INTRODUCTION Human dental pulp stem cells (hDPSCs), a promising source for autologous transplantation in regenerative medicine, have been shown to be able to differentiate into neural precursors. Optogenetics is considered as an advanced biological technique in neuroscience which is able to control the activity of genetically modified stem cells by light. The purpose of this study is to investigate the neurogenic differentiation of hDPSCs following optogenetic stimulation. METHODS The hDPSCs were isolated by mechanical enzymatic digestion from an impacted third molar and cultured in DMEM/F12. The cells were infected with lentiviruses carrying CaMKIIa-hChR2 (H134R). Opsin-expressing hDPSCs were plated at the density of 5 × 104 cells/well in 6-well plates and optical stimulation was conducted with blue light (470 nm) pulsing at 15 Hz, 90% Duty Cycle and 10 mW power for 10 s every 90 minutes, 6 times a day for 5 days. Two control groups including non-opsin-expressing hDPSCs and opsin-expressing hDPSCs with no optical stimulation were also included in the study. A day after last light stimulation, the viability of cells was analyzed by the MTT assay and the morphological changes were examined by phase contrast microscopy. The expression of Nestin, Microtubule-Associated protein 2 (MAP2) and Doublecortin (DCX) were examined by immunocytochemistry. RESULTS Human DPSCs expressed the reporter gene, mCherry, 72 hours after lentiviral infection. The result of MTT assay revealed a significant more viability in optical stimulated opsin-expressing hDPSCs as compared with two control groups. Moreover, optical stimulation increased the expression of Nestin, Doublecortin and MAP2 along with morphological changes from spindle shape to neuron-like shape. CONCLUSION Optogenetics stimulation through depolarizing the hDPSCs can increase the cells viability and/or proliferation and also promote the differentiation toward neuron-like cells.

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光遗传学刺激对人牙髓干细胞的神经源性分化

引言 人牙髓干细胞 (hDPSCs) 是再生医学中自体移植的一种有前景的来源，已被证明能够分化为神经前体细胞。光遗传学被认为是神经科学中的一种先进生物技术，它能够通过光来控制转基因干细胞的活性。本研究的目的是研究光遗传学刺激后 hDPSCs 的神经源性分化。方法通过机械酶消化从受影响的第三磨牙中分离 hDPSCs，并在 DMEM/F12 中培养。细胞用携带 CaMKIIa-hChR2 (H134R) 的慢病毒感染。将表达视蛋白的 hDPSC 以 5 × 104 细胞/孔的密度铺在 6 孔板中，并用 15 Hz 脉冲的蓝光（470 nm）进行光刺激，90% 占空比和 10 mW 功率，每 90 分钟 10 秒，每天 6 次，持续 5 天。该研究还包括两个对照组，包括不表达视蛋白的 hDPSCs 和表达视蛋白的 hDPSCs，没有光刺激。最后一次光刺激后一天，通过 MTT 测定分析细胞的活力，并通过相差显微镜检查形态变化。通过免疫细胞化学检查巢蛋白、微管相关蛋白 2 (MAP2) 和双皮质素 (DCX) 的表达。结果 慢病毒感染 72 小时后，人类 DPSCs 表达报告基因 mCherry。MTT 测定的结果显示，与两个对照组相比，光刺激的表达视蛋白的 hDPSCs 的生存能力显着提高。此外，光刺激增加了巢蛋白的表达，双皮质素和 MAP2 以及从纺锤形到神经元样形状的形态变化。结论通过去极化hDPSCs的光遗传学刺激可以增加细胞的活力和/或增殖，并促进向神经元样细胞的分化。