性用锥虫蓝染色检测复苏后细胞活性：正常活细胞的胞膜结构完整，锥虫蓝不能进入胞内，因此不能被染上颜色，此现象为“锥虫蓝拒染”；而死细胞或胞膜不完整的细胞，因胞膜破坏而可被锥虫蓝染成蓝色。实验中大部分活细胞为锥虫蓝拒染，少数死细胞被染成蓝色，计算细胞活性率达90%以上，表明细胞复苏后活性良好(图1)。 2.2 脐血间充质干细胞的生长曲线最初1-3 d细胞生长处于延滞期，此段时间内细胞生长较为缓慢；从第3天开始到第6天止细胞进入对数生长期，此段时间内细胞生长极为迅速，细胞数量以几何级数增长；6 d后细胞生长进入平台期，相比对数生长期增速明显减缓，生长曲线总体呈“S”型(图2)。 2.3 脐血间充质干细胞的形态特征倒置相差显微镜下观察可见脐血间充质干细胞形态为大小不一的长梭形、纺锤形 (图3A)，折光性强。培养六七天后细胞基本达80%融合状态，此时可见细胞集落呈漩涡状或放射状生长(图3B)。 2.4 mNGF诱导过程中脐血间充质干细胞形态学变化加入mNGF进行诱导分化培养后，每日在倒置相差显微镜下观察，诱导第1天各实验组脐血间充质干细胞形态均未见明显变化，诱导第 2，3天可见各实验组细胞形态发生改变——细胞胞体逐渐回缩变圆，胞体一侧或双侧伸出突起，呈现出单极、双极类神经元样改变，随着诱导时间的延长，可见细胞突起逐渐伸长并发出次级突起，诱导至第7天可见各实验组中相邻细胞通过突触相互连接，其中质量浓度为100 μg/L mNGF诱导组最为明显，而对照组脐血间充质干细胞形态则无明显改变，罕见类似细胞(图4)。7 d诱导时间内所有实验组细胞生长状态均良好，未见明显的脱落、死亡现象。 2.5 免疫细胞化学染色鉴定结果对mNGF诱导第7天的脐血间充质干细胞进行免疫细胞化学染色，发现各mNGF诱导组细胞NSE、GFAP均阳性表达，阳性细胞胞浆被染为棕褐色，而对照组细胞仅部分胞浆着色且着色浅，为非特异性染色，故NSE、GFAP均不表达为阴性(图5)。 2.6 统计学分析 ①各mNGF诱导组细胞NSE阳性率差异有显著性意义(F=41.52，P < 0.01)，50，100，150，200 μg/L mNGF诱导组NSE阳性率分别为(21.7±3.2)%，(37.2±3.4)%，(25.7±2.5)%，(25.3±4.9)%； ②各mNGF诱导组细胞GFAP阳性率差异也有显著性意义(F=37.01，P < 0.01)，50，100，150，200 μg/L mNGF诱导组GFAP阳性率分别为(30.5±2.9)%，(52.7±5.2)%，(39.3±2.5)%，(36.5±4.2)%；③各mNGF诱导组组间比较，100 μg/L组NSE，GFAP细胞阳性率均高于50，150，200 μg/L组，差异有显著性意义(P < 0.05，图6)。"

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