Establishing Neuronal Cell Differentiation Models Using Dental Pulp Cells

Objectives: Dental pulp stem cells (DPSCs) have been suggested to have neuronal differentiation potential, although their in vitro neurogenesis efficiency is limited. All-trans retinoic acid (ATRA) induces the human SH-SY5Y neuroblastoma cell line to differentiate into neuronal-like cells. However, the neurogenic effects of ATRA on DPSCs are not well established. The aim of this study was to evaluate the ATRA-induced neurogenesis of SH-SY5Y cells in comparison to DPSCs.
Methods: SH-SY5Y cells and primary human DPSCs were cultured in DMEM/F12. Neuronal differentiation was studied using 10μM ATRA supplementation. SH-SY5Y cells were cultured in different FBS concentrations (3 and 10%). Cell viability and numbers were monitored using Trypan blue exclusion and Alamar Blue assays. Neuronal differentiation was assessed morphologically by microscopic image analysis of neurite outgrowth (average neurite length and number of cells bearing neurites) and immunocytochemical staining of β3-tubulin. RT-PCR was used to analyse gene expression of neuronal markers.
Results: SH-SY5Y showed extensive neurite outgrowth in approximately 70% of the cell population after culture in ATRA-supplemented media. ATRA-treated SH-SY5Y had significantly increased expression of ACHE, NES and RET, but reduction in DBH suggesting selective differentiation into cholinergic-like neurons. The neuronal-like cells also positively stained for the neuronal marker β3-tubulin. No significant differences were found in ATRA-induced differentiation in between 3% and 5% FBS concentrations, although significant neuritogenesis occurred in the control cultures at 3% FBS compared with 10% FBS. DPSCs in ATRA-supplemented DMEM/F12 displayed significantly greater neurite-like extensions compared with DPSCs cultured in standard alpha-MEM.
Conclusions: This study demonstrated that a singular supplementation with ATRA resulted in significant induction of neuronal-like differentiation of the neuroblastoma cell line and to a lesser, albeit significant, extent of primary human DPSCs. Further work is continuing to characterise and optimise these cell models for dental neurogenesis research.