Neuronal differentiation of murine dental pulp stem cells in vitro

The potential of dental pulp stem cells (DPSCs) in regenerative medicine is becoming more widely understood. The ability of murine DPSCs to become chondrocytes, osteocytes and adipocytes has recently been demonstrated by several groups, but their neurogenic potential has not been widely shown. In this study we demonstrate neuronal differentiation of murine DPSCs in vitro. Method: mDPSCs were cultured in vitro under neuroinductive conditions (n=12) for 14 days. Cultures were subsequently assessed for neuronal and synaptic markers by immunohistochemistry (IHC) or whole cell patch clamp analysis to investigate intracellular electrophysiology. mDPSCs were also seeded onto microelectrode arrays (n=7) to assess network activity. Results: The mDPSCs showed a neuronal phenotype from 5 days in neuroinductive media and developed a neuritic network of processors. IHC revealed approximately 70% of cells expressed the pan-neuronal marker ß-III tubulin and 23% GFAP (n=8). mDPSCs also expressed markers for nestin, neurofilament-M and Schwann cell marker, S100, but not peripheral neuronal marker, periaxin. Differentiated mDPSCs stained positive for GABA and acetylcholine markers with positive expression of GAD 65/67 and ChAT, respectively, but did not contain tyrosine hydroxylase- or vGlut-2-positive cells. Intracellular electrophysiological analysis revealed the presence of L-type voltage-gated calcium channels in many distally connected neuronal-like cells. No sodium or potassium currents were found and the cells did not support spontaneous action potentials. Many cells existed in interconnected networks such that their cell capacitance was greater than that of distally connected and isolated cells. IHC revealed high expression of gap junction protein connexin 43 and the addition of 100µM gap junction blocker, 2-APB, reduced capacitance of clustered cells. Conclusion: These data suggest the differentiation of mDPSCs into a heterogeneous population of neural-like cell networks that may represent an early stage of neural development, dominated by gap junction-mediated cell-to-cell communication.