Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Biomaterials. 2015 Jun;54:158-67. doi: 10.1016/j.biomaterials.2015.03.017. Epub 2015 Apr 3.
Neural stem cells (NSCs) demonstrate encouraging results in cell replacement therapy for neurodegenerative disorders and traumatic injury in the central nervous system. Monitor the survival and migration of transplanted cells would provide us important information concerning the performance and integration of the graft during the therapy time course. Magnetic resonance imaging (MRI) allow us to monitor the transplanted cells in a non-invasive way. The only requirement is to use an appropriate contrast agent to label the transplanted cells. Superparamagnetic iron oxide (SPIO) nanoparticles are one of the most commonly used contrast agent for MRI detection of transplanted cells. SPIO nanoparticles demonstrated to be suitable for labeling several types of cells including NSCs. However, the current methods for SPIO labeling are non-specific, depending mostly on electrostatic interactions, demanding relatively high SPIO concentration, and long incubation time, which can affect the viability of cells. In this study, we propose a specific and relatively fast method to label NSCs with SPIO nanoparticles via DNA hybridization. Two short single stranded DNAs (ssDNAs), oligo[dT]20 and oligo[dA]20 were conjugated with a lipid molecule and SPIO nanoparticle respectively. The labeling process comprises two simple steps; first the cells are modified to present oligo[dT]20 ssDNA on the cell surface, then the oligo[dA]20 ssDNA conjugated with SPIO nanoparticles are presented to the modified cells to allow the oligo[dT]20-oligo[dA]20 hybridization. The method showed to be non-toxic at concentrations up to 50 μg/mL oligo[dA]20-SPIO nanoparticles. Presence of SPIO nanoparticles at cell surface and cell cytoplasm was verified by transmission electron microscopy (TEM). SPIO labeling via DNA hybridization demonstrated to not interfere on NSCs proliferation, aggregates formation, and differentiation. NSCs labeled with SPIO nanoparticles via DNA hybridization system were successfully detected by MRI in vitro as well in vivo. Cells transplanted into the rat brain striatum could be detected by MRI scanning up to 1 month post-transplantation.
神经干细胞（NSCs）在中枢神经系统退行性疾病和创伤的细胞替代治疗中显示出令人鼓舞的结果。监测移植细胞的存活和迁移将为我们提供有关移植在治疗过程中的性能和整合的重要信息。磁共振成像（MRI）允许我们以非侵入性的方式监测移植细胞。唯一的要求是使用适当的对比剂来标记移植细胞。超顺磁氧化铁（SPIO）纳米颗粒是用于 MRI 检测移植细胞的最常用的对比剂之一。SPIO 纳米颗粒已被证明适合标记包括 NSCs 在内的几种类型的细胞。然而，目前 SPIO 标记的方法是非特异性的，主要依赖于静电相互作用，需要相对较高的 SPIO 浓度和较长的孵育时间，这可能会影响细胞的活力。在这项研究中，我们提出了一种通过 DNA 杂交特异性且相对快速地标记 NSCs 的方法。两条短的单链 DNA（ssDNA），oligo[dT]20 和 oligo[dA]20 分别与脂质分子和 SPIO 纳米颗粒偶联。标记过程包括两个简单的步骤；首先，细胞被修饰以在细胞表面呈现 oligo[dT]20 ssDNA，然后将与 SPIO 纳米颗粒偶联的 oligo[dA]20 ssDNA 呈现给修饰的细胞，以允许 oligo[dT]20-oligo[dA]20 杂交。该方法在高达 50μg/mL oligo[dA]20-SPIO 纳米颗粒的浓度下显示出无毒。通过透射电子显微镜（TEM）证实了细胞表面和细胞质中存在 SPIO 纳米颗粒。通过 DNA 杂交进行的 SPIO 标记不会干扰 NSCs 的增殖、聚集形成和分化。通过 DNA 杂交系统用 SPIO 标记的 NSCs 在体外和体内均成功通过 MRI 检测。移植到大鼠纹状体的细胞在移植后 1 个月内可通过 MRI 扫描检测到。
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