Background and purpose: Cerebrospinal fluid (CSF) has a broad set of molecules which is essential for neurogenesis. Human dental pulp stem cells (hDPSCs) are putatively neural crest cell-derived that can differentiate into neurons and glial cells under appropriate neurotrophic factors. The aim of this study was to induce differentiation of hDPSCs into neuroglial phenotypes using Retinoic acid (RA) and CSF.

Materials and methods: The hDPSCs were isolated by mechanical enzymatic digestion from an impacted third molar and cultured. 2 × 10⁵ cells were treated by 10^-7 µM Retinoic acid (RA group) for 8 days, CSF (CSF group) for 8 days and pre-induced with RA for 4 days followed by inducing with CSF for 4 days (RC group). Nestin, βIII-tubulin and GFAP immunostaining were used for evaluating the differentiated cells. Axonal outgrowth was detected using Bielschowsky's silver impregnation method and Nissl bodies were stained in differentiated cells by Cresyl violet. Data analysis was performed in SPSS V.16 applying One-way ANOVA and Chi-square test.

Results: The morphology of differentiated cells in treated groups significantly changed after 3-5 days. The immunocytochemistry results showed that nestin, the neuroprogenitor marker, was observed in all groups. Whereas, a high percentage of nestin positive cells and ΒIII-tubulin, as mature neural markers, were seen at the pre-induction and induction stage, respectively. Nissl bodies were detected as dark-blue particles in the cytoplasm of treated cells.

Conclusion: The findings suggest that the RA as pre-inducer and CSF as inducer could be used for in vitro differentiation of neuroglial cells from hDPSCs.