Laser biostimulation of mouse bone marrow mesenchymal stromal cells: effects on cell proliferation and Notch-1 expression.

Introduction and Aims. Mesenchymal stromal cells (MSCs) are a promising cell candidate in regenerative medicine. Their proliferative potential can be increased by low-level laser irradiation (LLLI), but the mechanisms involved remain to be clarified. Here we investigated the effects of 635 nm diode laser on mouse MSC proliferation paying particular attention to the ability of the laser light to stimulate Notch-1 signaling pathway, a key determinant of MSC self-renewal.We also searched for an involvement of KCa1.1 ion channels, previously shown to be a target of the laser action, in the laser- mediated effects on Notch-1 regulation. Material and methods. Irradiated and control mouse bone marrow MSCs were assayed for cell viability and proliferation by MTS assay, time lapse videomicroscopy EdU incorporation. Expression of Notch-1 and its target gene Hes-1 were evaluated in MSCs cultured in the absence or presence of 0.5 mM Ba2+, to inhibit KCa1.1 channels, using RT-PCR, Western blotting and confocal immunofluorescence. Results. Compared with non-irradiated MSCs, those irradiated with diode laser showed increased proliferation; this phenomenon was associated with the up-regulation and activation of Notch-1, as also demonstrated by increased nuclear expression of Hes1 by the irradiated cells. Of interest, KCa1.1 channel inhibition was able to prevent the stimulatory effects induced by laser irradiation on MSC growth and Notch-1 expression, providing clues to advance our knowledge on the mechanisms leading to stimulation of cell proliferation by laser light. Of note, diode laser did not affect MSC viability or caused morphological alterations. Conclusions. Our findings demonstrate that the mitogenic effects observed in the diode laser-irradiated MSCs involve stimulation of Notch-1 signaling through KCa1.1 channel activation and suggest that LLLI may be a valid and safe approach for preconditioning of MSCs prior their transplantation for regenerative medicine purposes.