In vitro differentiation of the hypothalamic KNDy neuron, a master regulator for reproduction, from mouse embryonic stem cells

BACKGROUND: Hypothalamic kisspeptin neurons are master regulators of reproduction in mammals, including humans and rodents, via direct stimulation of gonadotropin-releasing hormone (GnRH) neurons. Therefore, they play a central role in puberty onset and gonadal function in mammals. Arcuate kisspeptin neurons express both neurokinin B (NKB) and dynorphin A (Dyn). Thus, these neurons are also referred to as KNDy neurons, and accumulating evidence suggests that KNDy neurons are responsible for tonic GnRH/gonadotropin release and the consequent folliculogenesis/steroidogenesis. Notably, mutations in genes encoding kisspeptin (KISS1), kisspeptin receptor (GPR54), or NKB (TAC3) in humans, or deletion of these genes in rodents, cause hypogonadotropic hypogonadism. Therefore, cellular models of KNDy neurons are useful for elucidating the pathogenesis of reproductive disorders and for developing novel therapies for these disorders. Here, we established a method to differentiate KNDy neurons from mouse embryonic stem cells (mESCs). METHODS: mESCs were incubated in culture medium containing agents that enhance Sonic Hedgehog signaling, a ventralizing signal, to induce the differentiation of ventral hypothalamic organoids. Kisspeptin-expressing cells were generated by adding a Notch inhibitor, dispersing the aggregates, and transferring them into a two-dimensional culture system. Kisspeptin-, NKB-, and Dyn-expressing cells were identified by immunohistochemistry, and kisspeptin secretion into the culture medium was quantified by enzyme-linked immunosorbent assay. RESULTS: Immunoreactivity for kisspeptin, NKB, and Dyn was detected in differentiated organoids derived from mESCs. Furthermore, kisspeptin secretion was evident in culture supernatants of differentiated KNDy neurons. CONCLUSIONS: This is the first report demonstrating the differentiation of KNDy neurons from pluripotent stem cells, which can be applied to create cellular models for various diseases caused by KNDy peptide deficiency and may contribute to the development of novel therapeutic approaches and understanding cellular mechanisms regulating mammalian reproduction.