Mahidol University, Faculty of Medicine Ramathibodi Hospital, Chakri Naruebodindra Medical Institute, Samut Prakan, Thailand.
Mahidol University, Faculty of Dentistry, Department of Anatomy, Bangkok, Thailand.
J Appl Oral Sci. 2021 Oct 1;29:e20210296. doi: 10.1590/1678-7757-2021-0296. eCollection 2021.
Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons.
After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied.
Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs.
These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.
人牙髓干细胞（DPSCs）已被用于再生受损的神经组织。然而，将 DPSCs 诱导为神经干细胞/祖细胞（NSPCs）或神经球的方法高度多样化，导致许多神经元分化结果。本研究旨在验证一种将 DPSCs 诱导为神经球和神经元的最佳方案。
在分离和鉴定间充质干细胞特性后，将 DPSCs 培养在 NSPC 诱导培养基和培养容器中。改变培养时间、解离方法和 DPSCs 的传代数。
神经球的形成需要一种抑制细胞附着的特殊表面。5 天是产生增殖性神经球的最适时间，它们强烈表达神经球标志物 Nestin。用 Accutase 酶解离后，神经球的再形成率明显高于其他方法。DPSCs 的传代数不影响神经球的形成，但影响神经元分化。我们发现，表达神经元标志物β-微管蛋白 III 并具有神经元形态的细胞在 DPSCs 的早期传代中明显增多。
这些结果为进一步研究和神经退行性疾病治疗提供了从 DPSCs 获得高效神经球和神经元分化的指导方针。
