Department of Vascular Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Chin Med J (Engl). 2011 Jan;124(2):298-303.
The development of regenerative therapies using derivatives of embryonic stem (ES) cells would be facilitated by a non-invasive method to monitor transplanted cells in vivo, for example, magnetic resonance imaging of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Although ES cells have been labeled with SPIO particles, the potential adverse effects of the label have not been fully examined. The objective of this study was to determine whether SPIO labeling affects murine ES cell viability, proliferation, or ability to differentiate into functional endothelial cells (ECs).
Cross-linked iron oxide (CLIO, an SPIO) was conjugated with human immunodeficiency virus transactivator of transcription (HIV-Tat) peptides, and murine ES cells were labeled with either CLIO-Tat, CLIO, or HIV-Tat. After labeling, ES cells were cultured for 4 days and Flk-1(+) ES cells identified and sorted by immunocytochemistry and fluorescence activated cell sorting (FACS). Flk-1(+) cells were replated on fibronectin-coated dishes, and ECs were obtained by culturing these for 4 weeks in endothelial cell growth medium supplemented with vascular endothelial growth factor (VEGF). ES cell viability was determined using trypan blue exclusion, and the proportion of SPIO(+) cells was evaluated using Prussian blue staining and transmission electron microscopy. After differentiation, the behavior and phenotype of ECs were analyzed by reverse transcription-polymerase chain reaction, flow cytometry, immunocytochemistry, DiI-labeled acetylated low-density lipoprotein (AcLDL) uptake, and Matrigel tube formation assay.
CLIO-Tat was a highly effective label for ES cells, with > 96% of cells incorporating the particles, and it did not alter the viability of the labeled cells. ECs derived from CLIO-Tat(+) ES cells were very similar to murine aortic ECs in their morphology, expression of endothelial cell markers, ability to form vascular-like channels, and scavenging of AcLDL from the culture medium.
CLIO-Tat is a highly effective label for ES cells and does not adversely affect cell viability, differentiation, or behavior. CLIO-Tat could be a useful marker for the non-invasive monitoring of transplanted stem cells.
利用胚胎干细胞（ES）衍生物开发再生疗法，将有助于通过非侵入性方法在体内监测移植细胞，例如，用超顺磁氧化铁（SPIO）纳米颗粒标记的细胞的磁共振成像。尽管 ES 细胞已用 SPIO 颗粒标记，但该标记的潜在不良反应尚未得到充分检查。本研究的目的是确定 SPIO 标记是否会影响鼠 ES 细胞的活力、增殖或分化为功能性内皮细胞（EC）的能力。
交联氧化铁（CLIO，一种 SPIO）与人类免疫缺陷病毒转录激活物（HIV-Tat）肽缀合，并用 CLIO-Tat、CLIO 或 HIV-Tat 标记鼠 ES 细胞。标记后，将 ES 细胞培养 4 天，并通过免疫细胞化学和荧光激活细胞分选（FACS）鉴定和分选 Flk-1（+）ES 细胞。Flk-1（+）细胞在纤连蛋白包被的培养皿上再接种，并在含有血管内皮生长因子（VEGF）的内皮细胞生长培养基中培养 4 周获得 ECs。通过台盼蓝排斥法测定 ES 细胞活力，并通过普鲁士蓝染色和透射电子显微镜评估 SPIO（+）细胞的比例。分化后，通过逆转录-聚合酶链反应、流式细胞术、免疫细胞化学、DiI 标记乙酰化低密度脂蛋白（AcLDL）摄取和 Matrigel 管形成测定分析 ECs 的行为和表型。
CLIO-Tat 是 ES 细胞的一种非常有效的标记物，>96%的细胞吸收了这些颗粒，并且不会改变标记细胞的活力。源自 CLIO-Tat（+）ES 细胞的 ECs 在形态、内皮细胞标志物的表达、形成血管样通道的能力以及从培养基中清除 AcLDL 的能力方面与鼠主动脉 ECs 非常相似。
CLIO-Tat 是 ES 细胞的一种非常有效的标记物，不会对细胞活力、分化或行为产生不利影响。CLIO-Tat 可作为移植干细胞非侵入性监测的有用标记物。
