EdU 标记对间充质干细胞的影响。
Effects of EdU labeling on mesenchymal stem cells.
机构信息
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA 94143, USA.
出版信息
Cytotherapy. 2013 Jan;15(1):57-63. doi: 10.1016/j.jcyt.2012.10.010.
BACKGROUND
Thymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been used for tracking mesenchymal stem cells (MSCs). In the present study, we tested whether EdU was cytotoxic and whether it interfered with differentiation, cytokine secretion and migration of MSCs.
METHODS
EdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10(-8) mol/L of EdU for 48 h. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay.
RESULTS
EdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express β-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein and to form capillary-like structures as well as the expression of vWF, eNOS and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1.
DISCUSSION
At the recommended dosage of 10(-8) mol/L, EdU is non-toxic to ADSCs. EdU label did not interfere with differentiation, cytokine secretion or migratory response to SDF-1 by ADSCs.
背景
胸苷类似物 5-乙炔基-2-脱氧尿苷（EdU）最近被用于追踪间充质干细胞（MSCs）。在本研究中，我们测试了 EdU 是否具有细胞毒性，以及它是否会干扰 MSCs 的分化、细胞因子分泌和迁移。
方法
通过将脂肪来源的干细胞（ADSCs）与 10（-8）mol/L 的 EdU 孵育 48 小时来进行 EdU 标记。通过与叠氮化物缀合的 Alexa594 的反应来检测 EdU 的掺入。通过细胞活力和彗星实验分别比较标记和未标记的 ADSCs 的增殖和凋亡。通过形态学、标志物表达和功能比较它们向神经元样和内皮分化的情况。通过细胞因子抗体阵列比较它们分泌的细胞因子谱。通过迁移实验比较它们对归巢因子 SDF-1 的反应。
结果
EdU 大约 70%掺入到 ADSCs 的核中。在 EdU 标记和未标记的 ADSCs 之间，增殖和凋亡率没有显著差异。异丁基甲基黄嘌呤诱导 EdU 标记和未标记的 ADSCs 均呈现神经元样形态，并表达 β-III 微管蛋白。内皮生长培养基-2（EGM2）诱导 EdU 标记和未标记的 ADSCs 均分化为内皮细胞，包括摄取低密度脂蛋白和形成毛细血管样结构的能力，以及 vWF、eNOS 和 CD31 的表达。EdU 标记和未标记的 ADSCs 表现出相同的细胞因子分泌谱和对 SDF-1 的相同迁移反应。
讨论
在推荐剂量 10（-8）mol/L 下，EdU 对 ADSCs 无毒。EdU 标记不干扰 ADSCs 的分化、细胞因子分泌或对 SDF-1 的迁移反应。
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