Comparison of three kinds of mesenchymal stem cells differentiating into nerve cells under co-culture induction

doi:10.3969/j.issn.2095-4344.2017.17.015

Abstract

Abstract:

BACKGROUND: Scholars have been trying to create a microenvironment similar to the human body, which can induce the directional differentiation of mesenchymal stem cells from human bone marrow, placenta and umbilical cord blood.
OBJECTIVE: To compare the neuronal differentiation of human bone marrow mesenchymal stem cells, human placental mesenchymal stem cells and human umbilical cord blood mesenchymal stem cells induced by co-culture with nerve cells.
METHODS: Human bone marrow mesenchymal stem cells, human placental mesenchymal stem cells and human umbilical cord blood mesenchymal stem cells cultured in vitro were co-cultured with nerve cells using the Transwell system. The morphological changes of three kinds of cells in the co-culture system were detected. After co-culture for 4-5 days, immunofluorescence staining was used to measure the expression of neuron-specific enolase in cells. Mesenchymal stem cells only cultured in low glucose DMEM medium were used as controls.
RESULTS AND CONCLUSION: These three kinds of mesenchymal stem cells were extended, and interconnected processes were detective. The positive expression of neuron-specific enolase was highest in the human umbilical cord blood mesenchymal stem cells followed by human placental mesenchymal stem cells and human bone marrow mesenchymal stem cells in order. In the control group, none of the three kinds of mesenchymal stem cells have neuronal morphology, and the expression of neuron specific enolase was negative for the immunofluorescence staining. To conclude, microenvironment provided by nerve cells can induce these three kinds of mesenchymal stem cells to differentiate into neurons.

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: Mesenchymal Stem Cells, Coculture Techniques, Neurons, Cell Transdifferentiation, Tissue Engineering

CLC Number:

R394.2

Xu Li-li, Wang Hong-yuan, Li Xue-da, Liu Bing, Zheng Fang-fang, Yang Nai-long. Comparison of three kinds of mesenchymal stem cells differentiating into nerve cells under co-culture induction[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2714-2721.

Figures/Tables 2

2.1 人骨髓间充质干细胞形态细胞在第2天时开始贴壁生长，细胞两端有较长的突起，细胞核呈卵圆形，细胞克隆样生长，集落大小不一，细胞刚开始生长较缓慢，2周时融合可达85%左右。传代之后细胞生长均匀分布，为长梭形形态(图1)，有两三天的倍增时间。经过反复传代，其纯度可超过90%。 2.2 人骨髓间充质干细胞表面标志物表达通过流式细胞仪检测，人骨髓间充质干细胞CD44强表达，阳性率大于99%；不表达CD34，阳性率<1% (图2)。 2.3 人胎盘间充质干细胞的形态在第7天可见少量的细胞贴壁生长，第10天可见贴壁细胞增多(图3A)，第18天可见细胞融合约75%，细胞呈梭形、圆形和不规则形等形态，并混杂其他细胞杂质，传代之后细胞生长速度显著增快，三四天可传1代。传代之后细胞形态比较均一，以长梭形、平行生长为主，也有细胞表现为火焰、漩涡状生长，第3代之后细胞的纯度显著增高(图3B)。 2.4 人胎盘间充质干细胞表面标志物表达通过流式细胞仪检测，人胎盘间充质干细胞CD44及CD29强表达，两者阳性率均大于99%；CD34、CD106不表达，阳性率均< 1%(图4，5)。 2.5 人胎盘间充质干细胞超微结构通过透射电镜可看到细胞为长梭形，核仁较明显，核异染色质比较少，胞浆中有扩张的微管、微丝以及粗面内质网，且可看到内质网分泌微丝；少数细胞的核质比较大，胞浆较少，胞浆中的细胞器较少，符合间充质干细胞的特点(图6)。 2.6 脐血间充质干细胞形态细胞在第2天开始贴壁生长，细胞核呈卵圆形，细胞两端有较长的突起，克隆样生长，集落形成大小不一，细胞一开始生长较慢，2周后融合可达85%左右。细胞的形态呈多样化，如星形、小圆形、不规则形等。形态与人骨髓间充质干细胞相似，但体积较小，随着培养时间的延长，细胞的体积会逐渐变大并伸展生长(图7)。 2.7 脐血间充质干细胞表面标志物表达通过流式细胞仪检测，脐血间充质干细胞CD44及CD29均呈强表达；CD34不表达，符合间充质干细胞的特征。 2.8 脐血间充质干细胞超微结构通过透射电镜可见脐血间充质干细胞的细胞核呈类圆形以及不规则形，核膜清晰，核仁明显，染色质较粗且位于核膜周围。细胞质中细胞器的种类及数量众多，如微管、微丝、线粒体、粗面内质网等。通过扫描电镜可以看到在盖玻片表面贴壁生长的间充质干细胞，细胞呈长梭形，突起较长，可在细胞的表面看见微绒毛(图8)。 2.9 神经细胞形态培养第2天，大部分神经细胞已经贴壁，胞体形态多呈纺锤形，伸出小的突起。有些细胞的胞体形态呈体积较大的扁平形，并伸出数量不等的棒状大突起，表现为胶质细胞。培养6 d后，神经细胞胞体变大，细胞呈圆形及椭圆形，胞体周围伸展延长而成为单极、双极或者多极的突起(图9)。神经元特异性烯醇化酶染色阳性。 2.10 共培养细胞形态变化 2.10.1 人骨髓间充质干细胞与神经细胞共培养结果共培养体系中，在第3天即可观察到人骨髓间充质干细胞的形态变化，扁平的细胞缩小，胞体多呈圆形并形成较多突起，细胞形态逐渐成为星状(图10)。第4天之后星状细胞变多，并且可以互相连接，形态具有神经样特征，免疫荧光染色显示神经元特异性烯醇化酶表达阳性(图11)，阳性率平均为(32.7±11.5)%，表现为神经样细胞的特征；对照组培养约8 d后，大部分人骨髓间充质干细胞的形态仍旧扁而宽，没有形成神经样形态结构，免疫荧光染色显示神经元特异性烯醇化酶表达阴性。 2.10.2 人胎盘间充质干细胞与神经细胞共培养结果共培养体系中，在第3天即可观察到人胎盘间充质干细胞逐渐由之前的扁平变得细长，且可以看见丝状突触，部分细胞呈星状(图12)，对照组人胎盘间充质干细胞没有显著的改变。共培养4 d后免疫荧光染色显示神经元特异性烯醇化酶表达阳性(图13)，阳性率平均(35.3±13.5)%，表现出神经样细胞特性。 2.10.3 脐血间充质干细胞与神经细胞共培养结果共培养体系中，在第3天可见脐血间充质干细胞由之前的扁平形逐渐缩小，胞体多数呈圆形，并且形成了突起，部分细胞形态呈星状或者网格状(图14)。第6天之后星状细胞逐渐变多并互相连接，表现出神经样细胞的特征，免疫荧光染色显示神经元特异性烯醇化酶表达阳性(图15)，阳性率平均(37.5±12.7)%，具有神经样细胞的特性；而对照组脐血间充质干细胞培养第8天时，大多数细胞仍是长梭样形态，没有形成神经样的形态结构，免疫荧光染色显示神经元特异性烯醇化酶表达阴性。"

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