Biological characterization and in vitro biocompatibility of human placenta derived mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.05.017

Abstract

Abstract:

BACKGROUND: At present bone marrow mesenchymal stem cells act as the main seed cells in bone tissue engineering, but only 0.001%-0.01% cells are in the bone with difficulty in cell separation and purification.
OBJECTIVE: To explore the biological characterization of human placenta derived mesenchymal stem cells and biocompatibility with three-dimensional porous hydroxyapatite ceramic scaffold.
METHODS: Human placenta derived mesenchymal stem cells were morphologically observed and identified using flow cytometry, followed by osteogenic, adipogenic, chondrogenic induction for 3 weeks. Afterwards, the potential of multi-directional differentiation was identified by alizarin red S, oil red O and toluidine blue staining. DAPI staining was used to observe the adhesion of cells on the surface of the hydroxyapatite ceramic scaffold under scanning electron microscope.
RESULTS AND CONCLUSION: The human placenta derived mesenchymal stem cells showed long spindle shape and uniform size under the microscope; they highly expressed CD29 and D90, but did not express CD45 and CD106. Following induction, mineralized nodules were observed by alizarin red S staining, lipid droplets by oil red O staining and blue-dyed toluidine blue staining. These cells adhered well to the scaffold surface, indicting they are suitable for bone 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: Placenta, Mesenchymal Stem Cells, Hydroxyapatites, Cell Adhesion, Tissue Engineering

CLC Number:

R394.2

Wu Jia, Wen Yong-mei, Lv Xin-rong, Mu Yan-dong. Biological characterization and in vitro biocompatibility of human placenta derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 755-759.

Figures/Tables 1

2.1 人胎盘来源间充质干细胞的形态第3代人胎盘来源间充质干细胞复苏培养5 d后，细胞长满培养瓶底部，显微镜下观察细胞呈集簇状生长，螺旋状、漩涡状排列，呈典型的长梭形，类似成纤维细胞样形态，细胞大小均匀一致，生长状况良好(图2)。人胎盘来源间充质干细胞平均3-5 d传代1次。 2.2 流式细胞仪检测人胎盘来源间充质干细胞表面标志人胎盘来源间充质干细胞高表达CD29，CD90等间充质干细胞特征性表面抗原，低表达造血细胞表面抗原CD45、CD106等[9-10](图3)，表达量分别为98.14%，99.34%，0.29%，0.43%。 2.3 人胎盘来源间充质干细胞的多向分化潜能鉴定 2.3.1 成骨分化潜能人胎盘来源间充质干细胞在成骨诱导培养基中培养1周，细胞形态由长梭形变为多边形。培养1-3周，人胎盘来源间充质干细胞形成钙化结节，随着时间的延长，钙化结节增大，并且分布也比较均匀，用茜素红S染色液对细胞进行染色，可以观察到细胞被染成红色的大小不等的钙化结节(图4A)，表明分离得到的人胎盘来源间充质干细胞具有成骨细胞分化能力。 2.3.2 成脂分化潜能人胎盘来源间充质干细胞在成脂诱导培养基中培养7-10 d，细胞核增大。诱导2到3周，细胞逐渐变圆，胞质内出现脂滴，并且脂滴逐渐增大，数量增多。经成脂诱导培养基培养3周，用油红O对细胞进行染色，可以观察到细胞被分化为大小不一的脂肪滴，脂肪滴被染成红色(图4B)[11]，呈葡萄串状，表明分离得到的人胎盘来源间充质干细胞具有向脂肪细胞分化的能力。 2.3.3 成软骨分化潜能人胎盘来源间充质干细胞经成软骨诱导培养基诱导培养3周，出现软骨基质。对细胞进行甲苯胺蓝染色，可以看到软骨被染成蓝色(图4C)，表明分离得到的人胎盘来源间充质干细胞具有向软骨细胞分化的能力。 2.4 人胎盘来源间充质干细胞与支架复合共培养共培养5 d时，呈梭形的细胞围绕在羟基磷灰石陶瓷支架边缘生长，排列一致，随着时间的递增，细胞数目增多(图5)。 2.5 人胎盘来源间充质干细胞在支架表面的黏附情况将接种1，4 h后的细胞进行DAPI染色(图6)，人胎盘来源间充质干细胞在羟基磷灰石陶瓷支架表面黏附良好，随着共培养时间的延长，黏附在支架表面的细胞明显增多。 2.6 扫描电镜观察人胎盘来源间充质干细胞在支架表面黏附形态人胎盘来源间充质干细胞在羟基磷灰石陶瓷支架表面生长良好，保持长梭形的形态特点，部分细胞伸出伪足，有利于细胞更加紧密黏附于支架表面(图7)。"

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