Stem Cell-derived GnRH Neurons: Optimization and Characterization

干细胞衍生的 GnRH 神经元：优化和表征

• 批准号: 9331170
• 负责人: Ei Terasawa-Grilley
• 金额: $ 22.95万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9331170
• 关键词: Adult; Aging; Agonist; Alpha Cell; Amyotrophic Lateral Sclerosis; Animals; Antimitotic Agents; Behavior; Blood Circulation; Brain; CRISPR/Cas technology; Calcium Oscillations; Cell Line; Cells; Characteristics; Clinical Management; Clinical Research; Contraceptive methods; Development; Disease; Disease model; Embryo; Embryonic Development; Ensure; Exhibits; Failure; Female; Funding; Generations; Genes; Germ Cells; Glaucoma; Goals; Gonadal Steroid Hormones; Gonadal structure; Gonadotropin Hormone Releasing Hormone; Hormones; Human; Huntington Disease; Hypothalamic structure; Immunohistochemistry; In Vitro; Incidence; Individual; Infertility; Institutes; Kallmann Syndrome; Label; Life; Menopause; Methods; Modeling; Molecular; Monkeys; Mus; Neurobiology; Neurons; Ovulation; Parkinson Disease; Patients; Pattern; Periodicals; Periodicity; Physiologic pulse; Pituitary Gland; Play; Population; Primates; Process; Production; Property; Proteins; Puberty; Rattus; Recording of previous events; Replacement Therapy; Reporting; Research; Resources; Sheep; Somatic Cell; Specimen; Stem Cell Research; Stem cells; Syndrome; System; Terminal Disease; Testing; Time; Tissues; Translational Research; Tumor-Derived; Universities; Wisconsin; basal forebrain; embryonic stem cell; human embryonic stem cell; induced pluripotent stem cell; inhibitor/antagonist; male; median eminence; migration; nonhuman primate; notch protein; public health relevance; relating to nervous system; reproductive; reproductive function; reproductive neuroendocrinology; success; symptomatic improvement; tool

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.

抽象的： 干细胞研究的最新进展为晚期患者的治疗工具带来了希望 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。虽然 生殖功能缺失并不危及生命，但对于特发性患者来说这是一个严重的问题 促性腺激素低下性腺功能减退症患者，没有功能性促性腺激素释放激素 (GnRH) 下丘脑神经元。因此，她们无法怀孕并且需要激素 终其一生的治疗。在此应用中，我们建议建立生成高度 来自胚胎干细胞 (ESC) 和诱导多能干细胞的 GnRH 神经元富集群 (iPSC) 源自人类 (h)，并表征 ESC/iPSC 衍生的 GnRH 神经元的特性。我们 将检验源自 hESC/hiPSC 的 GnRH 神经元表现出类似功能特征的假设 正如我们之前在非人类灵长类动物中报道的那样，在初级 GnRH 神经元中看到的那些。在目标 1 中，我们将 建立从 hESC 和 hiPSC 生成 GnRH 神经元的效率更高的方法。为了 生成高度富集或接近纯的 GnRH 神经元群体，我们将提高效率 基底前脑祖细胞并应用抗有丝分裂剂，例如缺口抑制剂。我们还将生成 使用 CRISPR/Cas9 荧光标记的 GnRH 神经元。在目标 2 中，我们将检查 GnRH 神经元是否衍生 hESC/hiPSC 以脉冲方式释放 GnRH，并表现出细胞内周期性同步 钙振荡 [Ca2+]i，与灵长类 GnRH 神经元中报道的相似。 PI 在研究方面的专业知识 非人类灵长类动物的 GnRH 神经生物学以及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。 拟议项目的结果将为开发 R01 项目提供基础，从而形成地面制动单元 在无排卵猴中使用 iPSC 诱导的 GnRH 神经元进行替代疗法，作为人类模型 患者。此外，PI 相信，从干细胞中产生纯 GnRH 神经元将使我们能够 建立特发性低促性腺激素性性腺功能减退症的疾病模型，并帮助开发新工具 不孕症的临床管理，以及相反的生育控制的替代安全方法。