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终端患者的治疗工具提供了希望 帕金森氏症，亨廷顿，肌萎缩性侧面硬化症和青光眼等疾病。虽然 缺乏生殖功能不是威胁生命的疾病，这是一个严重的特发性问题 性腺功能低下患者，没有功能性促性腺激素释放的同性恋患者 （GnRH）下丘脑中的神经元。因此，他们无法构想和需要同性子 一生的治疗。在此应用中，我们建议建立生成高度的方法 来自胚胎干细胞（ESC）和诱导多能干细胞的富含GnRH神经元的群体种群 （IPSC）源自人（H）并表征ESC/IPSC衍生的GNRH神经元的特性。我们 将检验以下假设：hESC/HIPSC暴露功能特征的GNRH神经元相似 正如我们先前在非人类隐私中报道的那样，对于原发性GNRH神经元中的人来说。在目标1中，我们将 建立从HESC和HIPSC产生GNRH神经元的效率较高的方法。为了 产生高度富集或几乎纯净的GnRH神经元，我们将提高效率 基本的前脑祖细胞并应用抗魔法剂，例如缺口抑制剂。我们还将生成 使用CRISPR/CAS9使用荧光标记的GNRH神经元。在AIM 2中，我们将检查GNRH神经元是否得出 以脉动的方式从hESC/HIPSC释放GNRH，并在细胞内周期性同步 钙振荡[Ca2+] I，类似于灵长类GNRH神经元中报道的钙。 PI在研究方面的专业知识 非人类隐私的GNRH神经生物学以及干细胞研究中的历史和丰富资源 威斯康星大学确保该项目的成功。 拟议项目的结果将为开发R01项目提供基础，导致地面制动单元格 用IPSC诱导的GNRH神经元替换疗法，作为人类的模型 患者。此外，PI认为，从干细胞中的纯GnRH神经元产生将使我们能够 偏心疾病模型的特发性性腺功能障碍性性腺功能减退，并有助于开发新工具 不育的临床管理以及相反的生育能力控制方法。