PDF(Pubmed)
Abstract：
BACKGROUND: The enteric nervous system (ENS) is comprised of neurons, glia, and neural progenitor cells that regulate essential gastrointestinal functions. Advances in high-efficiency enteric neuron culture would facilitate discoveries surrounding ENS regulatory processes, pathophysiology, and therapeutics.
METHODS: Development of a simple, robust, one-step method to culture murine enteric neurospheres in a 3D matrix that supports neural growth and differentiation.
RESULTS: Myenteric plexus cells isolated from the entire length of adult murine small intestine formed ≥3000 neurospheres within 7 days. Matrigel-embedded neurospheres exhibited abundant neural stem and progenitor cells expressing Sox2, Sox10 and Msi1 by day 4. By day 5, neural progenitor cell marker Nestin appeared in the periphery of neurospheres prior to differentiation. Neurospheres produced extensive neurons and neurites, confirmed by Tubulin beta III, PGP9.5, HuD/C, and NeuN immunofluorescence, including neural subtypes Calretinin, ChAT, and nNOS following 8 days of differentiation. Individual neurons within and external to neurospheres generated depolarization induced action potentials which were inhibited in the presence of sodium channel blocker, Tetrodotoxin. Differentiated neurospheres also contained a limited number of glia and endothelial cells.
METHODS: This novel one-step neurosphere growth and differentiation culture system, in 3D format (in the presence of GDNF, EGF, and FGF2), allows for ∼2-fold increase in neurosphere count in the derivation of enteric neurons with measurable action potentials.
CONCLUSIONS: Our method describes a novel, robust 3D culture of electrophysiologically active enteric neurons from adult myenteric neural stem and progenitor cells.
METHODS: Development of a simple, robust, one-step method to culture murine enteric neurospheres in a 3D matrix that supports neural growth and differentiation.
RESULTS: Myenteric plexus cells isolated from the entire length of adult murine small intestine formed ≥3000 neurospheres within 7 days. Matrigel-embedded neurospheres exhibited abundant neural stem and progenitor cells expressing Sox2, Sox10 and Msi1 by day 4. By day 5, neural progenitor cell marker Nestin appeared in the periphery of neurospheres prior to differentiation. Neurospheres produced extensive neurons and neurites, confirmed by Tubulin beta III, PGP9.5, HuD/C, and NeuN immunofluorescence, including neural subtypes Calretinin, ChAT, and nNOS following 8 days of differentiation. Individual neurons within and external to neurospheres generated depolarization induced action potentials which were inhibited in the presence of sodium channel blocker, Tetrodotoxin. Differentiated neurospheres also contained a limited number of glia and endothelial cells.
METHODS: This novel one-step neurosphere growth and differentiation culture system, in 3D format (in the presence of GDNF, EGF, and FGF2), allows for ∼2-fold increase in neurosphere count in the derivation of enteric neurons with measurable action potentials.
CONCLUSIONS: Our method describes a novel, robust 3D culture of electrophysiologically active enteric neurons from adult myenteric neural stem and progenitor cells.
摘要：
背景：肠神经系统（ENS）由神经元组成，glia,和调节基本胃肠功能的神经祖细胞。高效肠神经元培养的进展将促进围绕ENS调节过程的发现，病理生理学,和治疗学。
方法：开发一个简单的，健壮,在支持神经生长和分化的3D基质中培养鼠肠神经球的一步法。
结果：从成年鼠小肠的整个长度分离的肌间神经丛细胞在7天内形成≥3000个神经球。到第4天，基质胶嵌入的神经球表现出大量表达Sox2，Sox10和Msi1的神经干细胞和祖细胞。到第5天,神经祖细胞标记Nestin在分化之前出现在神经球的周围。神经球产生大量的神经元和神经突，经微管蛋白βIII证实，PGP9.5,HuD/C,和NeuN免疫荧光，包括神经亚型Calretinin,Chat,分化8天后和nNOS。神经球内部和外部的单个神经元产生去极化诱导的动作电位，这些动作电位在钠通道阻滞剂存在下被抑制，河豚毒素.分化的神经球还含有有限数量的神经胶质和内皮细胞。
方法：这种新颖的一步神经球生长和分化培养系统，3D格式(在GDNF存在的情况下，EGF,和FGF2），在具有可测量的动作电位的肠神经元的衍生中，允许神经球计数增加约2倍。
结论：我们的方法描述了一种新颖的，来自成人肌间神经干细胞和祖细胞的电生理活性肠神经元的强大3D培养。
方法：开发一个简单的，健壮,在支持神经生长和分化的3D基质中培养鼠肠神经球的一步法。
结果：从成年鼠小肠的整个长度分离的肌间神经丛细胞在7天内形成≥3000个神经球。到第4天，基质胶嵌入的神经球表现出大量表达Sox2，Sox10和Msi1的神经干细胞和祖细胞。到第5天,神经祖细胞标记Nestin在分化之前出现在神经球的周围。神经球产生大量的神经元和神经突，经微管蛋白βIII证实，PGP9.5,HuD/C,和NeuN免疫荧光，包括神经亚型Calretinin,Chat,分化8天后和nNOS。神经球内部和外部的单个神经元产生去极化诱导的动作电位，这些动作电位在钠通道阻滞剂存在下被抑制，河豚毒素.分化的神经球还含有有限数量的神经胶质和内皮细胞。
方法：这种新颖的一步神经球生长和分化培养系统，3D格式(在GDNF存在的情况下，EGF,和FGF2），在具有可测量的动作电位的肠神经元的衍生中，允许神经球计数增加约2倍。
结论：我们的方法描述了一种新颖的，来自成人肌间神经干细胞和祖细胞的电生理活性肠神经元的强大3D培养。
