Li Yueying, Liu Tie, Van Halm-Lutterodt Nicholas, Chen JiaYu, Su Qingjun, Hai Yong
Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China.
Department of Orthopedics, Beijing Chao-Yang Hospital, Capital Medical University, GongTiNanLu 8#, Chaoyang District, Beijing, 100020, China.
Stem Cell Res Ther. 2016 Feb 17;7:31. doi: 10.1186/s13287-016-0290-7.
An attempt was made to reprogram peripheral blood cells into human induced pluripotent stem cell (hiPSCs) as a new cell source for cartilage repair.
We generated chondrogenic lineage from human peripheral blood via hiPSCs using an integration-free method. Peripheral blood cells were either obtained from a human blood bank or freshly collected from volunteers. After transforming peripheral blood cells into iPSCs, the newly derived iPSCs were further characterized through karyotype analysis, pluripotency gene expression and cell differentiation ability. iPSCs were differentiated through multiple steps, including embryoid body formation, hiPSC-mesenchymal stem cell (MSC)-like cell expansion, and chondrogenic induction for 21 days. Chondrocyte phenotype was then assessed by morphological, histological and biochemical analysis, as well as the chondrogenic expression.
hiPSCs derived from peripheral blood cells were successfully generated, and were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. Flow cytometric analysis showed that MSC markers CD73 and CD105 were present in monolayer cultured hiPSC-MSC-like cells. Both alcian blue and toluidine blue staining of hiPSC-MSC-chondrogenic pellets showed as positive. Immunohistochemistry of collagen II and X staining of the pellets were also positive. The sulfated glycosaminoglycan content was significantly increased, and the expression levels of the chondrogenic markers COL2, COL10, COL9 and AGGRECAN were significantly higher in chondrogenic pellets than in undifferentiated cells. These results indicated that peripheral blood cells could be a potential source for differentiation into chondrogenic lineage in vitro via generation of mesenchymal progenitor cells.
This study supports the potential applications of utilizing peripheral blood cells in generating seed cells for cartilage regenerative medicine in a patient-specific and cost-effective approach.
人们尝试将外周血细胞重编程为人类诱导多能干细胞（hiPSC），作为软骨修复的新细胞来源。
我们使用无整合方法通过hiPSC从人类外周血中生成软骨细胞系。外周血细胞要么从人类血库获取，要么从志愿者新鲜采集。将外周血细胞转化为iPSC后，通过核型分析、多能性基因表达和细胞分化能力对新获得的iPSC进行进一步表征。iPSC经过多个步骤进行分化，包括胚状体形成、hiPSC间充质干细胞（MSC）样细胞扩增以及软骨诱导21天。然后通过形态学、组织学和生化分析以及软骨细胞表达评估软骨细胞表型。
成功从外周血细胞中生成了hiPSC，并通过多能性标志物的荧光免疫染色和体内畸胎瘤形成进行表征。流式细胞术分析表明，单层培养的hiPSC-MSC样细胞中存在MSC标志物CD73和CD105。hiPSC-MSC软骨生成微球的阿尔新蓝和甲苯胺蓝染色均呈阳性。微球的胶原蛋白II和X的免疫组织化学染色也呈阳性。硫酸化糖胺聚糖含量显著增加，软骨生成微球中软骨生成标志物COL2、COL10、COL9和聚集蛋白聚糖的表达水平显著高于未分化细胞。这些结果表明，外周血细胞可能是通过生成间充质祖细胞在体外分化为软骨细胞系的潜在来源。
本研究支持以患者特异性且具有成本效益的方法利用外周血细胞生成软骨再生医学种子细胞的潜在应用。
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