Molecular control of spermatogonial stem cell fate for achieving cell therapy

精原干细胞命运的分子控制以实现细胞治疗

• 批准号: 8526486
• 负责人: Christina Tenenhaus Dann
• 金额: $ 28.38万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-08 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8526486
• 关键词: Adult; Affect; Autologous Transplantation; Biological Assay; Cell Count; Cell Culture Techniques; Cell Therapy; Cells; Data; Development; Disease; Dose; Down-Regulation; Doxycycline; Embryo; Engineering; Equilibrium; Exogenous Factors; Foundations; Frequencies; Generations; Germ Cells; Goals; Human; In Vitro; Infertility; Knowledge; Lead; Link; Literature; Lysine; Malignant neoplasm of testis; Mediating; Modeling; Molecular; Mus; Mutation; POU Domain; Patients; Physiological; Play; Pluripotent Stem Cells; Post-Translational Protein Processing; Post-Translational Regulation; Process; Production; Proteins; Publishing; Regenerative Medicine; Regulation; Rodent; Role; Somatic Cell; Spermatogenesis; Spermatogonia; Stem cells; Testing; Testis; Therapeutic; Trans-Activators; Transgenic Mice; Transitional Cell; Translating; Transplantation; Ubiquitination; Vision; base; blastomere structure; cell type; cellular engineering; chemotherapy; embryonic stem cell; gene therapy; improved; in vivo; innovation; insight; mouse model; new technology; overexpression; pluripotency; prevent; progenitor; self-renewal; stem; stem cell differentiation; stem cell fate; transcription factor; tumorigenesis; tumorigenic; ubiquitin-protein ligase

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.

描述（由申请人提供）：精子干细胞（SSC）是独一无二的，因为它们是成年细胞在多元效力的阈值上有依赖的成年细胞，具有自发重新编程为体外多能性的能力而无需添加外源性因素。 SSC培养和将SSC重编程为多能细胞是两种新技术，它们具有巨大的希望，可以创建用于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疗法。但是，SSC培养物中“真实”干细胞的罕见性以及SSC重编程的效率低下是实现治疗目标的主要障碍。在这里，我们通过操纵转录因子OCT4来测试一种克服这些障碍的策略，同时检查了SSC调节的基本问题。对OCT4水平的精美控制对于维持胚胎细胞中的多能性至关重要 导致差异化。基于我们对小鼠SSC中OCT4功能的研究，以及用于ES细胞中OCT4翻译后控制的新兴模型，我们提出了以下假设：SSC命运是通过翻译后修饰的微调OCT4蛋白水平来确定的。第一个目的是确定OCT4水平在体外如何影响SSC命运，并与实现基于SSC的治疗直接相关。使用强力霉素剂量依赖性的SSC进行了使用SSC进行过表达OCT4的初步数据，这表明OCT4的含量足以增加重编程；同样，增加的OCT4在体外抑制SSC分化，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。我们将通过测试SSC中E3泛素连接酶WWP2的功能开始，确定控制OCT4泛素化的顺式和反式作用因子。第三个目的是确定异常的OCT4水平如何影响体内的SSC命运。通过创建一种转基因小鼠以有条件地表达OCT4，我们将测试OCT4水平在精子发生过程中“拒绝”以允许精子分化的想法，这一过程仍然不确定。同样，OCT4的过表达与肿瘤发生有关，我们的小鼠模型对于确定OCT4过表达是否在生殖细胞肿瘤发生中起因果作用很有用。该提议的高度创新性方面是：（1）通过稳定的OCT4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SSC中的Oct4功能和调节，而不是ES细胞。我们的研究将影响对SSC，多能细胞和肿瘤细胞之间关系的基本理解，这可能是由于精子命运的不利影响。此外，这些研究之所以重要，是因为它们将提供知识和机械洞察力的基础，这些基础适用于增加SSC培养物中干细胞百分比以及通过调节OCT4蛋白来重编程的频率。