Generation of caudal-type serotonin neurons and hindbrain fate organoids from hPSCs

Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on the 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 (NPCs) and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid (RA), 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 have also suggested 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. To investigate the regional identity of the 5-HT neurons, we performed a transcriptome analysis by comparing the microarray profiles from undifferentiated hESCs (group E), non-patterned neurons (group N; differentiation day 25), and 5-HT neurons (group S; differentiation day 30). For each sample group (S, N, and E), samples were collected from three independent cultures that each consisted of 3 wells of 24-well plate. Total RNA was isolated using TRIZOL (Life Technology), and RNA quality was checked by OD 260/280 ratio and analyzed by Agilent 2100 Bioanalyzer, Transcriptome analysis was executed using The Affymetrix Whole Transcript (WT) Expression Array, cDNA was synthesized using the GeneChip WT Amplification kit.