Isolation, Characterization and Neuronal Differentiation of Human Dental Pulp Stem Cells (DPSCs) from a Permanent Tooth

Dental pulp stem cells (DPSCs), mesenchymal stem cells found in deciduous and permanent teeth, possess multilineage differentiation potential, including neuronal commitment. Their ability to differentiate into neuronal phenotypes holds promise for neurodegenerative disease therapies, cell-based treatments, and tissue engineering. This study investigated the neuronal differentiation potential of DPSCs isolated from the first molar of a healthy 46-year-old female donor. Cells were cultured in a chemically defined medium (ITS 1%: Insulin, Transferrin, Selenium) with or without growth factors (BDNF, GDNF). Phenotypic characterization was performed via Western blot, immunofluorescence, RT-PCR, and flow cytometry. Undifferentiated DPSCs expressed mesenchymal (CD105, CD90, CD73, CD13) and neural/stem cell markers (βIII-tubulin, GFAP, Nestin, SOX2, Oct3/4, NANOG) but lacked hematopoietic markers (CD45, CD34, CD31, CD14). After 15 to 35 days in vitro (DIV) in ITS medium plus GDNF, DPSCs exhibited upregulated mature neuronal markers (NeuN, NF-M) and downregulated mesenchymal markers, confirming neuronal commitment. Electrophysiological analysis (whole-cell patch-clamp) revealed more hyperpolarized resting membrane potentials (RMP) in GDNF-treated cells (15 DIV) compared to controls (DMEM/F12 + 10% FBS), alongside increased membrane capacitance (Cm) and input resistance (Rin), suggesting progressive neuronal maturation. While action potentials were not observed, these electrophysiological shifts indicate early functional differentiation. In conclusion, prolonged GDNF exposure (15–35 DIV) in a chemically defined medium promoted DPSCs differentiation into a mature neuronal phenotype, marked indicated by NeuN expression and altered membrane properties. These findings highlight DPSCs as a viable tool for regenerative medicine and neurodegenerative disease research.