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

Qixin Chen; Changyong Yuan; Shan Jiang; Boon Chin Heng; Ting Zou; Zhongshan Shen; Penglai Wang; Chengfei Zhang

doi:10.3892/etm.2021.9978

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

Exp Ther Med. 2021 Jun;21(6):546. doi: 10.3892/etm.2021.9978. Epub 2021 Mar 24.

Authors

Qixin Chen^{1

2}, Changyong Yuan², Shan Jiang¹, Boon Chin Heng³, Ting Zou¹, Zhongshan Shen⁴, Penglai Wang², Chengfei Zhang¹

Affiliations

¹ Restorative Dental Sciences, Endodontology, Faculty of Dentistry, The University of Hong Kong, Hong Kong 999077, SAR, P.R. China.
² Department of Implant Dentistry, The Affiliated Stomatological Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, P.R. China.
³ School of Stomatology, Peking University, Beijing 100081, P.R. China.
⁴ Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, Jiangsu 221004, P.R. China.

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.

Keywords: apical papilla; differentiation; neurogenesis; small molecule; stem cells.

Grants and funding

Funding: This work was funded by The Health and Medical Research Fund (Hong Kong) - Full Grant (grant no. 06172396; to CZ).