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.

摘要： 干细胞研究的最新进展为晚期乳腺癌患者的治疗工具提供了希望。 疾病，如帕金森氏病、亨廷顿氏病、肌萎缩侧索硬化症和青光眼。虽然 缺乏生殖功能不是一个危及生命的条件，这是一个严重的问题，特发性 低促性腺激素性性腺功能减退症患者，没有功能性促性腺激素释放激素 下丘脑中的促性腺激素释放激素（GnRH）神经元。因此，他们不能怀孕，需要激素 一辈子的治疗。在本申请中，我们提出建立用于产生高度非线性的方法。 来自胚胎干细胞（ESC）和诱导多能干细胞的GnRH神经元的富集群体 （h）并表征ESC/iPSC衍生的GnRH神经元的性质。我们 将检验来自hESC/hiPSC的GnRH神经元表现出类似于 与我们先前在非人类灵长类动物中报道的原代GnRH神经元中的结果一致。在目标1中， 建立从hESC和hiPSC产生GnRH神经元的更高效率的方法。为 产生高度富集或接近纯的GnRH神经元群体，我们将提高促性腺激素释放激素的效率。 基底前脑祖细胞，并应用抗有丝分裂剂，如notch抑制剂。我们还将产生 使用CRISPR/Cas9荧光标记的GnRH神经元。在目标2中，我们将检查是否GnRH神经元衍生 从hESC/hiPSC以脉冲方式释放GnRH，并表现出细胞内GnRH的周期性同步化。 钙振荡[Ca 2 +]i，类似于灵长类GnRH神经元中报道的那些。PI在以下研究方面的专业知识 非人灵长类的GnRH神经生物学以及干细胞研究的历史和丰富资源 威斯康星州大学确保所提出的项目的成功。 建议项目的结果将为R 01项目的开发提供基础，导致地面制动单元 在无排卵猴中用iPSC诱导的GnRH神经元替代疗法，作为人类模型 患者此外，PI相信从干细胞产生纯的GnRH神经元将使我们能够 建立特发性低促性腺激素性腺功能减退症的疾病模型，并帮助开发新的工具， 不孕症的临床管理，以及相反，生育控制的替代安全方法。