Biological characteristics and dopaminergic neural-like cell differentiation potential of human amniotic membrane-derived mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2014.23.013

Abstract

Abstract:

BACKGROUND: Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered to be one kind of adult stem cells that can be easily obtained in large quantities without using an invasive method. Because of their low immunogenicity, anti-inflammatory properties, multipotency of differentiation and without ethical issue, human amniotic membrane-derived mesenchymal stem cells have been proposed as a good candidate to be used in cell therapy and regenerative medicine. However, the biological properties and the differentiation capacity of human amniotic membrane-derived mesenchymal stem cells are still poorly characterized.

OBJECTIVE: To establish a practical method for isolation and purification of human amniotic membrane-derived mesenchymal stem cells, and to study the biological characteristics and dopaminergic neural-like cell differentiation potential of the human amniotic membrane-derived mesenchymal stem cells.

METHODS: Human amniotic membrane-derived mesenchymal stem cells were disassociated and isolated from the amniotic membrane by trypsin and collagenase based enzymic digestion, and purified by percoll mediated density gradient centrifugation. Expressions of surface antigens and transcription factors of the human amniotic membrane-derived mesenchymal stem cells were determined by flow cytometry and western blot assays. Based on the osteogenic and adipogenic induction, the multipotent differentiation capability of human amniotic membrane-derived mesenchymal stem cells was determined. Induction of neural cell differentiation of human amniotic membrane-derived mesenchymal stem cells was conducted in Neurabasal conditioning medium with ATRA supplement. Neural cell associated bio-markers were determined by immunofluoresence staining and confocal microscope.

RESULTS AND CONCLUSION: In this study, we performed a practical method to isolate and purify human amniotic membrane-derived mesenchymal stem cells and amniotic epithelial cells simultaneously, with high cells yield. We demonstrated a group of constitutive expressions of neural antigens and embryonic associated transcription factor proteins (OCT-4, SOX-2 and KLF4) in fresh isolated human amniotic membrane-derived mesenchymal stem cells as well as in human amniotic membrane-derived mesenchymal stem cells after in vitro passage, which suggested that the human amniotic membrane-derived mesenchymal stem cells not only possessed intrinsic tendency to neural cell differentiation, but also maintained their stem cell characteristics after in vitro passage. We stimulated the human amniotic membrane-derived mesenchymal stem cells in the neurobasal-A and B27 based conditioning medium to induce neural cell differentiation. The induced human amniotic membrane-derived mesenchymal stem cells displayed an up-regulation of expression in panel of neural and dopaminergic associate molecules (β-tubulin III, neuron-specific nuclear protein, tyrosine hydroxylase, glial fibrillary acidic protein, myelin basic protein and nestin) by flow cytometry and immunofluorescence staining, which demonstrated the multipotent differentiation capability and dopaminergic neuron-like differentiation potential of the human amniotic membrane-derived mesenchymal stem cells.

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

全文链接：

Key words: stem cells, amnion, mesenchymal stem cells, epithelial cells, cell differentiation, dopamine, neurons

CLC Number:

R394.2

Zhou Wen-ran, Li Xin, Wang Wen-bo, Xie Yan-xia, Tang Na, Yan Ying. Biological characteristics and dopaminergic neural-like cell differentiation potential of human amniotic membrane-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3682-3690.

Figures/Tables 3

2.1 人羊膜间充质干细胞体外培养中的形态特点原代培养：经Ⅱ型胶原酶消化和Percoll纯化后收集的有核细胞在培养24 h后，有少部分细胞贴壁。刚贴壁的细胞呈圆形，折光性强。随后细胞胞质逐渐展开，折光性减弱，逐渐形成扁平单层细胞，呈漩涡状生长或成簇生长， 7 d细胞融合度达到80%。传代培养：传代细胞生长迅速，接种4 h后细胞贴壁，形态、大小均一，两三天可倍增，细胞可以稳定生长传代。用于诱导分化的第4代羊膜间充质干细胞形态(图1)。 2.2 人羊膜间充质干细胞相关抗原的表达原代羊膜间充质干细胞(图2)和第4代羊膜间充质干细胞(图3)细胞表面抗原和神经细胞相关抗原表达的特点。羊膜间充质干细胞不表达造血细胞特异性抗原CD45，CD14，CD11c和CD34(图中未展示)。原代和第4代羊膜间充质干细胞均表达间充质干细胞特异性标志抗原CD44，CD73和CD105，传代后的羊膜间充质干细胞上述抗原表达趋向均一，表达比例也有所增加。神经细胞相关抗原巢蛋白、CD133、β-微管蛋白Ⅲ和胶质纤维酸性蛋白在原代羊膜间充质干细胞中表达比例分别为2.44%、10.19%、47.06%和73.07%，而在第4代羊膜间充质干细胞中表达比例分别为3.66%、29.72%、71.08%、97.17%。髓鞘碱性蛋白、酪氨酸羟化酶和神经元特异性核蛋白等神经细胞标记物在原代羊膜间充质干细胞中表达比例为1.58%、2.22%、13.95%，在第4代羊膜间充质干细胞中表达比例分别为0.26%、0.14%和0.14%。 2.3 原代和体外培养第4代羊膜间充质干细胞表达与胚胎干细胞密切相关的转录因子蛋白 Western Blot方法检测结果显示，上述与胚胎干细胞生物学特征密切相的转录因子蛋白OCT-4和SOX-2在原代和体外培养第4代羊膜间充质干细胞中均有明显表达。KLF4在原代羊膜间充质干细胞中有微弱表达，在第4代羊膜间充质干细胞中表达明显(图4)。 2.4 人羊膜间充质干细胞向成骨细胞的诱导分化人羊膜间充质干细胞在诱导为骨细胞的初始阶段，细胞形态保持长梭形。随着诱导时间的延长，细胞增殖速度减慢，细胞趋向聚集，逐渐汇合呈铺路石状，细胞由长梭形变为胞体较小的立方形，同时在高倍镜下可见细胞呈复层生长。随后出现多角形成骨样细胞，胞质颜色变深，含粗大颗粒，胞核明显，胞外基质分泌逐渐增多，胶原堆积、钙盐沉积。诱导培养至10-12 d，细胞中有不透光钙结节形成(图5A)，钙结节茜素红染色阳性(图5B)。 2.5 人羊膜间充质干细胞向成脂细胞的诱导分化第4代羊膜间充质干细胞在成脂条件培养基诱导下，形态逐渐趋向圆形，胞体逐渐变大。在诱导第12天的细胞中可见大的脂肪滴，细胞介于不成熟脂肪细胞和成熟脂肪细胞之间阶段的标志(图5C)，用0.3%油红(Oil Red-O)染色，显微镜下可见阳性细胞(图5D)。"

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