Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany.
Cell Mol Neurobiol. 2011 May;31(4):635-43. doi: 10.1007/s10571-011-9659-4. Epub 2011 Feb 18.
Human neurons derived from stem cells can be employed as in vitro models to predict the potential of neurochemicals affecting neurodevelopmental cellular processes including proliferation, migration, and differentiation. Here, we developed a model of differentiating human neurons from well characterized human embryonal carcinoma stem cells (NT2). NT2 cells were induced to differentiate into neuronal phenotypes after 2 weeks of treatment with retinoic acid in aggregate culture. Nestin positive progenitor cells migrate out of NT2 aggregates and differentiate into βIII-tubulin expressing neuronal cells. Culturing the NT2 cells for an additional 7-14 days resulted in increased percentage of βIII-tubulin expressing cells, elaborating a long neurite that positively stained for axonal marker (Tau) and presynaptic protein (synapsin). We then asked whether neurite outgrowth from NT2 cells is modulated by bioactive chemicals. Since the cAMP/PKA pathway has been widely investigated as a regulator of neurite outgrowth/regeneration in several experimental systems, we used chemical activators and inhibitors of cAMP/PKA pathway in the culture. The adenylyl cyclase activator, forskolin, and cell-permeable analog of cAMP, 8-Br-cAMP increased the percentage of neurite bearing cells and neurite extension. Application of the protein kinase A inhibitors, H-89 and Rp-cAMP, blocked neurite formation. Taken together, NT2 aggregates undergo migration, differentiation, and neurite elaboration and can be used as a model of differentiating human neurons to screen neurochemicals and to understand cellular mechanisms of human nerve cell development.
人干细胞来源的神经元可作为体外模型，预测影响神经发育细胞过程（包括增殖、迁移和分化）的神经化学物质的潜力。在这里，我们从经过充分表征的人胚胎癌细胞（NT2）中开发了一种分化人神经元的模型。在集落培养中用维甲酸处理 2 周后，NT2 细胞被诱导分化为神经元表型。巢蛋白阳性祖细胞从 NT2 聚集物中迁移出来并分化为表达βIII-微管蛋白的神经元细胞。将 NT2 细胞再培养 7-14 天，导致表达βIII-微管蛋白的细胞比例增加，延长的轴突正向染色（Tau）和突触前蛋白（突触素）。然后，我们询问 NT2 细胞的突起生长是否受生物活性化学物质的调节。由于 cAMP/PKA 途径已在多种实验系统中作为调节突起生长/再生的调节剂进行了广泛研究，因此我们在培养物中使用了 cAMP/PKA 途径的化学激活剂和抑制剂。腺苷酸环化酶激活剂 forskolin 和 cAMP 的细胞渗透性类似物 8-Br-cAMP 增加了有突起细胞的百分比和突起延伸。蛋白激酶 A 抑制剂 H-89 和 Rp-cAMP 的应用阻断了突起形成。总之，NT2 聚集物经历迁移、分化和突起延伸，可作为分化人神经元的模型，用于筛选神经化学物质并理解人神经细胞发育的细胞机制。
