title: Generation of kisspeptin-responsive GnRH neurons from human pluripotent stem cells.
abstract: 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.
authors.affiliation: Centre for Endocrinology, Queen Mary University of London, London, EC1M 6BQ, UK.
authors.forename: Ariel
authors.initials: A
authors.lastname: Poliandri
authors.affiliation: Centre for Endocrinology, Queen Mary University of London, London, EC1M 6BQ, UK.
authors.forename: Duncan
authors.initials: D
authors.lastname: Miller
authors.affiliation: Centre for Endocrinology, Queen Mary University of London, London, EC1M 6BQ, UK.
authors.forename: Sasha
authors.initials: S
authors.lastname: Howard
authors.affiliation: The Heart Centre, Queen Mary University of London, London, EC1M 6BQ, UK.
authors.forename: Muriel
authors.initials: M
authors.lastname: Nobles
authors.affiliation: Centre for Endocrinology, Queen Mary University of London, London, EC1M 6BQ, UK.
authors.forename: Gerard
authors.initials: G
authors.lastname: Ruiz-Babot
authors.affiliation: The Heart Centre, Queen Mary University of London, London, EC1M 6BQ, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Harmer
authors.affiliation: The Heart Centre, Queen Mary University of London, London, EC1M 6BQ, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Tinker
authors.affiliation: School of Healthcare Science, The Manchester Metropolitan University, Manchester, M1 5GD, UK.
authors.forename: Tristan
authors.initials: T
authors.lastname: McKay
authors.affiliation: Centre for Endocrinology, Queen Mary University of London, London, EC1M 6BQ, UK.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Guasti
authors.affiliation: Centre for Endocrinology, Queen Mary University of London, London, EC1M 6BQ, UK. Electronic address: l.dunkel@qmul.ac.uk.
authors.forename: Leo
authors.initials: L
authors.lastname: Dunkel
mesh_terms: D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D000066449:Human Embryonic Stem Cells / Q000378:metabolism; D006801:Humans; D059648:Kisspeptins / Q000378:metabolism*; D058953:Neural Stem Cells / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000378:metabolism*
chemical_list: D059648:Kisspeptins; D007987:Gonadotropin-Releasing Hormone; D002118:Calcium
keywords: Embryonic stem cells; GnRH neurons; Kisspeptin; Pluripotent stem cells