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

描述（申请人提供）：精原干细胞（SSC）是哺乳动物精子发生的基础。在啮齿类动物中，SSC池由A型精原细胞组成，其平衡自我更新和分化分裂以精确地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，将啮齿动物SSC数据外推到包括人类在内的高等物种是很有吸引力的。然而，在人类和非人类灵长类动物（NHP）中，精子发生来自Adark（Ad）和Apale（Ap）精原细胞，干细胞的身份受到争议。物种之间的根本差异突出了研究NHP SSC生物学的必要性，NHP SSC与人类生理学具有更大的相关性。为了便于灵长类动物睾丸精原干细胞的研究，需要一种体内功能检测方法。在初步研究中，我们开发了灵长类动物到裸鼠异种移植作为一种生物学试验，以评估SSC功能。此外，我们确定了一种导致恒河猴长期不育的白消安治疗方案，以促进自体和同源睾丸细胞移植。该模型将用于测试恒河猴SSC的完全再生潜力和干细胞/生态位相互作用。我们将在本申请中利用恒河猴-裸鼠异种移植试验来表征幼年和成年的SSC活性（目的1），并使用荧光激活细胞分选仪鉴定恒河猴SSC的表型特征（目的2）。目标2的进展将使恒河猴精原干细胞的分离、富集和分子鉴定成为可能。由于恒河猴和小鼠之间的进化距离，恒河猴精原干细胞将植入并持续存在于小鼠曲细精管中，但不会产生完整的精子发生。因此，将在目标3中进行自体和同源恒河猴间移植，以检测目标2中鉴定的推定SSC富集恒河猴睾丸细胞群的完全再生潜力。这一目标还将提供有关白消安处理的睾丸中干细胞生态位的质量、自体与同源供体细胞的免疫耐受性以及冷冻保存对SSC再生潜力的影响的信息。最后，我们的进展表征NHP SSC（从目标1-3）将指导我们的初步研究目标4，以表征人类精原干细胞。这些研究将提供有关非人灵长类动物睾丸中干细胞和干细胞生态位的基本信息，并为将来研究人类精原干细胞的生物学和治疗潜力奠定基础。公共卫生相关性：这些研究将为研究恒河猴精原干细胞的生物学和分子特征提供实验框架，并将结果转化为促进人类精原干细胞的研究。这些研究还将建立一个非人灵长类不育模型，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病例有意义。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.