A System for Culturing Mammalian Spermatogonial Cells

哺乳动物精原细胞培养系统

• 批准号: 9447188
• 负责人: INA DOBRINSKI
• 金额: $ 22.98万
• 依托单位: UNIVERSITY OF CALGARY
• 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 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9447188
• 关键词: Address; Adolescent; Adult; Animal Model; Autophagocytosis; Autophagosome; Biological Assay; Biological Markers; Bioreactors; Cell Culture Techniques; Cell Separation; Cell Survival; Cell Transplantation; Cell physiology; Cells; Cellular Stress; Confocal Microscopy; Cytotoxic agent; Data; Development; Digestion; Dissociation; Enzymes; Excision; Exposure to; Family suidae; Fertility; Foundations; Germ Cells; Germ Lines; Goals; Homologous Transplantation; Human; In Situ; In Vitro; Infertility; Label; Maintenance; Male Infertility; Malignant Neoplasms; Mechanics; Mitochondria; Monitor; Mus; Organoids; Pathway interactions; Patients; Phylogenetic Analysis; Physiological; Physiology; Population; Production; Proliferating; Proteins; Rattus; Rodent; Somatic Cell; Spermatogenesis; Spermatogonia; Standardization; Stem cells; Structure; Suspension Culture; Suspensions; System; Technology; Testing; Testis; Tissues; Toxicology; Toxin; Translating; Translations; Transplantation; Tubular formation; Undifferentiated; WNT Signaling Pathway; Work; Xenograft procedure; base; biological adaptation to stress; cancer therapy; cell type; clinical translation; experimental study; fertility preservation; human pluripotent stem cell; improved; in vivo; inhibitor/antagonist; male; male fertility; member; novel; novel strategies; phthalates; preservation; response; restoration; screening; sperm cell; stressor; tool

Project Summary The long term goal of this project is to establish an efficient system for the culture of human spermatogonial stem cells (SSCs). Availability of safe and efficient strategies to propagate SSCs in culture will aid in the development of new options for male factor infertility as well as preservation and restoration of fertility in juvenile and adult patients undergoing potentially sterilizing treatments for cancer. This project will use pigs as an established, accessible animal model phylogenetically and physiologically more similar to humans than rodents. Key findings will be translated to human SSCs. Lack of appropriate tools to monitor SSC function, to generate sufficient numbers of SSCs for fertility preservation, and to develop alternatives to transplantation for screening of SSC function are identified as significant barriers for translation to new treatment options for male infertility (PAR 16-114). Three specific aims address these barriers: 1) To characterize autophagy as a functional biomarker in SSCs, 2) To investigate stirred suspension bioreactors as a novel approach to SSC culture, and 3) To explore testicular organoid formation as a short term functional assay for SSCs. Aim 1 will explore activation of the autophagic pathway in SSCs in response to stressors associated with removal from the testis microenvironment, culture in vitro and exposure to toxic insults. This is expected to establish autophagy as a functional biomarker to improve culture conditions and as a toxicological bioassay for germ line stem cells. Aim 2 will focus on establishing culture in stirred suspension bioreactors as an original scalable approach to maintain and expand SSCs in vitro. Our team members are pioneers of this approach, having established its use for culture of mouse and human pluripotent stem cells in an undifferentiated state, and it has shown early promise for SSC culture. Experiments also address strategies to isolate undifferentiated spermatogonia from the donor testis while minimizing cell stress, thus initiating a suitable cell population for culture. As homologous transplantation is not a viable bioassay for stem cell potential in non-rodent SSCs, support of spermatogenesis in de novo formed testis tissue after grafting to mouse host, as previously validated by our team, will serve as a functional endpoint. Aim 3 will explore testicular organoid formation in microwell culture as an alternative bioassay for transplantation. We will draw on expertise of our collaborator who invented culture of functional organoids in microwells. Formation of organoids with appropriate cell associations will be monitored by confocal microscopy and correlated with support of spermatogenesis after grafting in vivo. If parameters identified in microwell culture are predictive of the ability of SSCs to support sperm production after transplantation, microwell organoid culture could serve as a functional short-term screening assay for SSCs. Taken together, our experimental results will provide an integrated system to isolate, culture and functionally analyze non-rodent SSCs with clinical translation towards the preservation and restoration of human fertility.

项目概要 该项目的长期目标是建立一个高效的人类文化体系 精原干细胞（SSC）。在文化中传播 SSC 的安全有效策略的可用性将 帮助开发针对男性因素不孕症的新选择以及保留和恢复生育能力 在接受潜在的癌症绝育治疗的青少年和成年患者中。该项目将使用猪 作为一种已建立的、易于使用的动物模型，在系统发育和生理学上比人类更相似 啮齿动物。主要发现将转化为人类 SSC。缺乏适当的工具来监控 SSC 功能， 产生足够数量的SSC来保存生育力，并开发移植的替代方案 SSC 功能筛查被认为是转化为男性新治疗方案的重大障碍 不孕症（第 16-114 段）。解决这些障碍的三个具体目标：1）将自噬描述为 SSC 中的功能生物标志物，2) 研究搅拌悬浮生物反应器作为一种新方法 SSC 培养，3) 探索睾丸类器官的形成作为 SSC 的短期功能测定。 目标 1 将探索 SSC 中自噬途径的激活，以响应与去除相关的应激源 来自睾丸微环境、体外培养和接触有毒物质。预计这将建立 自噬作为改善培养条件的功能生物标志物和作为种系毒理学生物测定 干细胞。目标 2 将侧重于在搅拌悬浮生物反应器中建立培养物作为原始可扩展的 体外维持和扩展 SSC 的方法。我们的团队成员是这种方法的先驱， 确立了其在未分化状态下培养小鼠和人类多能干细胞的用途，并且已 展现了 SSC 文化的早期前景。实验还提出了隔离未分化细胞的策略 来自供体睾丸的精原细胞，同时最大限度地减少细胞应激，从而启动合适的细胞群 文化。由于同源移植不是一种可行的非啮齿类 SSC 干细胞潜力生物测定方法， 正如之前所验证的，移植到小鼠宿主后从头形成的睾丸组织支持精子发生 由我们的团队设计，将作为功能端点。目标 3 将探索微孔中睾丸类器官的形成 培养作为移植的替代生物测定。我们将利用发明者的合作者的专业知识 在微孔中培养功能性类器官。具有适当细胞关联的类器官的形成将是 通过共聚焦显微镜监测并与体内移植后精子发生的支持相关。如果 微孔培养中确定的参数可预测 SSC 支持精子产生的能力 移植后，微孔类器官培养可以作为 SSC 的功能性短期筛选试验。 总而言之，我们的实验结果将提供一个集成的系统来分离、培养和功能化 分析非啮齿动物 SSC 并进行临床转化，以保存和恢复人类生育能力。