Molecular Stress Response of Mitochondria during Electrostimulation Evoking Stem Cell Differentiation Revealed by Fluorescence Imaging Combined with SERS Spectra

Stem cells are a class of multipotential cells with the capability of self-replication, which can differentiate into multiple functional cells under extra stimulus. The differentiation of stem cells has important implications for tissue regeneration. Therefore, controllable regulation of dental pulp stem cell (DPSC) behaviors is critical for repairment and regeneration of damaged teeth tissues. Rapid promotion of DPSCs, directed differentiation, and revealing molecular events within the organelle level during the cell differentiation process are in great demand for regeneration of teeth, which remains a great challenge. Herein, we developed a highly effective and uncomplicated stimulation platform to promote the DPSCs for odontogenic differentiation based on impulse electrical stimulation and revealed the molecular stress response of mitochondria during cell differentiation based on fluorescence imaging combined with surface-enhanced Raman spectroscopy (SERS). Our approach can greatly shorten the DPSC differentiation time from usually more than 20 days to only about 3 days under 0.8 V for 5 min every day than drug stimulation. Notably, the glycogen and adenosine triphosphate levels within mitochondria were apparently elevated, which is conducive to improving the progression of cell differentiation. Simultaneously, the expression of mitofusin1 and mitofusin2 within mitochondria was significantly down-regulated during the differentiation process. Mechanistically, the molecular insights into mitochondria within DPSCs were clearly revealed through SERS spectra. It demonstrated that the expression of phenylalanine was significantly reduced, whereas the contents of tryptophan within mitochondria were promoted during the cell differentiation process. This study provides a comprehensive and clinically feasible strategy for the rapid promotion of DPSCs-directed differentiation and reveals the molecular dynamic changes within mitochondria, which broadens the biomedical cognition of electrical stimulation for dental pulp stem cell differentiation and provides a potential application for teeth tissue regeneration in the future.