The School of Life Sciences and the Centre for Health Technologies, University of Technology Sydney, Sydney, Australia.
Translational Cancer Research Group, University of Technology Sydney, Sydney, Australia.
PLoS One. 2019 Jul 18;14(7):e0220013. doi: 10.1371/journal.pone.0220013. eCollection 2019.
Due to their ease of isolation, gene modification and tumor-homing properties, mesenchymal stem cells (MSCs) are an attractive cellular vehicle for the delivery of toxic suicide genes to a variety of cancers in pre-clinical models. In addition, the incorporation of suicide genes in stem cell-derived cell replacement therapies improves their safety profile by permitting graft destruction in the event of unexpected tumorigeneses or unwanted differentiation. Due to the functional requirement of ATP for the Firefly luciferase gene Luc2 to produce light, luciferase-based reporting of cytotoxicity can be engineered into potential cell therapies. Consequently, we nucleofected mammalian expression plasmids containing both the Luc2 and the yeast fusion cytosine deaminase uracil phosphoribosyltransferase (CDUPRT) genes for expression in murine MSCs to assess luciferase as a reporter of suicide gene cytotoxicity, and MSC as vehicles of suicide gene therapy. In vitro bioluminescence imaging (BLI) showed that following the addition of the non-toxic prodrug fluorocytosine (5-FC), CDUPRT-expressing MSCs displayed enhanced cytotoxicity in comparison to Luc2 reporter MSC controls. This study demonstrates the utility of luciferase as a reporter of CDUPRT-mediated cytotoxicity in murine MSC using BLI.
由于间充质干细胞 (MSCs) 易于分离、基因修饰和肿瘤归巢特性，因此它们是将毒性自杀基因递送到临床前模型中各种癌症的有吸引力的细胞载体。此外，将自杀基因整合到干细胞衍生的细胞替代疗法中可以通过允许在意外发生肿瘤形成或不希望的分化时破坏移植物来提高其安全性。由于萤火虫荧光素酶基因 Luc2 产生光需要 ATP 的功能要求，因此可以将基于荧光素酶的细胞毒性报告工程设计到潜在的细胞疗法中。因此，我们转染了含有 Luc2 和酵母融合胞嘧啶脱氨酶尿嘧啶磷酸核糖转移酶 (CDUPRT) 基因的哺乳动物表达质粒，以在鼠 MSCs 中进行表达，以评估荧光素酶作为自杀基因细胞毒性的报告基因，以及 MSCs 作为自杀基因治疗的载体。体外生物发光成像 (BLI) 显示，在添加非毒性前药氟胞嘧啶 (5-FC) 后，与 Luc2 报告 MSC 对照相比，表达 CDUPRT 的 MSC 显示出增强的细胞毒性。这项研究证明了使用 BLI 在鼠 MSC 中使用荧光素酶作为 CDUPRT 介导的细胞毒性的报告基因的实用性。
