RESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Neuronas/citología , Organoides/citología , Células Madre Pluripotentes/citología , Rombencéfalo/citología , Serotonina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Humanos , Inmunohistoquímica/métodos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Neuronas/metabolismo , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Rombencéfalo/metabolismo , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Tretinoina/farmacologíaRESUMEN
In this study, we found that undifferentiated human pluripotent stem cells (hPSCs; up to 30% of total cells) present in the cultures of neural stem or precursor cells (NPCs) completely disappeared within several days when cultured under neural differentiation culture conditions. Intriguingly, the disappearance of undifferentiated cells was not due to cell death but was instead mediated by neural conversion of hPSCs. Based on these findings, we propose pre-conditioning of donor NPC cultures under terminal differentiation culture conditions as a simple but efficient method of eliminating undifferentiated cells to treat neurologic disorders. In addition, we could establish a new neural differentiation protocol, in which undifferentiated hPSCs co-cultured with NPCs become differentiated neurons or NPCs in an extremely efficient, fast, and reproducible manner across the hESC and human-induced pluripotent stem cell (hiPSC) lines.