Directional differentiation of human amniotic mesenchymal stem cells into osteoblasts, chondrocytes and lipocytes in vitro

doi:10.3969/j.issn.2095-4344.2017.01.022

Abstract

Abstract:

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) are a kind of adult stem cells that can differentiate into osteocytes, chondrocytes and lipocytes under different conditions in vitro. Compared with other mesenchymal stem cells (MSCs), hAMSCs have many advantages like wide source, noninvasive isolation, low immunogenicity and short growth cycle, making hAMSCs an important source of seed cells in tissue engineering.
OBJECTIVE: To investigate whether hAMSCs have the characteristics of MSCs and the ability of differentiation
into osteoblasts, chondrocytes and lipocytes.
METHODS: Agreed by patients, the amniotic membrane from partus maturus parturients was separated, and hAMSCs were isolated by enzyme digestion. Passage 3 cells were cultured in osteogenic, chondrogenic or adipogenic medium induction.
RESULTS AND CONCLUSION: The alizarin red staining results showed multiple mineralized nodules after osteogenic induction, and the activity of alkaline phosphatase was significantly increased after induction. The toluidine blue staining showed positive results after chondrogenic induction, and a large amount of glycosaminoglycans were secreted from the cell matrix. After adipogenic differentiation, lipid droplets were observed in the cytoplasm of the cells shown by the oil red O staining. Real-time PCR results showed the mRNA expressions of relative genes at day 21 after osteogenic, chondrogenic and adipogenic induction upregulated significantly compared with the results at day 7 (P < 0.05). These experimental results demonstrate that hAMSCs have the basic characteristics of MSCs and possess the potential of differentiation into osteoblasts, chondrocytes and lipocytes, which could be selected as the seed cells for tissue engineering.

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: Cell Differentiation, Amnion, Tissue Engineering

CLC Number:

R394.2

Li Yu-wan, Zhu Xi-zhong, Jin Ying, Wang Sheng-min, Yang Ji-bin, Xiong Hua-zhang, Zou Gang, Liu Yi. Directional differentiation of human amniotic mesenchymal stem cells into osteoblasts, chondrocytes and lipocytes in vitro[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 122-127.

Figures/Tables 2

2.1 人羊膜间充质干细胞的形态倒置相差显微镜观察到原代人羊膜间充质干细胞呈椭圆形、多角形或不规则形态，贴壁生长；传代后细胞大多呈长梭形，部分可见漩涡状、鱼群状生长细胞(图1)。 2.2 人羊膜间充质干细胞的鉴定流式细胞术结果显示，人羊膜间充质干细胞呈CD73、CD90、CD105、CD44阳性，CD34，CD19，CD45，CD11b，HLA-DR阴性(图2)。 2.3 人羊膜间充质干细胞成骨诱导结果第3代人羊膜间充质干细胞成骨诱导7 d后，细胞逐渐汇合成铺短棒、铺路石状，细胞体积变大，密度增加，逐渐向成骨细胞转化，随诱导时间增加形成多个小圆形致密矿化结节，且逐渐变大，周围细胞密度较其他区域高，培养21 d后形成片状黑褐色骨化物质，透光性差，有黑沙样物质沉积于细胞周围，细胞间可见较多致密椭圆形、圆形团块并逐步增大，轮廓不清，团块中心区逐渐变暗。茜素红染色显示矿化结节较分散，单个结节呈橘红色沉淀(图3)。诱导21 d时细胞中碱性磷酸酶活性明显高于7 d(P < 0.01；图4)。 2.4 人羊膜间充质干细胞成软骨诱导结果成软骨诱导7 d后，可观察到人羊膜间充质干细胞细胞生长速度减缓，同时细胞形态发生明显改变，由长梭形逐渐变短、消散，核浆比降低，21 d后，软骨细胞状形态趋势更加明显(图5)。甲苯胺蓝染色可见诱导后细胞基质中分泌大量的糖胺聚糖；对照组细胞甲苯胺蓝染色后未见明显改变。 2.5 人羊膜间充质干细胞成脂诱导结果成脂诱导7 d后，细胞排列状态紊乱，细胞由长梭形变圆并开始出现脂滴样物质，诱导21 d后，细胞浆可见脂滴存在(图6)。 2.6 诱导后相关基因的表达与7 d相比，诱导21 d时细胞中硬骨素，RUNX-2，碱性磷酸酶，Ⅱ型胶原，过氧化物酶体增生物激活受体，脂蛋白脂酶表达水平较高(P < 0.05；表2)。"

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