Zhang Linxia, Chan Christina
Department of Chemical Engineering and Materials Science, Michigan State University, USA.
J Vis Exp. 2010 Mar 22(37):1852. doi: 10.3791/1852.
MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.
间充质干细胞是一类成体干细胞，是具有治疗应用前景的细胞来源。这些细胞可从骨髓中分离出来，由于其贴壁特性，能很容易地与造血干细胞（HSC）分离。本方案描述了如何从大鼠股骨和胫骨中分离间充质干细胞。通过磁性细胞分选进一步富集分离出的细胞，使其针对两种间充质干细胞表面标志物CD54和CD90。然后通过流式细胞术分析确认表面标志物CD54和CD90的表达。还检测造血干细胞标志物CD45，以检查分选后的间充质干细胞是否不含造血干细胞。间充质干细胞天然具有很大的异质性。存在具有不同形状、增殖和分化能力的细胞亚群。这些亚群均表达已知的间充质干细胞标志物，且尚未鉴定出针对不同亚群的独特标志物。因此，分离不同亚群的另一种方法是使用克隆环分离单克隆来源的细胞。然后可以分别培养和评估来自单克隆的细胞。
