Transplantation of Testis Stem Cells in Large Animals

描述（由申请人提供）：本研究的长期目标是建立生殖系干细胞（GSCs）移植作为产生人类和动物疾病的转基因大型动物模型的方法，并提供可访问的系统来研究非啮齿动物中GSCs的生物学。通过雄性生殖系引入基因改造将缩短生产动物模型所需的时间，并且可能比目前的体细胞核移植方法更有效。睾丸干细胞的独特之处在于，它是成年个体中唯一一种分裂并向后代提供基因的细胞类型，使其成为基因改造的理想目标。猪被用作非啮齿动物模型，因为它对生物医学研究很重要。由于猪的体型和生理，在猪身上获得的结果有望适用于其他大型动物物种和人类。本更新项目的目的是：1)为猪GSCs的分离和扩增提供一个有效的系统；2)通过生殖细胞移植产生携带目标基因缺失的猪；3)利用离体睾丸细胞异种移植研究睾丸微环境对多能干细胞形态发生和生殖细胞分化的影响；4)研究微环境对猪GSCs向体细胞系转分化的诱导作用。实验将探索GSCs在搅拌生物反应器悬浮培养中的富集和扩增，与表达VP22-c-Myc融合蛋白的稳定饲养系共培养中对生殖细胞增殖的瞬时刺激，以及最近开发的转录激活因子样效应核酸酶（TALEN）技术在GSCs中的应用，以进行靶向基因修饰。通过生殖细胞移植产生携带杜氏肌营养不良症（DMD）基因靶向突变的猪模型将提供原理证明，该项目开发的新策略代表了产生功能丧失大型动物模型的有效方法。此外，该猪模型将为DMD的研究提供宝贵的资源。在之前的资助期内，分离细胞移植到小鼠宿主后，功能睾丸组织的新生形态发生得到了发展。它允许在体内培养系统中从分离的细胞中重建精子发生，这一过程目前在体外是不可能的。该系统将作为猪诱导多能干细胞生殖谱系分化的生物测定，并将使我们能够在睾丸形态发生和干细胞生态位建立过程中检查初级纤毛对睾丸体细胞的功能作用。最后，该项目将采用独特的体外和体内试验，探索暴露于诱导微环境后，GSCs直接分化为上皮细胞或血管内皮细胞。总之，本研究中采用的同源生殖细胞移植和睾丸细胞移植的互补策略将在大型动物模型中实现有效的生殖系基因缺失，并将为生殖和再生医学中使用GSCs的工作建立临床前平台。

DESCRIPTION (provided by applicant): The long-term goal of this research is to establish transplantation of germ line stem cells (GSCs) as an approach to generate genetically modified large animal models of human and animal diseases, and to provide accessible systems to study the biology of GSCs in non-rodent animals. Introduction of genetic modification through the male germ line will shorten the time necessary to produce animal models and is potentially more efficient than the current method of somatic cell nuclear transfer. The testis stem cell is unique in that it is the only cell type in an adult individual that divides and contributes genes to subsequent generations, making it an ideal target for genetic modification. The pig is used as a non-rodent model because of its importance for biomedical research. Due to their size and physiology, results obtained in pigs are expected to be applicable to other large animal species and humans. The aims of this renewal project are 1) to provide an efficient system for isolation and expansion of porcine GSCs; 2) to generate pigs carrying a targeted gene deletion through germ cell transplantation; 3) to study the effects of the testicular microenvironment on morphogenesis and germ cell differentiation from pluripotent stem cells using xenografting of isolated testis cells; and 4) to investigate the inductive effect of the microenvironment on transdifferentiation of porcine GSCs to somatic lineages. Experiments will explore enrichment and expansion of GSCs in stirred bioreactor suspension culture, transient stimulation of germ cell proliferation in co-culture with stable feeder lines expressing a VP22-c-Myc fusion protein, and application of recently developed transcription activator-like effector nuclease (TALEN) technology to GSCs for targeted gene modification. Generation of a pig model carrying a targeted mutation in the Duchenne's muscular dystrophy (DMD) gene through germ cell transplantation will provide proof-of- principle that the novel strategy developed in this project represents an efficient approach for the generation of loss-of-function large animal models. In addition, this pig model will provide a valuable resource for the study of DMD. De novo morphogenesis of functional testis tissue after grafting of isolated cells to mouse hosts was developed in the previous funding period. It allows reconstitution of spermatogenesis from isolated cells in an in vivo culture system, a process that is currently not possible in vitro. Thi system will serve as a bioassay for germ lineage differentiation from porcine induced pluripotent stem cells, and will enable us to examine the functional role of primary cilia on testicular somati cells during testicular morphogenesis and establishment of the stem cell niche. Finally, the project will employ unique in vitro and in vivo assays to explore direct differentiation of GSCs into epithelial or vascular endothelial cells following exposure to an inductive microenvironment. Overall, the complementary strategies of homologous germ cell transplantation and testis cell grafting to be employed in this proposal will enable efficient germ line gene deletion in a large animal model and will establish a pre-clinical platform for work with GSCs in reproductive and regenerative medicine.