Abstract: Recent advancements in stem cell research have provided hope for treatment tools in patients with terminal diseases, such as Parkinson’s, Huntington’s, amyotrophic lateral sclerosis, and glaucoma. Although the absence of reproductive function is not a life-threatening condition, it is a serious problem for Idiopathic Hypogonadotropic Hypogonadism patients, who do not have functional gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus. Consequently, they are not able to conceive and need hormone treatments for their entire lives. In this application we propose to establish methods for generating a highly enriched population of GnRH neurons from embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) derived from humans (h) and to characterize the properties of ESC/iPSC-derived GnRH neurons. We will test the hypothesis that GnRH neurons derived from hESC/hiPSC exhibit functional characteristics similar to those seen in primary GnRH neurons, as we reported previously in non-human primates. In Aim 1 we will establish methods yielding a higher efficiency of generating GnRH neurons from hESC and hiPSC. For generation of a highly enriched or nearly pure population of GnRH neurons, we will increase efficiency for basal forebrain progenitor cells and apply antimitotic agents, such as a notch inhibitor. We will also generate fluorescent-labeled GnRH neurons using CRISPR/Cas9. In Aim 2, we will examine if GnRH neurons derived from hESC/hiPSC release GnRH in a pulsatile manner and exhibit periodical synchronization of intracellular calcium oscillations [Ca2+]i, similar to those reported in primate GnRH neurons. The PI’s expertise in studies of GnRH neurobiology in non-human primates and the history and rich resources in stem cell research in the University of Wisconsin ensure the success of the proposed project. Results from proposed project will provide a basis for developing R01 projects, leading to ground braking cell replacement therapy with iPSC-induced GnRH neurons in anovulatory monkeys, as a model for human patients. Moreover, the PI believes that generation of the pure GnRH neurons from stem cells will allow us to estrablish a disease model of Idiopathic Hypogonadotropic Hypogonadism and help to develop new tools for clinical management of infertility and, conversely, alternative safe methods of fertility control.

Project Narrative / Public Health Relevance Statement Understanding the control mechanism of GnRH release is fundamentally important for the onset of puberty during development, cyclic ovulation during adult life, and menopause after aging. The proposed study generating a highly enriched population of GnRH neurons from human embryonic stem cells and induced pluripotent stem cells will open up a major avenue for research in reproductive neuroendocrinology. This includes disease modeling of Idiopathic Hypogonadotropic Hypogonadism, and the development of new tools for clinical management of infertility and, conversely, alternative safe methods of fertility control.