Small molecules efficiently reprogram apical papilla stem cells into neuron‑like cells

Abstract

Stem cell‑based therapy may provide a novel approach for neural tissue regeneration. A small molecule cocktail‑based culture protocol was previously shown to enhance neurogenic differentiation of stem cells from dental tissues. The present study aimed to investigate the early phase of small molecule‑induced neurogenic differentiation of stem cells from the apical papilla (SCAP). SCAP were cultured in neural‑induction medium or neural‑induction medium with small molecules (NIMS‑SCAP) and examined for their cell morphologies. Expression levels of neural progenitor cell‑related markers, including Nestin, paired‑box gene 6 (Pax6) and Sry‑related HMG box 2 (Sox2), were examined using western blotting and immunocytofluorescence. Expression of differentiated neuron‑related markers, including neurofilament protein (NFM), neuron‑specific nuclear protein (NeuN) and microtubule‑associated protein (MAP)‑2, were also examined using western blotting, while NFM and MAP2 gene expression and cell proliferation were assessed using reverse transcription‑quantitative (RT‑q)PCR and Cell Counting Kit (CCK)‑8 assays, respectively. SCAP morphology was affected by small molecules after as little as 30 min. Specifically, Nestin, Pax6 and Sox2 expression detected using western blotting was increased by day 3 but then decreased over the course of 7 days with neural induction, while immunocytofluorescence revealed expression of all three markers in NIMS‑SCAP. The protein levels of NFM, NeuN and MAP2 on day 7 were significantly upregulated in NIMS‑SCAP, as detected using western blotting, while NFM and MAP2 gene expression levels detected using RT‑qPCR were significantly increased on days 5 and 7. Proliferation of NIMS‑SCAP ceased after 5 days. Electrophysiological analysis showed that only SCAP cultured in NIMS had the functional activity of neuronal cells. Thus, small molecules reprogrammed SCAP into neural progenitor cells within the first 3 days, followed by further differentiation into neuron‑like cells.