Molecular control of spermatogonial stem cell fate for achieving cell therapy

描述（由申请方提供）：精原干细胞（SSC）的独特之处在于它们是处于多能性阈值的成体细胞，具有在体外自发重编程为多能性的能力，而无需添加外源因子。SSC培养和将SSC重编程为多能细胞是两种新技术，它们在创造治疗多种疾病的患者特异性疗法方面具有很大的前景。我们的长期目标是阐明SSC自我更新和分化以及SSC重编程现象的分子机制，并将这些发现转化为基于细胞的治疗方法。然而，SSC培养物中"真正的"干细胞的稀有性和SSC重编程的低效率是实现治疗目标的主要障碍。在这里，我们测试的策略，克服这些障碍，通过操纵的转录因子，OCT 4，同时检查SSC监管的基本问题。OCT 4水平的精确控制对于维持具有畸变的胚胎细胞的多能性至关重要 导致分化。基于我们对小鼠SSC中OCT 4功能的研究和ES细胞中OCT 4翻译后控制的新兴模型，我们提出以下假设：SSC命运部分由通过翻译后修饰微调OCT 4蛋白水平决定。第一个目的是确定OCT 4水平如何影响体外SSC命运，并与实现基于SSC的治疗直接相关。使用以多西环素剂量依赖性方式过表达OCT 4的SSC的初步数据表明，增加的OCT 4足以增加重编程;此外，增加的OCT 4在体外抑制SSC分化，并且预测SSC数量相应增加。第二个目的是检查调节SSC中OCT4蛋白水平的机制。我们将确定顺式和反式作用的因素控制OCT 4泛素化开始测试的功能E3泛素连接酶，Wwp2，在精原干细胞。第三个目的是确定异常的OCT 4水平如何影响体内SSC命运。通过建立一个转基因小鼠条件性过表达OCT 4，我们将测试的想法，OCT 4水平是"下调"在精子发生过程中，允许精原细胞分化，一个过程，仍然不明确。此外，OCT4过表达与肿瘤发生有关，我们的小鼠模型将有助于确定OCT4过表达是否在生殖细胞肿瘤发生中起致病作用。该提案的高度创新性方面是：（1）用稳定的OCT 4提高重编程效率;（2）专注于SSC重编程而不是体细胞重编程;（3）分析OCT 4在SSC而不是ES细胞中的功能和调节。我们的研究将影响对精原细胞、多能细胞和可能由精原细胞命运失调引起的致瘤细胞之间关系的基本理解。此外，这些研究是重要的，因为它们将提供知识和机制洞察力的基础，这些知识和机制洞察力将适用于增加SSC培养物中干细胞的百分比和通过OCT 4蛋白调节重编程的频率。 公共卫生相关性：设想了一种用于再生医学的策略，其中精原干细胞（SSC）可以从睾丸分离并用于患者特异性治疗，或者直接用于在化疗后补充睾丸生殖细胞，或者间接用于在移植之前将SSC重编程为其他类型的细胞以治疗各种疾病。了解支配SSC命运和重编程过程的分子机制是实现这一愿景的关键一步，也将与治疗不孕症和睾丸癌有关。

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. PUBLIC HEALTH RELEVANCE: A strategy for regenerative medicine is envisioned wherein spermatogonial stem cells (SSCs) could be isolated from the testes and used in patient-specific therapy either directly, to replenish testicular germ cells following chemotherapy, or indirectly, y reprogramming SSCs to other types of cells prior to transplantation for treatment of a variety of diseases. Understanding the molecular mechanisms governing SSC fate and the reprogramming process is a crucial step to achieving this vision and will also be relevant to treating infertility and testicular cancer.