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。在啮齿动物身上， SSC池由单精原细胞(AS)组成，它平衡自我更新和 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，因此很容易推断啮齿动物的SSC数据 更高级的物种，包括人类。然而，在人类和非人类灵长类动物(NHP)中， 精子发生起源于Adark(Ad)和Apale(AP)精原细胞以及干细胞的特性 是有争议的。物种之间的根本差异突出了研究的必要性 NHP SSCs的生物学，这与人类生理学有更大的相关性。为了方便 为了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……里面 初步研究，我们发展了灵长类到裸鼠的异种移植作为一种生物学实验 评价SSc功能。此外，我们确定了一种可导致长期- 恒河猴的不孕症促进自体和同源睾丸细胞移植。 这个模型将被用来测试恒河猴干细胞和干细胞/利基的全部再生潜力。 互动。我们将开展恒河猴到裸鼠的异种移植实验。 青少年和成人SSC活性特征(AIMS 1)及其表型鉴定的应用 用荧光激活细胞分选仪研究恒河猴干细胞的特性(目标2)。AIM 2的进展 这将使恒河猴干细胞的分离、浓缩和分子鉴定成为可能。因为. 恒河猴与小鼠的进化距离，恒河猴SSCs将在小鼠体内移植并存活 生精小管，但不会产生完全的精子发生。因此，自体和 在目标3中，将进行恒河猴到恒河猴的同源移植，以测试完整的再生 目标2中确定的假定的SSC富集型恒河猴睾丸细胞群的潜力。该目标还将 提供有关经白消安治疗的睾丸中干细胞利基的质量的信息， 自体与同种供体细胞的免疫耐受性及冷冻保存的影响 关于SSC再生潜力。最后，我们对NHP SSC的描述(来自目标1-3)的进展将 指导我们在目标4中的初步研究，以确定人类精原干细胞的特征。 这些研究将提供有关干细胞和干细胞生态位的基本信息 非人灵长类动物的测试，并为将来将这些发现转化为 人干细胞的生物学和治疗潜力的研究。项目叙事 建议的研究将提供实验框架，以研究生物和 恒河猴精原干细胞(SSCs)的分子特征及其转化 促进人类SSCs的研究。这些研究还将建立一个非人类灵长类动物模型 并检测SSCs的再生潜力以建立供者精子发生 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 对某些男性不育病例治疗的启示。

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.