Real-Time, Non-Invasive Monitoring of Neuronal Differentiation Using Intein-Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell-Based Biosensors

초록

Real-time and non-invasive monitoring of neuronal differentiation helps to increase understanding of neuronal development and develop stem cell therapies for neurodegenerative diseases. Conventional methods such as RT-PCR, western blotting, and immunofluorescence (IF), lack single-cell-level resolution and require invasive procedures, fixation, and staining. These limitations hinder accurate monitoring progress of neural stem cell (NSC) differentiation and understanding its functions. Herein, a novel approach is reported to non-invasively monitor neuronal differentiation in real-time using cell-based biosensors (CBBs) that detects hippocalcin, biomarker of neuronal differentiation. To construct the hippocalcin sensor proteins, two different hippocalcin bioreceptors are fused to each split-intein, carrying split-nuclear localization signal (NLS) peptides, respectively, and fluorescent protein is introduced as reporter. CBBs operated in the presence of hippocalcin to generate functional signal peptides, which promptly translocated the fluorescence signal to the nucleus. The NSC-based biosensor shows fluorescence signal translocation only upon neuronal differentiation and not undifferentiated stem cells or glial cells. Furthermore, this approach allows monitoring of neural differentiation at earlier stages than detected using IF staining. It is believed that novel CBBs offer an alternative to current techniques by capturing the dynamics of differentiation progress at the single-cell-level and providing a tool to evaluate how NSCs efficiently differentiate into neurons. Novel cell-based biosensor (CBB) is developed to monitor neuronal differentiation in real-time. CBB reports early neurogenesis through translocation of fluorescence signal before expression of TuJ1, key neuronal marker. The ability to capture differentiation dynamics at single-cell-level improves the capability to examine and control neuronal development. Its non-invasiveness can be valuable to investigate neurodegenerative diseases and promote advances in cell therapy. image

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