A novel way to isolate MSCs from umbilical cords
A novel way to isolate MSCs from umbilical cords
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从脐带中分离间充质干细胞的新方法
DOI：
10.1002/eji.201142356
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发表时间：
2012-08-01
影响因子：
5.4
通讯作者：
Hou, Yayi
中科院分区：
文献类型：
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作者：
Liu, Liu;Zhao, Xiaoyin;Hou, Yayi
间充质干细胞（MSC）不仅具有广泛的自我更新潜力，可以调节免疫细胞激活[1]，而且它们很容易在体外扩增和储存，被认为具有“免疫特权”。 MSC 调节免疫细胞（包括 T 和 B 淋巴细胞、树突状细胞和自然杀伤细胞）的功能，抑制其增殖并改变其细胞因子分泌。我们小组和其他人之前的数据[2-4]证明了间充质干细胞在治疗包括自身免疫性疾病在内的多种动物疾病模型中具有免疫抑制和抗炎作用。由于这些独特的品质，间充质干细胞成为基于干细胞的治疗中有吸引力的候选者。四十年前，Friedenstein 等人[5]首次成功从骨髓中分离出成纤维细胞样集落，即间充质干细胞。该分离方法基于骨髓来源的成纤维细胞样细胞粘附到塑料细胞培养板上，并且伴随而来的是骨髓来源的造血细胞缺乏粘附。贴壁分离的MSCs细胞形态均一，可长期培养扩增，且成本低，是一种实用的生产方法。但据报道，由于MSCs在组织中很少见，采用传统的细胞贴壁法分离MSCs效果不佳；骨髓中MSCs的比例小于1:10,000个单核细胞[6, 7]。因此，很难从骨髓中获得足够的间充质干细胞用于医疗用途。此外，母胎界面是间充质干细胞的重要来源，一些研究小组已从脐带、胎盘和蜕膜中分离出间充质干细胞[8-10]。脐带组织被认为是临床废物，是最稳定、最容易获得的间充质干细胞来源，值得研究。Florian等[11]报道称，细胞分离方案对骨髓源性祖细胞的功能活性有重大影响，凸显了优化 MSC 分离方案的重要性。据报道，分离 MSC 的方法有四种：(i) 流式细胞术分选 [12]；(ii) 磁力分离 [13]；(iii) 密度梯度离心 [14]；(ii) 磁力分离 [13]。 (iv) 细胞塑料粘附法 [15, 16]。由于 MSC 没有特定的表面标记，因此很难将 MSC 从消化的细胞沉淀中分离出来 [17]。尽管一些报告描述了使用 CD133、CD271 和 CD105 作为分离 MSC 的标记物 [18-20]，但该方法还需要一些阴性标记物（CD3、CD14、CD19、CD38 和 CD66b）来辅助分选。此外，流式细胞分选和磁力分离对细胞活力影响很大，需要精密的设备。因此，这些方法尚未得到广泛应用。另一方面，密度梯度离心也需要精密的设备和合适的分离介质，因此不适合MSC的大规模生产。
Mesenchymal stem cells (MSCs) not only possess extensive self-renewal potential and can modulate immune cell activation [1], but also they are easily expanded and stored ex vivo, and are considered to be “immune privileged.” MSCs modulate the function of immune cells, including T and B lymphocytes, dendritic cells, and natural killer cells, inhibit their proliferation and alter their cytokine secretion. Previous data from our group and others [2–4] demonstrated the immunosuppressive and antiinflammatory effects of MSCs in the treatment of several animal disease models including autoimmune diseases. As a result of these unique qualities, MSCs are attractive candidates in stem cell-based therapy. The first successful isolation of fibroblast-like colonies from bone marrow, that is, MSCs, was described four decades ago by Friedenstein et al.[5]. The isolation method was based on the adherence of bone marrow-derived, fibroblast-like cells to the plastic cell culture plate, and a concomitant lack of adherence of bone marrow-derived hematopoietic cells. MSCs separated by adherence have uniform cell morphology and long-term culture expansion, and the cost of this method is low that makes it desirable as a practical method of production. However, it is reported that MSC separation using the traditional cell-adherent method will not yield good results because MSCs are rare in tissue; the proportion of MSCs in the bone marrow is less than 1: 10,000 mononuclear cells [6, 7]. Therefore, it is difficult to obtain enough MSCs from bone marrow for medical use. In addition, the maternal–fetal interface is an important source of MSCs, and several groups have isolated MSCs from umbilical cord, placenta, and decidua [8–10]. Umbilical cord tissue, which is considered as a clinical waste, is the most stable and readily available source of MSCs and of worth studying.Florian et al.[11] reported that cell isolation protocols have a major impact on the functional activity of bone marrowderived progenitor cells, highlighting the importance of optimizing MSC isolation protocols. There are four methods reported to separate MSCs:(i) flow cytometrical sorting [12];(ii) magnetic separation [13];(iii) density gradient centrifugation [14]; and (iv) the cell plastic adherence method [15, 16]. Since there are no specific surface markers for MSCs, it is difficult to separate MSCs from the digested cell pellet [17]. Although several reports describe the use of CD133, CD271, and CD105 as markers to separate MSCs [18–20], this method also requires several negative markers (CD3, CD14, CD19, CD38, and CD66b) to assist sorting. In addition, flow cytometrical sorting and magnetic separation have large effects on cell viability and require sophisticated equipment. Therefore, these methods are not yet widely used. Density gradient centrifugation, on the other hand, also requires sophisticated equipment and a suitable separation medium and is therefore not suitable for large-scale production of MSCs.