Abstract
BackgroundLuminescent 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.ResultsWe 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.ConclusionsOur 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.
Highlights
Luminescent reporter proteins are vital tools for visualizing cells and cellular activity
Overexpression of the luciferin synthesis pathway is required for continuous luminescence in iPSCs The previously published pCMVlux vector [12] harbors a synthetic lux operon consisting of viral 2A element linked luxCDABEF genes under the control of a CMV promoter and has only been shown to function effectively in a handful of immortalized cancer cell lines [11, 12]. pCMVlux functionality was confirmed via observation of autobioluminescence following transfection into HEK293 cells (2.09 × 105 (± 4.03 × 103) photons/s)
This approach failed to produce autobioluminescence (20 (± 62) photons/s; p = 0.309 compared to untransfected control) after transient transfection and following qPCRbased analysis confirming genomic integration of the luxCDABEF genes in stably transfected isolates
Summary
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. 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 advantageous. Biological processing results in dynamic uptake and clearance rates between experiments [5, 6], and chemical interaction produces artifacts in high-throughput operations [7] These restrictions limit the functionality of luciferases when working with precious samples or those that are destined for further experimentation
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