Biological features and differentiation of human umbilical cord mesenchymal stem cells at different passages

doi:10.3969/j.issn.2095-4344.2017.13.009

Abstract

Abstract:

BACKGROUND: As a kind of emerging seed cells in the field of tissue engineering and regenerative medicine,
human umbilical cord mesenchymal stem cells are applied depending on whether it can remain stable biological characteristics and multi-directional differentiation capacity after long-term passage in vitro, which needs further experimental studies.
OBJECTIVE: To investigate differences of vital biological features of human umbilical cord mesenchymal stem cells over different passages.
METHODS: Human umbilical cord mesenchymal stem cells taken from Wharton's jelly were isolated and cultured using tissue explant method. The proliferation abilities of cells cultured for 3-6 generations were detected using both cell counting method and MTT method. Expressions of surface markers of the cells after expanding over nine passages were comprehensively determined by flow cytometry. Karyotype analysis of cells cultured for 3-6 generations was assessed using cell staining method. The 3rd generation of cells were used to detect differentiation abilities into nerve cells, osteoblasts and hepatocytes.
RESULTS AND CONCLUSION: Proliferation curve of cultured cells was “S” shape, and it was in the slow growth phase within 1-2 days, in the increased logarithmic growth phase within 2-4 days, in the plateau growth phase within 4-5 days, and in the declining growth phase within 5-6 days. Furthermore, two methods of testing results were consistent. Expression levels of cell surface markers CD90, CD44, CD73 were all above 90% while the expression levels of CD11b, CD18, CD34, CD43 and HLA-DR were very low at passages 1-9. No abnormality in cell karyotype was found in cells at passages 3-6. Human umbilical cord mesenchymal stem cells could differentiate into nerve cells, osteoblasts and hepatocytes. All theses experimental findings show that the human umbilical cord mesenchymal stem cells have high proliferation activity and multi-directional differentiation properties.

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: Stem Cells, Umbilical Cord, Mesenchymal Stem Cells, Tissue Engineering

CLC Number:

R394.2

Liu Yu-si, Wang Feng-chang, Xia Hai-xiong, Xu Jian-wei, Yang Yan, Cui Dong-bing, Wang Li-long, He Zhi-xu, Shu Li-ping. Biological features and differentiation of human umbilical cord mesenchymal stem cells at different passages[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(13): 2015-2022.

Figures/Tables 3

2.1 hUCMSCs的形态学鉴定采用组织块法分离培养5-7 d后，可见贴壁细胞从组织边缘爬出，原代细胞(P0)逐渐融合成片状生长，形态均一，由于组织块的存在不能完全贴满培养瓶，镜下观察细胞类似于成纤维细胞，成长梭形流水状生长(图1)，细胞增殖能力强，可稳定传至9代，P1-P9代细胞细胞形态规整，成长梭形流水状贴壁生长，细胞形态良好，并随着细胞的传代，细胞形态未发生变化。 2.2 hUCMSCs的增殖活性采用两种不同方法对hUCMSCs进行增殖学实验，即细胞计数法和MTT法。图2A为脐带间充质干细胞细胞计数法的增殖曲线，根据增殖曲线可看出，细胞在一二天时处于缓慢增长期，2-4 d时处于对数增长期，四五天时处于平台期，五六天时处于衰退期，不同代次细胞生长曲线相似。图2B示脐带间充质干细胞MTT法增殖曲线，细胞在一二天时处于缓慢增长期，2-4 d时处于对数增长期，四五天时处于平台期，五六天时处于衰退期，不同代次细胞生长曲线相似，其细胞增殖与细胞计数法基本相似，细胞增殖曲线呈“S”型。 2.3 hUCMSCs细胞表型分析流式细胞仪检测结果显示，hUCMSCs表达间充质干细胞表面标记CD90、CD44、CD73，且表达量均在90%以上；不表达或低表达CD11b、CD19、CD34、CD43及HLA-DR，其表达量呈现逐渐减低后逐渐增加的趋势，体外长期传代后，hUCMSCs 表面标记表达未发生显著改变(图3，表1)。 2.4 hUCMSCs核型分析为研究hUCMSCs随着传代代数的增加染色体是否发生畸变，采用染色体G显带法对第3-6代细胞进行了染色体核型分析(图4)。根据染色体的核型，可以看出核型为(46，XY)，并随着细胞代数的增加并未发现染色体的缺失、重复、倒位、异位、增多等畸形的改变，其核型均为正常人体的核型图。 2.5 hUCMSCs多向分化功能研究 2.5.1 hUCMSCs神经诱导分化第3代hHCMSCs神经预诱导24 h后，可见少量细胞略有变化，形态变为不规则。正式诱导1 h后部分细胞胞质向核回缩，胞体变小变短，呈不规则形或圆形，折光性强。诱导4 h后细胞胞体伸出长突起，有些可形成双极或多极突起，细胞突起向周围呈放射状，或相互连结形成网状，出现类似神经细胞的形态改变。经免疫组织化学检测胶质纤维酸性蛋白、神经元特异性烯醇化酶、微管相关蛋白的表达，hHCMSCs细胞核着色，阳性表达胶质纤维酸性蛋白、神经元特异性烯醇化酶、微管相关蛋白，细胞核呈棕褐色(图5)。 2.5.2 hUCMSCs成骨诱导分化第3代hHCMSCs成骨诱导14 d，细胞聚集汇合且呈多层重叠生长，形成圆形钙化结节沉积，周围折光性增强，茜素红染色着色呈红色为阳性表达(图6)。 2.5.3 hUCMSCs肝样诱导分化第3代hHCMSCs肝样诱导18 d后，光镜下可见细胞折光性增强，具有较多形态改变，细胞大小不均，呈多边形，类似肝细胞的形态学特征；核大而圆，居中，胞质内颗粒丰富，经免疫组织化学检测白蛋白和角蛋白的表达，诱导组细胞细胞核着色，阳性表达白蛋白和角蛋白，细胞核呈棕褐色(图7)。"

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