A System for Culturing Mammalian Spermatogonial Cells

项目摘要 该项目的长期目标是建立一个有效的人类文化体系。 精原干细胞(SSC)。安全和高效的策略在培养中传播SSCs将 协助制定男性因素不育症以及保存和恢复生育能力的新选择 在接受潜在绝育癌症治疗的青少年和成年患者中。这个项目将使用猪 作为一种成熟的、可访问的动物模型，在系统发育和生理上与人类更相似，而不是 啮齿动物。关键发现将被转化为人类SSCs。缺乏适当的工具来监控SSC功能，以 产生足够数量的干细胞以保持生育能力，并开发移植的替代方案 SSC功能筛查被认为是转化为新的男性治疗方案的重要障碍 不孕症(标准杆16-114杆)。解决这些障碍的三个具体目标：1)将自噬描述为 SSCs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的方法 SSC培养；3)探讨睾丸类器官形成作为SSCs短期功能检测方法。 目的1将探索自噬通路在SSCs对与移除相关的应激源的响应中的激活 从睾丸微环境、体外培养和暴露于有毒的侮辱。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生殖系的毒理生物测定 干细胞。目标2将专注于在搅拌悬浮生物反应器中建立培养物作为一种原始的可扩展 干细胞的体外维持和扩增方法。我们的团队成员是这种方法的先驱，拥有 建立了在未分化状态下用于培养小鼠和人类多能干细胞的方法，并已 对SSC文化表现出了早期的希望。实验还解决了隔离未分化的策略 从供体睾丸中分离出精原细胞，同时将细胞压力降至最低，从而为 文化。由于同种移植不是对非啮齿动物SSCs中干细胞潜能的可行生物测试， 如先前证实的，移植到小鼠宿主后新生形成的睾丸组织对精子发生的支持 由我们的团队提供，将作为功能终点。目标3将在微孔中探索睾丸器官的形成 培养作为移植生物检测的替代方法。我们将利用我们的合作者的专业知识 微孔培养功能有机物。具有适当细胞缔合的有机物的形成将是 通过共聚焦显微镜监测，并与体内移植后对精子发生的支持相关。如果 微孔培养中发现的参数可以预测SSCs支持精子产生的能力 移植、微孔器培养可作为SSCs功能的短期筛选方法。 综上所述，我们的实验结果将提供一个完整的分离、培养和功能系统 分析非啮齿动物SSCs的临床翻译，以保存和恢复人类生育能力。

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.