Stem Cell-derived GnRH Neurons: Optimization and Characterization

摘要： 干细胞研究的最新进展为晚期糖尿病患者的治疗手段带来了希望。 疾病，如帕金森氏症、亨廷顿氏症、肌萎缩侧索硬化症和青光眼。尽管 生殖功能缺失不是危及生命的疾病，而是特发性疾病的严重问题 没有功能性促性腺激素释放激素的促性腺激素减退症患者 下丘脑(GnRH)神经元。因此，她们不能怀孕，需要激素 为他们的一生提供治疗。在本应用程序中，我们建议建立方法来生成高度 胚胎干细胞来源的促性腺激素释放激素神经元和诱导的多能干细胞 目的：研究来源于人(H)的促性腺激素释放激素(GnRH)神经元的特性。我们 将检验来自hESC/hiPSC的GnRH神经元表现出类似的功能特征的假设 与我们之前在非人类灵长类动物中报道的初级GnRH神经元中的GnRH类似。在目标1中，我们将 建立从hESC和hiPSC产生更高效率的GnRH神经元的方法。为 产生高度丰富或几乎纯净的GnRH神经元群体，我们将提高 并应用抗有丝分裂药物，如缺口抑制剂。我们还将生成 CRISPR/Cas9荧光标记促性腺激素释放激素神经元在目标2中，我们将检查促性腺激素释放激素神经元是否源自 HESC/HiPSC以脉动方式释放GnRH，表现出细胞内周期性同步性 钙振荡[Ca~(2+)]i，与已报道的灵长类促性腺激素释放激素神经元相似。国际和平协会在研究 非人灵长类促性腺激素释放激素神经生物学及其干细胞研究的历史和丰富的资源 威斯康星大学确保拟议项目的成功。 拟建项目的结果将为开发R01项目提供基础，从而导致地面制动单元 IPSC诱导的促性腺激素释放激素神经元替代疗法作为人促性腺激素释放激素模型 病人。此外，PI认为，从干细胞产生纯净的GnRH神经元将使我们能够 建立特发性促性腺激素减退症的疾病模型，并帮助开发新的治疗工具 不孕不育的临床治疗，反过来，替代安全的生育控制方法。

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.