Molecules enhance neurogenic differentiation of dental-derived adult stem cells

Abstract

Objectives: Dental pulp stem cells (DPSCs), stem cells from apical papilla (SCAPs) and gingival mesenchymal stem cells (GMSCs) exhibit high neurogenic potential. The aim of this study was therefore to utilize a cocktail of small molecules to enhance the neurogenic differentiation of DPSCs, SCAPs and GMSCs in vitro. Methods: Neural induction of DPSCs, SCAPs and GMSCs was conducted by the cocktail of eight small molecules (CHIR99021, Dorsomorphin, Forskolin, GO6983, Repsox, SP600125, Valproic acid, and Y-27632 ) over a duration of 14 days. The derived putative neural lineage cells were analysed by qRT-PCR, Western blotting, immunocytochemistry and the Fluo-4 AM calcium flux assay. Results: After 14 days of neural induction by the cocktail of small molecules, distinct morphological characteristic changes of the neural lineage were observed in all three dental-derived adult stem cells. Conversely, much less marked morphological changes were displayed by the corresponding untreated controls. As shown by qRT-PCR analyses, DPSCs and SCAPs treated with small molecules demonstrated significantly increased expression of mature neural markers NSE and NFM, as compared with the control groups (all p<0.05). However, GMSCs treated with small molecules exhibited significantly increased expression of early neural markers such as βIII-tubulin, Musashi 1, MASH1, and NGN2 (all p<0.05). The immunostaining data and western blot analysis further indicated that only significant upregulation of NSE expression by all three chemical-induced neural lineages compared with the corresponding untreated control. Consistently, the calcium transient (F/Fo) peaks for the cells treated with small molecules were significantly higher than the corresponding untreated control as indicated by Fluo-4 AM calcium flux assay (all p<0.05). Conclusions: The small-molecule cocktail enhance the commitment and differentiation of DPSCs, SCAPs and GMSCs into the neural lineage.