The ventral spinal population of V0 interneurons (INs) contributes to the coordinated movements directed by spinal central pattern generators (CPGs), including respiratory circuits and left-right alternation in locomotion. One challenge in studying V0 INs has been the limited number of cells that can be isolated from primary sources for basic research or therapeutic use. However, derivation from a pluripotent source, such as has been done recently for other IN populations, could resolve this issue. However, there is currently no protocol to specifically derive V0 interneurons from pluripotent cell types. To generate an induction protocol, mouse embryonic stem cells (mESCs) were grown in suspension culture and then exposed to retinoic acid (RA) and collected at different time points to measure mRNA expression of the V0 progenitor transcription factor marker, Dbx1, and postmitotic transcription factor marker, Evx1. The cultures were also exposed to the sonic hedgehog signaling pathway agonist purmorphamine (purm) and the Notch signaling pathway inhibitor N-{N-(3,5-difluorophenacetyl-L-alanyl)}-(S)-phenylglycine-t-butyl-ester (DAPT) to determine if either of these pathways contribute to V0 IN induction, specifically the ventral (V0_V) subpopulation. From the various parameters tested, the final protocol that generated the greatest percentage of cells expressing V0_V IN markers was an 8-day protocol using 4 days of suspension culture to form embryoid bodies followed by addition of 1 μM RA from days 4 to 8, 100 nM purm from days 4 to 6, and 5 μM DAPT from days 6 to 8. This protocol will allow investigators to obtain V0 IN cultures for use in in vitro studies, such as those examining CPG microcircuits, electrophysiological characterization, or even for transplantation studies in injury or disease models.

中文翻译：

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从小鼠胚胎干细胞诱导腹侧脊髓 V0 中间神经元

V0 中间神经元 (INs) 的腹侧脊髓群有助于由脊髓中央模式发生器 (CPGs) 指导的协调运动，包括呼吸回路和运动中的左右交替。研究 V0 INs 的一个挑战是可以从主要来源中分离出来用于基础研究或治疗用途的细胞数量有限。然而，从多能来源的衍生，例如最近对其他 IN 人群所做的，可以解决这个问题。然而，目前没有专门从多能细胞类型中获得 V0 中间神经元的协议。要生成诱导方案，Dbx1和有丝分裂后转录因子标记Evx1。培养物还暴露于声波刺猬信号通路激动剂purmorphamine (purm)和Notch信号通路抑制剂N-{N-(3,5-二氟苯乙酰-L-丙氨酰)}-(S)-苯基甘氨酸-叔丁基-酯（DAPT）以确定这些途径中的任何一个是否有助于 V0 IN 诱导，特别是腹侧（V0 _V）亚群。根据测试的各种参数，最终方案产生了最大百分比的表达 V0 _V的细胞IN 标记是一个为期 8 天的方案，使用 4 天的悬浮培养形成胚状体，然后从第 4 天到第 8 天添加 1 μM RA，从第 4 天到第 6 天添加 100 nM purm，从第 6 天到第 8 天添加 5 μM DAPT。该协议将允许研究人员获得用于体外研究的 V0 IN 培养物，例如检查 CPG 微电路、电生理学特征，甚至用于损伤或疾病模型的移植研究。