Thongsuk Amarin, Seemaung Peeratchai, Phanthong Phetcharat, Janebodin Kajohnkiart, Ruangsawasdi Nisarat, Gonmanee Thanasup, Phruksaniyom Chareerut, Chodchavanchai Tarinee, Balit Tatcha, White Kenneth L, Thonabulsombat Charoensri, Songsaad Anupong Thongklam
Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand.
Department of Pharmacology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand.
PLoS One. 2025 Sep 2;20(9):e0331120. doi: 10.1371/journal.pone.0331120. eCollection 2025.
Human dental pulp stem cells (hDPSCs) are promising adult stem cells that present multilineage differentiation ability. Interestingly, ergothioneine (ERGO) has the potential to uptake into the organic cation transporter N1 (OCTN1) to promote neuronal differentiation. Therefore, this study aims to demonstrate the effect of co-treatment of ergothioneine on the neuronal differentiation of hDPSCs.
The hDPSCs were established from the impacted third molars. Subsequently, the hDPSCs investigated the cell viability with ergothioneine at concentrations of 0-500 µM for 30 hours. The non-cytotoxic concentration of ergothioneine was synergistically induced with the neuronal induction medium. The characteristics of differentiated cells were verified as neuronal cells (d-hDPSCs) by identification of the Nissl substance. The optimal concentration of ergothioneine, which triggered the highest neuronal differentiation of hDPSCs, was further confirmed by neuronal phenotypes via immunofluorescent staining, gene expression, and the ability of neurotransmitter release by intracellular calcium oscillation.
The isolated cells from human dental pulp tissue were characterized as mesenchymal stem cells (MSCs), verified as hDPSCs. The cellular toxicity of ergothioneine was not observed up to 500 µM for 30 hours. The d-hDPSCs presented a neuronal-like shape and positively expressed the Nissl substance. Interestingly, the highest number of neuronal-like cells was detected at 500 µM of ergothioneine. These neuronal-like cells exhibited the synaptic vesicle glycoprotein 2A (SV2A) expression and dynamic change of intracellular Ca2+, suggesting potential functional neuronal characteristics. Furthermore, co-treatment of ergothioneine at 500 µM triggered neurogenic maturation by decreasing Nestin and NES expression and increasing Beta-III tubulin, TUBB3, and microtubule-associated protein 2 (MAP2) expression, respectively.
Co-treatment of ergothioneine at 500 µM can enhance neuronal differentiation, which has the potential to promote neurogenic maturation. Therefore, these findings suggest the alternative of using hDPSCs and the potential of ergothioneine co-treatment as stem cell-based therapy for further transplantation to cure various neurological diseases.
人牙髓干细胞（hDPSCs）是具有多向分化能力的有前景的成体干细胞。有趣的是，麦角硫因（ERGO）可通过有机阳离子转运体N1（OCTN1）摄取，以促进神经元分化。因此，本研究旨在证明麦角硫因联合处理对hDPSCs神经元分化的影响。
从阻生第三磨牙中分离培养hDPSCs。随后，hDPSCs用浓度为0 - 500 μM的麦角硫因处理30小时，检测细胞活力。麦角硫因的无细胞毒性浓度与神经元诱导培养基协同诱导。通过鉴定尼氏物质，将分化细胞的特征确认为神经元细胞（d - hDPSCs）。通过免疫荧光染色、基因表达以及细胞内钙振荡引起的神经递质释放能力等神经元表型，进一步确定触发hDPSCs最高神经元分化的麦角硫因最佳浓度。
从人牙髓组织分离的细胞被鉴定为间充质干细胞（MSCs），并确认为hDPSCs。在30小时内，高达500 μM的麦角硫因未观察到细胞毒性。d - hDPSCs呈现神经元样形态，并阳性表达尼氏物质。有趣的是，在500 μM麦角硫因处理组中检测到最多的神经元样细胞。这些神经元样细胞表现出突触囊泡糖蛋白2A（SV2A）表达以及细胞内Ca2 + 的动态变化，提示潜在的功能性神经元特征。此外，500 μM麦角硫因联合处理分别通过降低巢蛋白（Nestin）和NES表达以及增加β - III微管蛋白（TUBB3）和微管相关蛋白2（MAP2）表达，触发神经源性成熟。
500 μM麦角硫因联合处理可增强神经元分化，具有促进神经源性成熟的潜力。因此，这些发现提示了使用hDPSCs的可能性以及麦角硫因联合处理作为基于干细胞的疗法进一步移植治疗各种神经疾病的潜力。
