CBD Drives Neurogenic Differentiation in Mouse Dental Pulp Stem Cells

Airam Vivanco, MSc, DDS
University of Florida

Co-authors: Airam Nicole Vivanco-Estela^1,2, Daniele P. Ferrari³, Roger R. Fernandes¹, Bianca Andretto de Mattos², Mauricio dos-Santos-Pereira⁴, Francisco S. Guimarães², Karina F. Bombonato-Prado², Elaine Del-Bel², Glauce Crivelaro Nascimento^2
1University of Florida, ²University of Sao Paulo, ³University of British Columbia, ⁴Broad Institute of MIT and Harvard

Dental pulp stem cells (DPSCs) exhibit intrinsic neurogenic potential and represent a promising cell source for regenerative strategies targeting neurological disorders. Cannabidiol (CBD), a non-psychotomimetic phytocannabinoid, has emerged as a potent modulator of neurogenesis, neuronal survival, and redox homeostasis. However, its effects on neuronal differentiation of DPSCs remain poorly understood. Here, we investigated whether CBD enhances neuronal differentiation of DPSCs and modulates oxidative stress during this process. DPSCs (OD-21 cells) were subjected to neuronal induction using Neurobasal medium in the presence or absence of CBD (1, 2.5, and 10 µM). Cell viability, neuronal differentiation markers, and reactive oxygen species (ROS) production were evaluated at distinct stages of differentiation. CBD did not affect DPSC viability at any concentration or experimental period analyzed (24 h to 21 days), demonstrating low cytotoxicity under neurogenic conditions. Western blot and immunofluorescence analyses revealed that CBD differentially modulated neuronal markers according to the stage of differentiation. CBD at 2.5 µM significantly increased Nestin expression during the early phase of differentiation (24 h and 4 days), indicating enhanced neural progenitor commitment. In intermediate stages (7–14 days), CBD markedly upregulated NeuN expression, suggesting increased neuronal maturation and commitment. Among all concentrations tested, 2.5 µM consistently produced the strongest pro-neurogenic effects. At later stages (21 days), CBD (2.5 and 10 µM) significantly enhanced MAP-2 expression, consistent with acquisition of a mature neuronal phenotype. Neuronal induction with Neurobasal medium increased intracellular ROS production, whereas CBD treatment significantly attenuated oxidative stress throughout differentiation, particularly at days 4, 7, and 14. These findings indicate that CBD promotes neuronal differentiation of DPSCs in a time- and dose-dependent manner while maintaining redox homeostasis during neuronal commitment and maturation. These findings support the therapeutic potential of CBD as a pharmacological adjunct in stem cell-based regenerative approaches for neurodegenerative and neurological disorders.