It is crucial to accurately identify and monitor the progress of neuronal differentiation and the heterogeneity of cells to ensure the correct specification of cell types and minimize the risk of tumor formation in stem cell differentiation protocols. At present, conventional methods like RT-PCR, immunofluorescence staining, and western blotting are employed to detect NSC differentiation. These methods, while valuable, often suffer from limitations such as low sensitivity, population averaging effects, and potential artifacts during sample preparation and analysis. They are time-consuming, invasive, and offer limited resolution at the single-cell level. Furthermore, they only provide snapshots of differentiation at specific time points, failing to capture the gradual progression of the process. Therefore, there is an urgent need for a more efficient, accurate, and convenient real-time monitoring method to evaluate neuronal differentiation non-invasively.

To address these challenges, Prof. Ki-Bum Lee and his team at Rutgers University's Department of Chemistry and Chemical Biology have developed a new way to monitor neuronal differentiation in real-time and non-invasively. They achieved this by developing cell-based biosensors (CBBs) using NSCs [Figure 1]. These biosensors consist of genetically engineered live cells equipped with bioreceptors, split-intein, and reporters, enabling them to detect and quantify target molecules at different levels of complexity within biological systems. CBBs offer several advantages, such as non-invasive detection and continuous real-time monitoring, without the need for invasive procedures like cell fixation and staining. They can also detect unknown analytes, making them useful for studying the intricacies of neuronal differentiation.

Our research introduces an innovative method that employs advanced cell-based biosensor (CBB) method for monitoring neuronal differentiation progress through the translocation of fluorescence signals, targeting hippocalcin as a groundbreaking neuronal biomarker. This integration of CBB technology with neural stem cell studies enables the real-time observation of neuronal differentiation processes at the granularity of individual cells. This approach can provide a comprehensive understanding of the temporal progression and heterogeneity of the process. Importantly, our method enables the early detection of neuronal differentiation events with no invasive labeling techniques.

Furthermore, CBBs can monitor 3D human stem cell systems, such as spheroid cultures and brain organoids, to study human diseases and supplement animal models. Their non-invasive characteristics can overcome the inaccuracy in detection caused by the size of the IgG antibodies used in IF staining. The integration of CBBs into stem cell research represents a paradigm shift, opening new avenues for studying neuronal differentiation with unprecedented precision and efficiency. Our approach, which is effective in overcoming the limitations imposed by conventional techniques, can potentially speed up the development of stem cell therapies that are both safe and effective to treat neurological disorders. This can potentially transform the landscape of regenerative medicine and neuroscience over a more extended period.

PUBLICATION: This work was recently published in Advanced Functional Materials, “Real-Time, Non-Invasive Monitoring of Neuronal Differentiation Using Intein-Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell-Based Biosensors”, 2024,

AUTHORS: Euiyeon Lee, Hye Kyu Choi, Youngeun Kwon, Ki-Bum Lee

CORRESPONDENCE: Prof. Ki-Bum Lee (Rutgers University),

Ki Bum Lee Ph     

KBLEE Group Team: Dr. Euiyeon Lee, Dr. Hye Kyu Choi,

 Jin HaChoi

Hye Kyu Choi