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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 …