Isolation murine mesenchymal stem cells by positive selection
Isolation murine mesenchymal stem cells by positive selection
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DOI：
10.1007/s11626-007-9041-5
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发表时间：
2007-10-01
影响因子：
2.1
通讯作者：
Soleimani, Masoud
中科院分区：
文献类型：
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作者：
Nadri, Samad;Soleimani, Masoud
由于造血细胞和非MSCs的有害生长，通过塑料贴壁培养分离纯化小鼠骨髓间充质干细胞(MSCs)是困难的。在本工作中，通过阳性选择从小鼠骨髓中分离出均一群的CD34(+)MSCs。为此，处死C57BL/6小鼠，抽吸骨髓细胞，然后与抗CD34抗体偶联的磁珠孵育。取阳性的CD34(+)细胞用于流式细胞仪检测CD34抗原的表达，其余细胞在25 cm(2)培养瓶中传代培养。为了研究间充质的性质，将塑料贴壁培养的细胞诱导分化为成骨和成脂细胞系。用流式细胞仪检测细胞表面标志物的表达。结果表明，第1代(培养开始后1wk)即可获得纯化的成纤维细胞样CD34(+)细胞群。细胞表达CD34、CD44、SCA-1和VCAM-1抗原(标记物)，但不表达CD11b和CD45。它们能够分化为骨细胞和脂肪细胞。这项研究表明，我们的方法可以有效地从C57BL/6小鼠骨髓中分离出均一群的MSCs。我们已经证明，小鼠骨髓来源的CD34(+)细胞在体外具有可塑性贴壁特性和向骨系分化的能力，是MSCs。
Isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures is arduous because of the unwanted growth of hematopoietic cells and non-MSCs. In this work, homogenous populations of CD34(+) MSCs from mouse bone marrow were isolated via positive selection. For this purpose, C57Bl/6 mice were killed and bone marrow cells were aspirated before incubation with magnetic bead conjugated to anti-CD34 antibody. A sample of positively selected CD34(+) cells were prepared for flow cytometry to examine the expression of CD34 antigen and others were subcultured in a 25-cm(2) culture flask. To investigate the mesenchymal nature, the plastic adherent cultivated cells were induced to differentiate along osteoblastic and adipogenic lineages. Furthermore, the expression of some surface markers was investigated by flow cytometry. According to the result, purified populations of fibroblast-like CD34(+) cells were achieved in the first passage (1 wk after culture initiation). The cells expressed CD34, CD44, Sca-1, and Vcam-1 antigens (markers) but not CD11b and CD45. They were capable of differentiating into osteocytes and adipocytes. This study indicated that our protocol can result in the efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We have shown that murine bone marrow-derived CD34(+) cells with plastic adherent properties and capability of differentiating into skeletal lineages in vitro are MSCs.