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

描述(申请人提供)：精原干细胞(SSCs)是哺乳动物精子发生的基础。在啮齿动物中，SSC池由单个(AS)精原细胞组成，它们平衡自我更新和分化分裂，以准确满足生精系统的生物学需求。哺乳动物的精子发生是高度保守的，因此很容易将啮齿动物的SSC数据外推到包括人类在内的高等物种。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，干细胞的身份存在争议。物种之间的根本差异突出了研究NHP SSCs生物学的必要性，因为NHP SSCs与人类生理学具有更大的相关性。为了便于研究灵长类动物睾丸中的精原干细胞，有必要进行体内功能检测。在初步研究中，我们发展了灵长类到裸鼠的异种移植作为评估SSC功能的生物学实验。此外，我们确定了一种导致恒河猴长期不育的白花丹治疗方案，以促进自体和同种睾丸细胞移植。这个模型将被用来测试恒河猴干细胞的全部再生潜力和干细胞/利基的相互作用。我们将利用目前应用中的恒河猴至裸鼠异种移植实验来表征幼年和成年恒河猴SSC的活性(AIMS 1)，并使用荧光激活细胞分选器鉴定恒河猴SSCs的表型特征(AIMS 2)。AIM 2的进展将使恒河猴SSCs的分离、浓缩和分子鉴定成为可能。由于恒河猴与小鼠在进化上的距离，恒河猴SSCs将移植并存活在小鼠生精小管上，但不会产生完全的生精作用。因此，在目标3中，将进行自体和同源恒河猴到恒河猴的移植，以测试目标2中确定的假定的SSC富集型恒河猴睾丸细胞群的全部再生潜力。这一目标还将提供关于经白丹处理的睾丸干细胞生态位的质量、自体与同种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，我们对NHP SSCs的研究进展(来自目标1-3)将指导我们在目标4中对人类精原干细胞的初步研究。这些研究将提供有关干细胞和非人灵长类动物睾丸中干细胞生态位的基本信息，并为未来研究人类SSCs的生物学和治疗潜力奠定基础。公共卫生相关性：拟议的研究将提供实验框架，以研究恒河猴精原干细胞(SSCs)的生物学和分子特征，并将结果转化为促进对人类SSCs的研究。这些研究还将建立不育的非人类灵长类动物模型，并检查SSCs的再生潜力以建立捐赠者精子发生和/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会对治疗一些男性不育病例产生影响。

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.