Characterization of male germline stem cells in a rhesus model of infertility

摘要 精原干细胞是哺乳动物精子发生的基础。在啮齿类动物中， SSC库由平衡自我更新和 区分分区，以准确满足生精系统的生物学需求。 精子发生在哺乳动物中是高度保守的，因此推断啮齿动物的SSC数据很有吸引力 包括人类在内的高等物种然而，在人类和非人灵长类动物（NHP）中， 精子发生起源于Adark（Ad）和Apale（Ap）精原细胞，干细胞的身份 是有争议的物种之间的根本差异突出了调查的必要性 NHP SSC的生物学，与人类生理学有更大的相关性。方便 为了研究灵长类动物睾丸中的精原干细胞，需要进行体内功能测定。在 在初步研究中，我们开发了灵长类动物-裸鼠异种移植作为一种生物学试验， 评价SSC功能。此外，我们确定了一种白消安治疗方案，导致长期- 恒河猴长期不孕症，以促进自体和同种睾丸细胞移植。 该模型将用于测试恒河猴SSC和干细胞/生态位的完全再生潜力。 交互.我们将在目前的研究中利用恒河猴-裸鼠异种移植试验。 应用于表征幼年和成年的SSC活性（目的1）并鉴定表型 使用荧光激活细胞分选仪（Aim 2）对恒河猴SSC的特征进行了研究。目标2的进展 将使恒河猴精原干细胞的分离、富集和分子表征成为可能。因为 由于恒河猴与小鼠之间存在进化距离，恒河猴精原干细胞将在小鼠体内移植并持续存在， 生精小管，但不会产生完整的精子发生。因此，自体和 将在目标3中进行同源恒河猴到恒河猴移植，以测试完全再生的恒河猴。 目的2中鉴定的推定的SSC富集的恒河猴睾丸细胞群的潜力。这一目标还将 提供白消安处理睾丸中干细胞小生境质量的信息， 自体与同源供体细胞的免疫耐受性，以及冷冻保存的效果 关于SSC再生潜力最后，我们在表征NHP SSC（目标1-3）方面的进展将 指导我们在目标4中的初步研究，以表征人类精原干细胞。 这些研究将提供有关干细胞和干细胞生态位的基本信息， 非人类灵长类动物的睾丸，并奠定基础，将这些发现转化为未来 研究人类SSC的生物学和治疗潜力。项目叙述 拟议的研究将提供实验框架，研究生物学和 恒河猴精原干细胞（SSCs）的分子特征，并将结果转化为 促进人类SSC的研究。这些研究还将建立一个非人类灵长类动物模型 并检查精原干细胞的再生潜力，以建立供体精子发生 和/或恢复生育能力。在恒河猴模型中负责任地发展这些想法可能 对治疗某些男性不育症的影响。

Abstract Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. In rodents, the SSC pool is comprised of type Asingle (As) spermatogonia that balance self-renewing and differentiating divisions to exactly meet the biological demand of the spermatogenic system. Spermatogenesis is highly conserved in mammals and it is tempting to extrapolate rodent SSC data to higher species, including humans. However, in humans and nonhuman primates (NHPs), spermatogenesis arises from Adark (Ad) and Apale (Ap) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between species highlight the need to investigate the biology of NHP SSCs, which have greater relevance to human physiology. To facilitate the investigation of spermatogonial stem cells in primate testes, an in vivo functional assay is needed. In preliminary studies, we developed primate- to-nude mouse xenotransplantation as a biological assay to evaluate SSC function. In addition, we identified a busulfan treatment regimen that causes long- term infertility in rhesus macaques to facilitate autologous and homologous testis cell transplantation. This model will be used to test the full regenerative potential of rhesus SSCs and stem cell/niche interactions. We will exploit the rhesus-to-nude mouse xenotransplantation assay in the current application to characterize SSC activity in juvenile and adult (Aims 1) and identify phenotypic characteristics of rhesus SSCs using a fluorescence-activated cell sorter (Aim 2). Progress in Aim 2 will enable the isolation, enrichment and molecular characterization of rhesus SSCs. Because of evolutionary distance between rhesus and mouse, rhesus SSCs will engraft and persist in mouse seminiferous tubules, but will not produce complete spermatogenesis. Therefore, autologous and homologous rhesus-to-rhesus transplantations will be performed in Aim 3 to test the full regenerative potential of putative SSC-enriched rhesus testis cell populations identified in Aim 2. This aim will also provide information about the quality of the stem cell niche in busulfan-treated testes, the immunological tolerance of autologous vs. homologous donor cells, and the effect of cryopreservation on SSC regenerative potential. Finally, our progress characterizing NHP SSCs (from Aims 1-3) will guide our initial studies in Aim 4 to characterize human spermatogonial stem cells. These studies will provide fundamental information about stem cells and the stem cell niche in nonhuman primate testes and lay the groundwork to translate these discoveries for future investigations of the biology and therapeutic potential of human SSCs. Project Narrative The proposed studies will provide the experimental framework to study the biology and molecular characteristics of rhesus spermatogonial stem cells (SSCs) and translate the results to facilitate the investigation of human SSCs. The studies will also establish a nonhuman primate model of infertility and examine the regenerative potential of SSCs to establish donor spermatogenesis and/or restore fertility. Responsible development of these ideas in the rhesus model may have implications for treating some cases of male infertility.