Functional Evidence for Neuronal Differentiation of Human PDLSCs In Vitro

Objectives: Recent studies have revealed the existence of stem cells in various human tissues including dental structures. We have shown that human dental pulp stem cells are able to differentiate into cells with distinct neuronal phenotypes with functional characteristics after exposure to specific signals (Kiraly et al., Neurochem Int, 55: 323-32, 2009). In the present study we aimed to prepare primary cultures with progenitor cells from the periodontal ligament of human impacted third molars (periodontal ligament stem cell, PDLSC) and to differentiate these cells into functional neurons. Methods: For neurogenic differentiation of PDLSC cultures, we used our recently described three step neuroinductive protocol consisting (1) epigenetic reprogramming, then (2) simultaneous PKC/PKA activation, followed by (3) incubation in a neurotrophic medium. Morphological changes were detected by phase contrast microscopy. The time-dependent appearance of neuronal cell markers was detected by immuncytochemistry and quantitative PCR, using specific antibodies and primers, respectively. To study the functional aspect of neuronal formation, differentiated cells were investigated for the presence of voltage-dependent cation channels by patch clamping using standard experimental protocols. Results: Our neuroinductive protocol resulted in transdifferentiation and the appearance of neuron-like cells in PDLSC cultures. Both at mRNA and at protein levels we also observed the time-specific expression of N-tubulin and NFM proteins, each of them is regarded as neurospecific marker. Patch clamp analysis showed the functional activity of voltage-dependent potassium channels in the differentiated cells. Conclusion: Our results suggest that the human periodontal ligament contains a cell population that is capable of neuronal differentiation. Membrane ion channel current studies show that the morphological changes of the differentiated cells are accompanied by the development of neuronal functional characteristics.

Acknowledgements: Supported by the Hungarian National Scientific Research Fund (OTKA 69008 and 72385).