Molecular control of spermatogonial stem cell fate for achieving cell therapy

描述(申请人提供)：精原干细胞(SSCs)是独一无二的，因为它们是处于多能性门槛的成年细胞，具有在体外自发重新编程为多能性的能力，而不需要添加外源性因素。干细胞培养和将干细胞重新编程为多能细胞是两项新技术，有望创造针对患者的治疗多种疾病的方法。我们的长期目标是阐明SSC自我更新和分化的分子机制以及SSC重编程现象，并将这些发现转化为基于细胞的治疗。然而，SSC培养中“真实”干细胞的稀有和SSC重新编程的低效是实现治疗目标的主要障碍。在这里，我们测试了一种通过操纵转录因子OCT4来克服这些障碍的策略，同时研究了SSC调控的基本问题。对OCT4水平的精确控制对于维持有异常的胚胎细胞的多能性至关重要 导致分化。基于我们对OCT4在小鼠SSCs中功能的研究，以及一个新出现的OCT4在ES细胞中的翻译后控制模型，我们提出了以下假设：SSC的命运部分取决于通过翻译后修饰微调OCT4蛋白水平。第一个目标是确定OCT4水平如何在体外影响SSC的命运，并与实现基于SSC的治疗直接相关。利用SSCs以剂量依赖的方式过表达OCT4的初步数据表明，OCT4的增加足以增加重编程；此外，OCT4的增加在体外抑制SSC的分化，预测SSC的数量相应增加。第二个目的是研究调节SSCs中OCT4蛋白水平的机制。我们将从测试SSCs中E3泛素连接酶WWP2的功能开始，识别控制OCT4泛素化的顺式和反式作用因子。第三个目标是确定OCT4的异常水平如何影响体内SSC的命运。通过创造一只有条件地过表达OCT4的转基因小鼠，我们将测试OCT4水平在精子发生期间被“下调”以允许精原细胞分化的想法，这一过程仍然定义不清。此外，OCT4的过度表达与肿瘤的发生有关，我们的小鼠模型将有助于确定OCT4的过度表达是否在生殖细胞肿瘤的发生中起关键作用。这一建议的创新之处在于：(1)通过稳定的OCT4来提高重编程效率；(2)关注SSC重编程而不是体细胞重编程；(3)分析OCT4在SSCs而不是ES细胞中的功能和调节。我们的研究将影响对SSCs、多能细胞和肿瘤细胞之间关系的基本理解，这些关系可能是由于精原命运的错误调节造成的。此外，这些研究具有重要意义，因为它们将提供知识基础和机制洞察力，适用于通过调节OCT4蛋白来增加干细胞在SSC培养中的百分比和重新编程的频率。

DESCRIPTION (provided by applicant): Spermatogonial stem cells (SSCs) are unique in that they are adult cells poised on the threshold of pluri- potency, having the ability to become spontaneously reprogrammed to pluripotency in vitro without addition of exogenous factors. SSC culturing and the reprogramming of SSCs into pluripotent cells are two new technologies that hold great promise to create patient-specific therapies for treating myriad diseases. Our long-term goals are to elucidate the molecular mechanisms underlying SSC self-renewal and differentiation and the phenomenon of SSC reprogramming, and to translate these discoveries into cell-based therapeutics. However, the rarity of the "true" stem cells in SSC cultures and the inefficiency of SSC reprogramming are major impediments to attaining the therapeutic goals. Here we test a strategy for overcoming these impediments through manipulation of the transcription factor, OCT4, while examining fundamental questions of SSC regulation. Exquisite control of OCT4 levels is critical for maintaining pluripotency in embryonic cells with aberrations leading to differentiation. Based on our studies on OCT4 function in mouse SSCs and an emerging model for post-translational control of OCT4 in ES cells we propose the following hypothesis: SSC fate is determined in part by fine-tuning OCT4 protein levels through post-translational modification. The first aim is to determine how OCT4 levels affect SSC fate in vitro and is directly relevant to achieving SSC-based therapy. Preliminary data using SSCs engineered to overexpress OCT4 in a Doxycycline dose-dependent manner suggest that in- creased OCT4 is sufficient to increase reprogramming; also, increased OCT4 suppresses SSC differentiation in vitro and the prediction is that SSC numbers are correspondingly increased. The second aim is to examine mechanisms for regulating OCT4 protein levels in SSCs. We will identify cis- and trans-acting factors controlling OCT4 ubiquitination beginning by testing the function of the E3 ubiquitin ligase, Wwp2, in SSCs. The third aim is to determine how aberrant OCT4 levels affect SSC fate in vivo. By creating a transgenic mouse to conditionally overexpress OCT4, we will test the idea that OCT4 levels are "turned down" during spermatogenesis to permit spermatogonial differentiation, a process that remains ill-defined. Also, OCT4 overexpression is linked to oncogenesis and our mouse model will be useful to determine whether OCT4 overexpression plays a causitive role in germ cell tumorogenesis. This proposal's highly innovative aspects are: (1) improving reprogramming efficiency with stabilized OCT4; (2) a focus on SSC reprogramming rather than somatic cell reprogramming; (3) analysis of OCT4 function and regulation in SSCs rather than ES cells. Our studies will impact fundamental understanding of the relationship between SSCs, pluripotent cells and the tumorigenic cells that may result from misregulation of spermatogonial fate. Furthermore, the studies are significant because they will provide a foundation of knowledge and mechanistic insight that will be applicable to increasing the percentage of stem cells in SSC cultures and the frequency of reprogramming through regulation of OCT4 protein.