Molecular control of spermatogonial stem cell fate for achieving cell therapy

描述（由申请人提供）：精原干细胞（ssc）是独特的，因为它们是处于多能性门槛的成年细胞，具有在体外自发重编程为多能性的能力，而无需添加外源因素。SSC培养和将SSC重编程为多能细胞是两项新技术，有望创造出治疗多种疾病的患者特异性疗法。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方法。然而，在SSC培养中“真正的”干细胞的稀缺性和SSC重编程的低效率是实现治疗目标的主要障碍。在这里，我们测试了通过操纵转录因子OCT4来克服这些障碍的策略，同时研究了SSC调控的基本问题。精细控制OCT4水平对于维持胚胎细胞的多能性至关重要

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.