Abstract：
OBJECTIVE: Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs.
METHODS: Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization.
RESULTS: PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations.
CONCLUSIONS: In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).
METHODS: Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization.
RESULTS: PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations.
CONCLUSIONS: In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).
摘要：
目的：牙髓干细胞（DPSC）包含具有广泛分化潜能的干细胞群，以及更多谱系承诺的祖细胞。这种异质性是实验和临床应用的重要障碍。这项研究的目的是从人DPSC中分离和表征同源的神经元祖细胞群体。
方法：使用磁激活细胞分选（MACS）技术从三个独立供体的牙髓中分离出聚唾液酸化神经细胞粘附分子（PSA-NCAM）神经祖细胞。使用一组神经和非神经标记的免疫荧光染色来表征磁性分离的PSA-NCAM+级分。然后在补充有神经营养因子的NeurobasalA中培养PSA-NCAM细胞：二丁酰基环AMP，神经营养蛋白-3，B27和N2补充剂诱导神经元分化。PSA-NCAM+和分化的PSA-NCAM+细胞均用于Ca2+成像研究以评估P2X3受体的功能以及膜去极化。
结果：使用磁激活细胞分选和抗PSA-NCAM微珠从hDPSC的异质群体中分离PSA-NCAM+神经祖细胞。流式细胞术分析表明,免疫磁性分选显著提高了PSA-NCAM+细胞的纯度。免疫荧光染色显示泛神经元和成熟神经元标志物的表达，PGP9.5和MAP2，分别以及成熟的感觉标记的弱表达，外周蛋白和胰岛1。ATP诱导的反应主要由未分化和分化细胞中的P2X3受体介导。在后者中观察到更大的幅度。此外,当加载快速电压响应荧光分子时，在分化之前和之后的细胞中也检测到膜去极化，FluoVolt™响应氯化钾。有趣的是,只有分化的PSA-NCAM+细胞能够自发的膜振荡。
结论：总之，DPSC含有具有增强的神经分化和功能性神经样特性的神经元祖细胞群,其可以用磁激活细胞分选(MACS)有效分离。
方法：使用磁激活细胞分选（MACS）技术从三个独立供体的牙髓中分离出聚唾液酸化神经细胞粘附分子（PSA-NCAM）神经祖细胞。使用一组神经和非神经标记的免疫荧光染色来表征磁性分离的PSA-NCAM+级分。然后在补充有神经营养因子的NeurobasalA中培养PSA-NCAM细胞：二丁酰基环AMP，神经营养蛋白-3，B27和N2补充剂诱导神经元分化。PSA-NCAM+和分化的PSA-NCAM+细胞均用于Ca2+成像研究以评估P2X3受体的功能以及膜去极化。
结果：使用磁激活细胞分选和抗PSA-NCAM微珠从hDPSC的异质群体中分离PSA-NCAM+神经祖细胞。流式细胞术分析表明,免疫磁性分选显著提高了PSA-NCAM+细胞的纯度。免疫荧光染色显示泛神经元和成熟神经元标志物的表达，PGP9.5和MAP2，分别以及成熟的感觉标记的弱表达，外周蛋白和胰岛1。ATP诱导的反应主要由未分化和分化细胞中的P2X3受体介导。在后者中观察到更大的幅度。此外,当加载快速电压响应荧光分子时，在分化之前和之后的细胞中也检测到膜去极化，FluoVolt™响应氯化钾。有趣的是,只有分化的PSA-NCAM+细胞能够自发的膜振荡。
结论：总之，DPSC含有具有增强的神经分化和功能性神经样特性的神经元祖细胞群,其可以用磁激活细胞分选(MACS)有效分离。
