Generation of kisspeptin-responsive GnRH-expressing neurons from human pluripotent stem cells. Homo sapiens

GnRH neurons are fundamental for reproduction in all vertebrates ultimately integrating all reproductive inputs. The inaccessibility of human GnRH-neurons has been a major impediment to studying the central control of reproduction and its disorders. Here, we report the efficient generation of kisspeptin responsive GnRH-secreting neurons by directed differentiation of human Pluripotent Stem Cells. The protocol involves the generation of intermediate Neural Progenitor Cells (NPCs) through long-term Bone morphogenetic protein 4 inhibition followed by terminal specification of these NPCs in a media containing FGF8 and a NOTCH inhibition. The resulting GnRH expressing and secreting neurons display a neuroendocrine gene expression pattern and present spontaneous calcium transients that can be stimulated by kisspeptin. These in vitro generated GnRH expressing cells provide a new resource for studying the molecular mechanisms underlying the development and function of GnRH neurons. Overall design: Hues7 embryonic stem cells (ESCs) where differentiated into Neural Progenitor Cells (NPCs). NPCs were expanded for 4 passages and finally differentiated into neurons for 21 (GnRH-EC 21) or 35 (GnRH-EC 35) days. Total RNA was extracted from ESCs, NPCs, GnRH-EC 21, and GnRH-EC 35, hybridized to an Illumina genome wide gene expression array (HT12v4) and data processed using Illumina's Genome Studio software.