Functional Evidence for Neuronal Differentiation of Human DPSCs In Vitro

Objectives: Increasing experimental evidence suggests that adult progenitor cells are able to transdifferentiate and form very different cell types. Recently, primary cultures with progenitor cells have been successfully prepared from the pulp of human impacted third molars (dental pulp stem cell culture, DPSC) in a number of different laboratories, including ours. Methods: We have studied the neurogenic differentiation potential of DPSC cultures using an optimized long term neuroinductive protocol based on the combination of intracellular cAMP elevation (with forskolin, IBMX and dbcAMP), PKC activation (TPA), cytokine induction (bFGF, EGF, NGF and NT3), and epigenetic reprogramming using 5-azacytidine. We followed the morphological changes by phase contrast microscopy. In addition, the time-dependent appearance of neuronal cell markers was detected by immuncytochemistry using specific antibodies. To study the functional aspect of neuronal formation, differentiated cells were investigated for the presence of voltage-dependent sodium channels by patch clamping using standard experimental protocols. Results: Our neuroinductive protocol resulted in the appearance of neuron-like cells in DPSC cultures. We also observed the time-specific expression of betaIII-tubulin and NeuN proteins, all of them are regarded as neurospecific ones. Patch clamp analysis showed the functional activity of voltage-dependent sodium channels in the differentiated cells. Conclusion: Our results suggest that the human dental pulp contains a cell population that is capable of neuronal differentiation. Functional investigation of this culture shows that the morphological changes are accompanied by the development of neuronal functional characteristics.

Acknowledgements: Supported by the Hungarian National Scientific Research Fund (OTKA 61543, 67250 and 69008) and the Hungarian Medical Research Council (ETT 332/2006).