Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%–40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5–8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission.

5-羟色胺 (5-HT) 神经元是中缝核的主要成分，来自腹侧后脑祖细胞。根据解剖位置和轴突投射，5-HT 神经元粗略地分为头端和尾端组。在这里，我们提出了一种从人类多能干细胞 (hPSC) 生成后脑 5-HT 神经元的新策略，该策略涉及腹侧型神经祖细胞的形成和后脑 5-HT 神经发育的刺激。一种尾化剂维甲酸被用来引导细胞进入后脑细胞命运。使用我们的方案，大约 30%–40% 的 hPSCs 成功发育成表达 5-HT 的神经元，其中大多数获得了尾部菱形体 (r5-8)。我们进一步修改了我们的单层分化系统，以产生富含 5-HT 神经元的后脑样类器官。我们还建议下游应用我们的 5-HT 单层和类器官培养物来研究神经元对肠道微生物群的反应。我们的方法可以成为未来与 5-HT 神经传递相关研究的有力工具。