Rho kinase inhibitor induced human dental pulp stem cells to differentiate into neurons

Neurological diseases due to neuron loss have become major public health problems. Current treatment reduces symptoms; however, there is no cure for neurological diseases. Therefore, stem cells may be an alternative therapy. Human dental pulp stem cells (hDPSCs) are an attractive source for cell-based approaches due to their high regenerative potential. The Rho kinase (ROCK) inhibitor Y-27632 promoted the neuronal differentiation of several stem cell types. However, its neuronal-inductive effect on hDPSCs has not been reported. Thus, the aim of our study was to investigate whether Y-27632 can induce the neuronal differentiation of hDPSCs.

hDPSCs were isolated from human third molars using an enzymatic method and were subsequently characterized. Cytotoxicity was evaluated using an MTT assay. The optimal concentration to induce neural differentiation was assessed using 1-50 μM Y-27632 as evaluated by Cresyl violet and immunofluorescence staining of neurofilaments and βIII-tubulin, respectively. Ten μM Y-27632 was used for neuronal induction for 72 h, and differentiation was confirmed based on the expression of neurogenic markers (MAP2, Brn3a, and ChAT) and intracellular calcium activity.

Our findings indicate that Y-27632 was not cytotoxic to hDPSCs and 10 μM Y-27632 was the lowest concentration that induced the morphological changes of hDPSCs into neuronal cells with Cresyl violet-positive staining and significantly enhanced the fluorescence intensity of neurofilament and βIII-tubulin. The neuronal genes' expression and intracellular calcium activity were upregulated after induction with Y-27632.

At the optimal concentration and time, Rho kinase inhibitor induces hDPSC differentiation into neuronal cells.