Transplantation of Testis Stem Cells in Large Animals

大型动物睾丸干细胞移植

• 批准号: 9885645
• 负责人: INA DOBRINSKI
• 金额: $ 23.91万
• 依托单位: UNIVERSITY OF CALGARY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9885645
• 关键词: Ablation; Adult; Alleles; Animal Model; Animals; Autophagocytosis; Award; Biological Markers; CRISPR/Cas technology; Cell Communication; Cell Culture System; Cell Differentiation process; Cell physiology; Cells; Chromosomes; Cilia; Complex; Cultured Cells; DNA Sequence Alteration; Data; Development; Disease model; Embryo; Environment; Epigenetic Process; Equilibrium; Family suidae; Fertilization in Vitro; Future Generations; Gene Dosage; Gene Targeting; Genes; Genetic; Germ; Germ Cells; Goals; Homologous Transplantation; Human; Immunodeficient Mouse; In Vitro; Insulin; Lead; Mediating; Metabolic; Metabolic Pathway; Modification; Monitor; Morphogenesis; Mus; Mutation; Nutrient; Organoids; Oxygen Consumption; Patients; Permanent neonatal diabetes mellitus; Phylogenetic Analysis; Physiological; Physiology; Play; Pre-Clinical Model; Protocols documentation; Recombinant adeno-associated virus (rAAV); Regenerative Medicine; Ribonucleoproteins; Rodent; Rodent Model; Role; Signal Pathway; Somatic Cell; Spermatogenesis; System; Testing; Testis; Time; Tissues; Toxic effect; Transgenes; Translating; Transplantation; Work; Xenograft procedure; base; cell type; design; ds-DNA; gene function; genome editing; germline stem cells; glucose metabolism; human disease; human model; improved; in vivo; induced pluripotent stem cell; male; metabolic phenotype; mutant; nonhuman primate; novel; novel strategies; nuclease; oxidative damage; pig genome; postnatal; pre-clinical research; reconstitution; repaired; screening; self-renewal; sertoli cell; sperm cell; stem cell biology; stem cell differentiation; stem cell niche; stem cell proliferation; stem cells; therapy design; tool; transmission process

Project Summary The long-term goals of this project are to establish germline stem cell (GSC)-based gene editing to generate large animal models of human diseases and for regenerative medicine (PAR-16-093), and to provide accessible systems to study the GSC niche in non-rodent animals. The testis stem cell is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Pigs are important models for pre-clinical research because they are phylogenetically and physiologically more similar to humans than rodents. Gene editing through GSCs rather than embryos will shorten the time necessary to produce germline gene edited animal models. It is also more broadly applicable to disease models where gene dosage and epigenetics have a role and smaller strains of pigs are required. The aims of this renewal project are 1) to establish a culture system that promotes porcine germ cell expansion in vitro; 2) to explore the role of testicular somatic cells in formation of a functional GSC niche; and 3) to develop a precise approach for targeted gene editing in porcine GSCs. While germline transmission of a genetic modification is possible with transplantation of primary pGSCs, efficiency of the approach would benefit from a robust in vitro system for expansion of edited cells prior to transplantation. To accomplish this goal, we will define the metabolic phenotype of pGSCs to inform design of appropriate culture conditions. Grafting to mouse hosts and transplantation to recipient pigs will monitor the ability of cultured cells to support spermatogenesis as functional endpoint. Germ cell function is controlled by interaction with the niche microenvironment. Our novel testicular organoid system and xenografting of testis cells will enable introduction of cell type-specific modifications and characterize stem cell-niche interactions. We will test the hypothesis that primary cilia on testicular somatic cells are essential for niche formation, and that the somatic environment modulates germ cell differentiation and supports germ lineage differentiation of porcine induced pluripotent stem cells (piPSCs). Finally, we will test the hypothesis that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated homology directed repair (HDR) will allow efficient targeted genome editing in GSCs for precise replication of human disease alleles. As HDR is limited to small DNA alterations (~1-50 bp), we will also explore Precise Integration into Target Chromosome (PITCh) for targeted gene editing to enable introduction of large transgenes and/or humanizing parts of the pig genome for regenerative medicine. Introduction of a mutant porcine insulin gene (INSC94Y) that matches the human INSC96Y mutation present in permanent neonatal diabetes mellitus will provide proof-of-principle that our novel strategies are feasible and efficient. Overall, this work will establish precise targeted germline gene editing in pigs, and further define stem cell-niche interactions.

项目摘要 该项目的长期目标是建立基于生殖干细胞（GSC）的基因编辑， 生成人类疾病和再生医学的大型动物模型（PAR-16-093），并提供 研究非啮齿类动物GSC生态位的可访问系统。睾丸干细胞是独一无二的;它是唯一的 成年男性中的一种细胞类型，可以分裂并为后代贡献基因，使其成为理想的靶点， 基因改造猪是临床前研究的重要模型，因为它们在遗传学上是相似的， 在生理上与人类比啮齿动物更相似。通过GSC而不是胚胎进行基因编辑 缩短生产种系基因编辑动物模型所需的时间。它的适用范围也更广 到疾病模型，其中基因剂量和表观遗传学起作用，需要较小的猪品系。的 本更新项目的目标是：1）建立促进猪生殖细胞扩增的培养体系 2）探索睾丸体细胞在功能性GSC生态位形成中的作用; 3） 在猪GSC中开发靶向基因编辑的精确方法。虽然生殖系传播的一个 基因修饰可能与原代pGSC的移植有关，该方法的效率将有利于 来自用于在移植之前扩增编辑的细胞的稳健的体外系统。为了实现这一目标，我们 将定义pGSC的代谢表型，以告知适当培养条件的设计。接枝至 小鼠宿主和移植到受体猪将监测培养的细胞支持 精子发生作为功能终点。生殖细胞的功能是通过与生态位的相互作用来控制的 微环境我们新的睾丸类器官系统和睾丸细胞异种移植将使引入 细胞类型特异性修饰和表征干细胞生态位相互作用。我们将检验这个假设， 睾丸体细胞上的初级纤毛对生态位的形成至关重要， 调节生殖细胞分化并支持猪诱导多能干细胞的生殖谱系分化 细胞（piPSC）。最后，我们将测试CRISPR/Cas9核糖核蛋白（RNP）介导的细胞凋亡的假设。 同源定向修复（HDR）将允许在GSC中进行有效的靶向基因组编辑，以精确复制 人类疾病等位基因。由于HDR仅限于小的DNA改变（~1-50 bp），我们还将探索精确 整合到靶染色体（PITCh）中，用于靶向基因编辑，以实现大转基因的引入 和/或将猪基因组的部分人源化用于再生医学。一种突变型猪胰岛素的引入 与永久性新生儿糖尿病中存在的人INSC 96 Y突变相匹配的基因（INSC 94 Y）将 提供了我们的新策略是可行和有效的原理证明。总的来说，这项工作将建立 在猪中进行精确的靶向生殖系基因编辑，并进一步定义干细胞-生态位相互作用。