Neurotrophic Effect of Dental Pulp Stem Cells on Rat Trigeminal Ganglion Neurons

Objectives: Dental pulp stem cells (DPSC) have gained significant interest for nerve repair therapy due to their significant neurogenic and neurotrophic potential. In this study we compared the trophic effects of DPSC and its conditioned medium (CM) on injured trigeminal ganglion (TG) neuronal cells.
Methods: TG and DPSC were isolated by enzymatic digestion from 4-6 weeks old rats. Primary co-cultures of DPSC and TG cells were established using Transwell inserts (1µm pore size) to evaluate DPSC paracrine effects. Neurotrophic factors (NGF, BDNF, NT-3, and GDNF) in DPSC-CM collected from 72 hr serum-free cultures were analysed by specific ELISAs. RT-PCR was used to analyse neuronal-associated gene expression of NeuN, MAP-2, βIII-tubulin, GAP-43, Synapsin-I and TRPV1 and immunocytochemistry to detect the neuronal markers NeuN, MAP-2 and βIII-tubulin. Specific neutralizing antibodies were used to study the role of neurotrophic factors detected in the DPSC-CM.
Results: DPSC and DPSC-CM significantly promoted TG neural survival with extensive neurite outgrowth and branching as evaluated by immunocytochemistry of neuronal markers. Interestingly, DPSC-CM was more effective in stimulating TG cell survival than co-cultures with DPSC or TG cultures treated with NGF. In comparison to controls, DPSC-CM significantly upregulated gene expression of several neuronal markers in addition to the thermosensitive vanilloid channel 1 (TRPV1). DPSC-CM was shown to contain substantial levels of NGF, BDNF, NT-3 and GDNF. Blockage of GDNF significantly impaired the promotion of neurite outgrowth by CM, whilst NT-3 inhibition resulted in a significant decrease in the number of surviving neurons. Interestingly, neuronal cell survival and neurite branching was not affected by NGF inhibition.
Conclusions: This study demonstrated that DPSC-CM being able to rescue and regenerate injured TG sensory neurons and may be considered as cell-free therapy for TG nerve repair.