Membrane Potential Hyperpolarization Controls the Osteogenic Differentiation of Human Dental Follicle Stem Cells by...

Kir2.1 channels are responsible for membrane hyperpolarization of many cell types. While these Kir2.1 channels are known to be necessary for proper bone development and play a critical role in osteogenesis, the underlying mechanisms remain poorly understood. Here, we examined the effect of Kir2.1-mediated membrane hyperpolarization on the osteogenic differentiation of human dental follicle stem cells (hDFCs), a type of mesenchymal stem cell (MSC), and explored the underlying mechanisms.

Levels of Kir2.1 and osteogenic marker expression were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Alkaline phosphatase (ALP) and Alizarin red staining were employed to evaluate ALP enzymatic activity and mineralized nodule formation, respectively. Intracellular Ca²⁺ levels were measured using fluorescent Ca²⁺ indicators with Ca²⁺ imaging.

Reversal of membrane hyperpolarization via modulation of extracellular K⁺ concentration ([K⁺]_e) was shown to suppress osteogenic differentiation of hDFCs, whereas the induction of membrane hyperpolarization through the opening of ATP-sensitive K⁺ channels had the opposite effect, enhancing hDFC osteogenesis. Kir2.1 channel expression was upregulated during the osteogenic differentiation of hDFCs. Inhibition of Kir2.1 using short hairpin RNA (shRNA) or a pharmacological inhibitor suppressed osteogenic differentiation. Hyperpolarizing the membrane by decreasing [K⁺]_e led to the elevation of intracellular Ca²⁺ levels, whereas this effect was eliminated by the removal of extracellular Ca²⁺, Kir2.1 inhibition, or treatment with La³⁺, a store-operated Ca²⁺ channel (SOC) blocker.

Our findings indicate that membrane hyperpolarization promotes osteogenic differentiation of hDFCs by increasing intracellular Ca²⁺ levels and that the Kir2.1 and SOC channels play important roles in this process.