Luminescent reporter proteins are vital tools for visualizing cells and cellular activity. Among the current toolbox of bioluminescent systems, only bacterial luciferase has genetically defined luciferase and luciferin synthesis pathways that are functional at the mammalian cell temperature optimum of 37 °C and have the potential for in vivo applications. However, this system is not functional in all cell types, including stem cells, where the ability to monitor continuously and in real-time cellular processes such as differentiation and proliferation would be particularly advantageous. We report that artificial subdivision of the bacterial luciferin and luciferase pathway subcomponents enables continuous or inducible bioluminescence in pluripotent and mesenchymal stem cells when the luciferin pathway is overexpressed with a 20–30:1 ratio. Ratio-based expression is demonstrated to have minimal effects on phenotype or differentiation while enabling autonomous bioluminescence without requiring external excitation. We used this method to assay the proliferation, viability, and toxicology responses of iPSCs and showed that these assays are comparable in their performance to established colorimetric assays. Furthermore, we used the continuous luminescence to track stem cell progeny post-differentiation. Finally, we show that tissue-specific promoters can be used to report cell fate with this system. Our findings expand the utility of bacterial luciferase and provide a new tool for stem cell research by providing a method to easily enable continuous, non-invasive bioluminescent monitoring in pluripotent cells.

中文翻译：

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通过自主生物发光成像实时跟踪干细胞活力、增殖和分化。


发光报告蛋白是可视化细胞和细胞活动的重要工具。在目前的生物发光系统工具箱中，只有细菌荧光素酶具有基因定义的荧光素酶和荧光素合成途径，这些途径在哺乳动物细胞最佳温度 37 °C 下发挥作用，并具有体内应用的潜力。然而，该系统并非在包括干细胞在内的所有细胞类型中都起作用，在干细胞中，连续实时监测细胞过程（例如分化和增殖）的能力将特别有利。我们报告，当荧光素途径以 20-30:1 的比例过度表达时，细菌荧光素和荧光素酶途径亚成分的人工细分能够在多能干细胞和间充质干细胞中实现连续或可诱导的生物发光。事实证明，基于比率的表达对表型或分化的影响最小，同时无需外部激发即可实现自主生物发光。我们使用这种方法来测定 iPSC 的增殖、活力和毒理学反应，并表明这些测定的性能与已建立的比色测定相当。此外，我们使用连续发光来追踪分化后的干细胞后代。最后，我们证明组织特异性启动子可用于通过该系统报告细胞命运。我们的研究结果扩大了细菌荧光素酶的用途，并通过提供一种轻松实现多能细胞中连续、非侵入性生物发光监测的方法，为干细胞研究提供了一种新工具。