Generation of kisspeptin-responsive GnRH neurons from human pluripotent stem cells

•A protocol that consistently and efficiently generates GnRH-expressing neurons from human Pluripotent Stem Cells.•An intermediate step involving Neural Progenitor Cells production allows for expansion before terminal differentiation.•Terminally differentiated GnRH-expressing neurons present spontaneous calcium transients.•Spontaneous calcium transients are increased by kisspeptin, both in frequency and amplitude.

GnRH neurons are fundamental for reproduction in all vertebrates, 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 Embryonic Stem Cells and induced-Pluripotent Stem Cells derived from a Kallman Syndrome patient and a healthy family member. 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 media containing Fibroblast Growth Factor 8 and a NOTCH inhibitor. 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.

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