Amplification and identification of human placental mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.1830

Abstract

Abstract:

BACKGROUND: As a seed cell for tissue engineering, placental mesenchymal stem cells are widely used in the treatment of clinical diseases and clinical research due to their multi-directional differentiation, low immunogenicity, and immunomodulation.
OBJECTIVE: To identify human placental mesenchymal stem cells from the Stem Cell Research Institute of Ningxia Medical University General Hospital according to the definition of mesenchymal stem cells, and to provide reliable protection for subsequent experiments.
METHODS: The human placental mesenchymal stem cells were obtained from the Stem Cell Research Institute of Ningxia Medical University General Hospital. Cells attached to plastic culture dishes or culture flasks were detected, and cell morphology was observed intermittently. The cultured third-generation human placental mesenchymal stem cells were tested for cell surface specific antigen by flow cytometry, and were cultured in adipogenic, osteogenic and chondrogenic induction media, respectively. After 21 days of induction, Oil red O, alizarin red and toluidine blue were used for cell staining to observe the differentiation ability of human placental mesenchymal stem cells into fat, bone and chondrocytes. The study protocol was approved by the Ethics Committee of Ningxia Medical University General Hospital (approval No. 2018-047).
RESULTS AND CONCLUSION: The third-generation human placental mesenchymal stem cells cultured in vitro showed a vortex-like adherent growth. The single cell was fusiform with fibroblast-like appearance. Human placental mesenchymal stem cells had a special cellular immunological phenotype: CD105, CD90, and CD73 were positively expressed, and CD34, CD45, CD14 and HLA-DR were negatively expressed. The human placental mesenchymal stem cells were induced to differentiate in vitro after 21 days, and were positive for alizarin red, oil red O, and toluidine blue staining. As per the definition of mesenchymal stem cells, human placental mesenchymal stem cells from the Stem Cell Research Institute of Ningxia Medical University General Hospital meet the characteristics of mesenchymal stem cells, providing good guarantee for further experiments.

Key words: human placental mesenchymal stem cells, hpMSC, subculture, adipogenic induction, osteogenic induction, chondrogenic induction, flow cytometry

CLC Number:

Ma Chen, Li Xiaoguo, Xu Mingjun, Ma Xiaona, Ma Xiaowei. Amplification and identification of human placental mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5293-5299.

Figures/Tables 2

2.1 hpMSCs的塑料黏附性及生长形态复苏培养第3代hpMSCs 24 h后细胞稳定贴壁生长，如图1A，单个细胞呈梭状，1周后细胞融合80%-90%呈漩涡状排列，如图1B。经传代的第4代细胞生长态势良好，大小均一，形态未发生改变，生长5 d细胞融合80%-90%继续传代，如图1C。第5代细胞形态略发生改变，变得细长，如图1D。 2.2 细胞表型分析流式细胞术分析hpMSCs强烈表达间充质干细胞表面经典特异性抗原CD73、CD90、CD105，阳性率分别为97.6%，98.7%，98.5%；造血标记物CD45、CD34、CD14、HLA-DR呈阴性表达，如图2。 2.3 人胎盘间充质干细胞诱导分化结果 2.3.1 成脂肪细胞诱导分化结果诱导7 d细胞形态发生改变呈扁圆形，体积增大，胞质中出现少量脂肪滴，如图3A；诱导14 d细胞形态进一步发生改变，胞质中脂肪滴增多，如图3B；诱导21 d显微镜下可见大量脂肪滴，如图3C；21 d阴性组hpMSCs呈梭形贴壁生长，如图3D。成脂肪诱导21 d油红O染色呈阳性，细胞染成红色，细胞核染成蓝色，如图4A；脂肪颗粒染成深红色，如图4B。 2.3.2 成骨细胞诱导分化结果诱导7 d细胞内胞浆减少，细胞形态明显发生改变，可见少量钙沉积，如图5A；14 d后细胞呈多边形，形态不规则，细胞连接紧密，钙沉积增多，如图5B；21 d细胞呈扁圆形多突起，细胞内几乎无胞浆，细胞与细胞之间缝隙连接，镜下可见散在钙结节，如图5C；阴性组hpMSCs形态未发生明显改变，如图5D。成骨细胞诱导21 d茜素红染色呈阳性，镜下可见橘红色大小不等的钙结节，如图6A，B。 2.3.3 成软骨细胞诱导分化结果诱导7 d，镜下观察细胞可发现局部区域细胞聚集，细胞形态发生改变，呈不规则排列，如图7A；第14天细胞呈不规则形，多突起，细胞聚集成簇的聚合体，如图7B；第21天局部细胞团块呈球形，多突起，如图7C；阴性组第21天hpMSCs呈梭形，形态未发生明显改变，如图7D。成软骨诱导21 d甲苯胺蓝染色呈阳性，细胞染成蓝色，局部成簇，如图8A；局部呈球状，如图8B。"

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