Interactions of Neuronally Induced Stem Cells from Apical Papilla Spheres, Stems Cells from Apical Papilla, and Human Umbilical Vascular Endothelial Cells on Vasculogenesis and Neurogenesis
Interactions of Neuronally Induced Stem Cells from Apical Papilla Spheres, Stems Cells from Apical Papilla, and Human Umbilical Vascular Endothelial Cells on Vasculogenesis and Neurogenesis
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DOI：
10.1016/j.joen.2023.10.006
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发表时间：
2023-12-12
影响因子：
4.2
通讯作者：
Zhang，Chengfei
中科院分区：
文献类型：
--
作者：
Basabrain，Mohammed S.;Zhong，Jialin;Zhang，Chengfei
方法采用流式细胞术和多向分化实验分离和鉴定SCAPs。通过小分子神经诱导培养基（NIM）神经诱导SCAP单层培养物和球体，并通过RT-qPCR和使用相差光和荧光显微镜评估第0、3和7天的神经基因表达和神经突形成。使用直接共培养或片上浆（pulp-on-chip）来研究iSCAP球与SCAPs和HUVEC的相互作用。RT-qPCR，荧光显微镜，β-tubulin III，α-SMA，和CD 31的免疫染色被用来研究神经基因表达，神经突的形成，和神经血管细胞interactions.ResultsNIM与小分子迅速诱导SCAP向神经样细胞分化。巢蛋白、β-微管蛋白III、MAP-2、NSE和NeuN的基因表达在iSCAP球中高于iSCAP中。与iSCAP相比，iSCAP球体形成更多和更长的神经突。iSCAP球、HUVEC和SCAPs直接共培养显著促进血管形成，沿着VEGF（P< 0.001）和多种神经标志物如Nestin（P< 0.01）、MAP-2（P<0.001）、S100（P< 0.001）和NG 2（P< 0.001）上调。iSCAP球、SCAPs和HUVEC在片上牙髓系统中培养可促进内皮细胞和神经细胞相互迁移，并促进α-SMA阳性和CD 31阳性细胞组装形成血管。
MethodsSCAPs were isolated and characterized using flow cytometry and multilineage differentiation assays. SCAP monolayer culture and spheres were neuronally induced by a small-molecule neural induction medium (NIM), and the neural gene expression and neurite formation at days 0, 3, and 7 were evaluated by RT-qPCR and using phase-contrast light and fluorescence microscopy. Direct coculture or pulp-on-chip was used to investigate iSCAP spheres' interaction with SCAPs and HUVECs. RT-qPCR, fluorescence microscopy, and immunostaining with β-tubulin III, α-SMA, and CD31 were used to study neural gene expression, neurite formation, and neurovascular cell interactions.ResultsNIM with small molecules rapidly induced SCAP differentiation toward neural-like cells. Gene expression of Nestin, β-tubulin III, MAP-2, NSE, and NeuN was higher in iSCAP spheres than in iSCAPs. iSCAP spheres formed more and longer neurites compared to iSCAPs. iSCAP spheres, HUVECs, and SCAPs direct coculture significantly enhanced vessel formation, along with upregulated VEGF (P< 0.001) and multiple neural markers, such as Nestin (P< 0.01), MAP-2 (P< 0.001), S100 (P< 0.001), and NG2 (P< 0.001). iSCAP spheres, SCAPs, and HUVECs cultured in a pulp-on-chip system promoted endothelial and neural cell migration toward each other and α-SMA-positive and CD31-positive cells assembling for the vascular constitution.Conclusions