Human embryonic stem cell neuronal differentiation models provide promising in vitro tools for the prediction of developmental neurotoxicity of chemicals. Such models mimic essential elements of human relevant neuronal development, including the differentiation of a variety of brain cell types and their neuronal network formation as evidenced by specific gene and protein biomarkers. However, the reproducibility and lengthy culture duration of cell models present drawbacks and delay regulatory implementation. Here we present a relatively short and robust protocol to differentiate H9-derived neural progenitor cells (NPCs) into a neuron-astrocyte co-culture. When frozen-stored NPCs were re-cultured and induced into neuron-astrocyte differentiation, they showed gene- and protein expression typical for these cells, and most notably they exhibited spontaneous electrical activity within three days of culture as measured by a multi-well micro-electrode array. Modulating the ratio of astrocytes and neurons through different growth factors including glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) did not compromise the ability to develop spontaneous electrical activity. This robust neuronal differentiation model may serve as a functional component of a testing strategy for unravelling mechanisms of developmental neurotoxicity.

人胚胎干细胞神经元分化模型在体外提供了有希望的用于预测化学品发育神经毒性的工具。这些模型模拟人类相关神经元发育的基本要素，包括各种脑细胞类型的分化及其神经元网络的形成，如特定基因和蛋白质生物标志物所证明的那样。然而，细胞模型的可重复性和漫长的培养持续时间存在缺陷并延迟监管实施。在这里，我们提出了一个相对较短和稳健的协议，将 H9 衍生的神经祖细胞 (NPC) 分化为神经元-星形胶质细胞共培养物。当冷冻保存的 NPC 被重新培养并诱导成神经元-星形胶质细胞分化时，它们显示出这些细胞典型的基因和蛋白质表达，最值得注意的是，它们在培养后的三天内表现出自发的电活动，这是通过多孔微电极阵列测量的。通过不同的生长因子调节星形胶质细胞和神经元的比例，包括神经胶质细胞系衍生神经营养因子 (GDNF)、脑源性神经营养因子 (BDNF) 和睫状神经营养因子 (CNTF)，并不会影响自发性电活动的发展。这种强大的神经元分化模型可以作为揭示发育神经毒性机制的测试策略的功能组成部分。和睫状神经营养因子 (CNTF) 没有损害发展自发电活动的能力。这种强大的神经元分化模型可以作为揭示发育神经毒性机制的测试策略的功能组成部分。和睫状神经营养因子 (CNTF) 没有损害发展自发电活动的能力。这种强大的神经元分化模型可以作为揭示发育神经毒性机制的测试策略的功能组成部分。