A new technique for the isolation and surface immobilization of mesenchymal stem cells from whole bone marrow using high-specific DNA aptamers
A new technique for the isolation and surface immobilization of mesenchymal stem cells from whole bone marrow using high-specific DNA aptamers
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DOI：
10.1634/stemcells.2006-0015
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发表时间：
2006-10-01
期刊：
影响因子：
5.2
通讯作者：
Wendel, Hans P.
中科院分区：
文献类型：
--
作者：
Guo, Ke-Tai;Schaefer, Richard;Wendel, Hans P.
成人间充质干细胞(AMSCs)是存在于骨髓中的一种干细胞群体，可在培养条件下分离、扩增和鉴定。由于缺乏特异性的表面标记，很难直接将MSCs从骨髓中分离出来。在这里，我们提出了一种使用高特异性核酸的新方法，称为适配子。以猪骨髓间充质干细胞为靶细胞，通过组合化学方法从一个巨大的随机文库中产生适配子，该文库被称为指数富集型配体系统进化(SELEX)。克隆和测序后，用包被链霉亲和素的磁性微球进行细胞分选实验，检测结合亲和力。我们还使用固定了适体的12孔板从细胞溶液中筛选出MSCs，并使用异硫氰酸酯标记的荧光素适配子高速荧光激活细胞分选从骨髓中分选MSCs。细胞表现出向成骨细胞和成脂细胞分化的潜能，具有典型的形态特征。表面标记染色显示贴壁细胞为CD29(+)、CD44(+)、CD45(-)、CD90(+)、SLA I类、SLA DQ(-)和SLA DR-。与现有的方法相比，本研究建立了一种新的、快速、高效的直接从猪骨髓中分离aMSCs的方法，即以适配子为探针来筛选aMSCs。这种适体的新应用可以极大地促进AMSC的分离和浓缩，从而提高AMSC体外分化后的细胞率，用于组织工程和再生医学等新兴领域的各种应用。
Adult mesenchymal stem cells (aMSCs) are a stem cell population present in bone marrow, which can be isolated and expanded in culture and characterized. Due to the lack of specific surface markers, it is difficult to separate the MSCs from bone marrow directly. Here, we present a novel method using high-specific nucleic acids called aptamers. Porcine MSCs were used as a target to generate aptamers by combinatorial chemistry out of a huge random library with in vitro technology called systematic evolution of ligands by exponential enrichment (SELEX). After cloning and sequencing, the binding affinity was detected using a cell-sorting assay with streptavidin-coated magnetic microbeads. We also used 12-well plates immobilized with aptamers to fish out MSCs from the cell solution and a fluorescein isothiocyanate-labeled aptamer to sort MSCs from bone marrow using high-speed fluorescence-activated cell sorting. The cells showed high potency to differentiate into osteogenic, as well as into adipogenic, lineages with typical morphological characteristics. Surface marker staining showed that the attached cells were CD29(+), CD44(+), CD45(-), CD90(+), SLA class I+, SLA DQ(-), and SLA DR-. Compared with existing methods, this study established a novel, rapid, and efficient method for direct isolation of aMSCs from porcine bone marrow by using an aptamer as a probe to fish out the aMSCs. This new application of aptamers can facilitate aMSC isolation and enrichment greatly, thereby enhancing the rate of aMSC-derived cells after in vitro differentiation for various applications in the emerging field of tissue engineering and regenerative medicine.