Transplantation of Testis Stem Cells in Large Animals

大型动物睾丸干细胞移植

基本信息

批准号：
9122506
负责人：
INA DOBRINSKI
金额：
$ 23.82万
依托单位：
UNIVERSITY OF CALGARY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9122506
关键词：
Adhesions Adult Animal Diseases Animal Model Animals Award Biological Assay Biology Biomedical Research Bioreactors Blood Vessels Cell Differentiation process Cell Line Cell Proliferation Cell Separation Cell Transplantation Cell Transplants Cells Chimeric Proteins Cilia Cloning Coculture Techniques Code Complement Development Duchenne muscular dystrophy Environment Epithelial Epithelial Cells Exposure to Family suidae Funding Gene Deletion Gene Mutation Gene Targeting Gene-Modified Generations Genes Genetic Germ Germ Cells Germ Lines Goals Hair follicle structure Health Human In Vitro Individual Investigation Knock-out Lead Mediating Mesenchymal Methods Modeling Modification Morphogenesis Mus Mutation Non-Rodent Model Phase Physiology Pluripotent Stem Cells Process Production Proliferating Property Protein Import Regenerative Medicine Reproductive Medicine Research Research Design Resources Rodent Rodent Model Role Seminal fluid Somatic Cell Spermatogenesis Stem cell transplant Stem cells Stimulation of Cell Proliferation Stromal Cells Suspension Culture System Technology Testis Time Tissues Transfection Transgenic Organisms Transplantation Vascular Endothelial Cell Viral Work Xenograft procedure base c-myc Genes cell type gain of function human disease in vivo induced pluripotent stem cell loss of function loss of function mutation male mutant nonhuman primate novel novel strategies postnatal pre-clinical reconstitution research study sertoli cell somatic cell nuclear transfer sperm cell stem cell biology stem cell niche tissue regeneration transcription activator-like effector nucleases transdifferentiation transmission process vector

项目摘要

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.

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（GSC）移植作为生成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 GSC 的生物学。通过雄性生殖系引入基因修饰将缩短产生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能够分裂并向后代贡献基因的细胞类型，使其成为基因改造的理想目标。由于猪对生物医学研究的重要性，猪被用作非啮齿动物模型。由于猪的体型和生理机能，在猪身上获得的结果预计也适用于其他大型动物物种和人类。该更新项目的目标是 1) 为猪 GSC 的分离和扩增提供有效的系统； 2）通过生殖细胞移植产生携带靶向基因缺失的猪； 3）利用分离的睾丸细胞异种移植，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向体细胞谱系转分化的诱导作用。实验将探索搅拌生物反应器悬浮培养中 GSC 的富集和扩增、与表达 VP22-c-Myc 融合蛋白的稳定饲养细胞系共培养中生殖细胞增殖的瞬时刺激，以及最近开发的转录激活剂样效应核酸酶的应用。 TALEN）技术对 GSC 进行靶向基因修饰。通过生殖细胞移植产生杜兴氏肌营养不良症（DMD）基因靶向突变的猪模型将提供原理证明，表明该项目中开发的新策略代表了产生功能丧失的有效方法大型动物模型。此外，该猪模型将为DMD的研究提供宝贵的资源。将分离的细胞移植到小鼠宿主后功能性睾丸组织的从头形态发生是在上一个资助期间开发的。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，并使我们能够检查初级纤毛在睾丸形态发生和干细胞生态位建立过程中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测定来探索 GSC 在暴露于诱导微环境后直接分化为上皮或血管内皮细胞。总体而言，本提案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能够在大型动物模型中有效地删除生殖系基因，并将为生殖和再生医学中的 GSC 建立一个临床前平台。