Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α
Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α
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DOI：
10.1038/nprot.2012.125
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发表时间：
2012-12-01
期刊：
影响因子：
14.8
通讯作者：
Matsuzaki, Yumi
中科院分区：
文献类型：
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作者：
Houlihan, Diarmaid D.;Mabuchi, Yo;Matsuzaki, Yumi
血小板衍生生长因子受体α (pdgfr - α)和干细胞抗原1 （Sca-1）最近被确定为小鼠间充质干细胞（MSCs）的选择性标记物。与标准培养选择的MSCs相比，pdgfr - α (+)Sca-1(+) (P α S) MSCs具有增强的生长潜力和强大的三谱系分化。此外，P α S MSCs的选择性分离避免了可能使其他方法复杂化的细胞污染。在这里，我们详细描述了我们使用流式细胞术分离P α S间充质干细胞的方案。简而言之，将胫骨和股骨分离并用杵臼粉碎。然后将粉碎的骨头切碎，在含0.2% （wt/vol）胶原酶的20 ml DMEM中于37℃下孵育1小时。红细胞溶解前对细胞悬液进行过滤，并与以下抗体孵育：异藻蓝蛋白（APC）偶联的pdgfr - α、fitc偶联的Sca-1、藻红蛋白（PE）偶联的CD45和Ter-119。在细胞分选器上构建适当的门以排除死亡细胞和谱系(CD45（+)Ter-119(+)）阳性细胞。然后每只小鼠可分离约10,000个P α S间充质干细胞。整个协议大约需要7小时才能完成。
Platelet-derived growth factor receptor alpha (PDGFR-alpha) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-alpha(+)Sca-1(+) (P alpha S) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of P alpha S MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate P alpha S MSCs using flow cytometry. In brief, the tibia and femora are isolated and crushed using a pestle and mortar. The crushed bones are then chopped and incubated for 1 h at 37 degrees C in 20 ml of DMEM containing 0.2% (wt/vol) collagenase. The cell suspension is filtered before red blood cell lysis and incubated with the following antibodies: allophycocyanin (APC)-conjugated PDGFR-alpha, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter-119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45(+)Ter-119(+))-positive cells. Approximately 10,000 P alpha S MSCs may then be isolated per mouse. The total protocol takes similar to 7 h to complete.