Neurogenic Differentiation of Human Dental Pulp Stem Cells by Optogenetics Stimulation

Background: The purpose of this study is to analyze the effect of optogenetics stimulation in propagation of human dental pulp stem cells (hDPSCs) and neurogenic induction. Optogenetics is considered as an advanced biological technique in neuroscience which is able to control the activity of genetically modified cells by light. Recently hDPSCs are being considered by researchers because of high potential of differentiation to neurons.Materials and Methods: The hDPSCs were isolated by me-chanical enzymatic digestion from an impacted third molar and cultured in DMEM/F12. The cells were infected with len-tiviruses carrying CaMKIIa-hChR2(H134R). Opsin-expressing hDPSCs were plated at the density of 5×104 cells/well in 6-well plates and optical stimulation was conducted with blue light (470 nm) pulsing at 15 Hz, 90% Duty Cycle and 10 mW power for 10 s every 90 minutes for 5 days. Morphology of cells were examined daily. Two control groups including native hDPSCs and opsin-expressing hDPSCs with no optical stimulation were also included in the study. A day after last light stimulation, the viability of cells was analyzed with the MTT assay and the expression of Nestin, as a neural progenitor marker, was exam-ined by immunocytochemistry.Results: Human DPSCs expressed the reporter gene, mCherry, 72 hours after lentiviral infection. The result of MTT assay re-vealed a significant more viability in optical stimulated opsin-expressing hDPSCs as compared with two control groups. The immunocytochemistry data showed an increase in Nestin ex-pression in optical stimulated cells compared with controls.Conclusion: Optogenetics stimulation through depolarizing the hDPSCs can increase the cells viability and/or proliferation and also promote the differentiation toward neural progenitors.