Transplantation of Testis Stem Cells in Large Animals

项目概要 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； it is the only 成年男性体内能够分裂并向后代贡献基因的细胞类型，使其成为理想的目标 genetic modification.猪是临床前研究的重要模型，因为它们在系统发育上和 在生理上比啮齿动物更类似于人类。通过 GSC 而不是胚胎进行基因编辑将 缩短产生种系基因编辑动物模型所需的时间。其适用范围也更加广泛 基因剂量和表观遗传学发挥作用并且需要较小品系的猪的疾病模型。的 该更新项目的目标是 1) 建立促进猪生殖细胞扩增的培养系统 in vitro; 2）探索睾丸体细胞在功能性GSC生态位形成中的作用； and 3) to 开发一种在猪 GSC 中进行靶向基因编辑的精确方法。虽然生殖细胞系传播 通过原代 pGSC 移植可以进行基因改造，该方法的效率将受益 来自强大的体外系统，用于在移植前扩增编辑的细胞。为了实现这一目标，我们 将定义 pGSC 的代谢表型，为适当培养条件的设计提供信息。 Grafting to 小鼠宿主和移植到受体猪中将监测培养细胞支持的能力 精子发生作为功能终点。生殖细胞功能由与生态位的相互作用控制 微环境。我们的新型睾丸类器官系统和睾丸细胞异种移植将能够引入 细胞类型特异性修饰并表征干细胞生态位相互作用。我们将检验以下假设： 睾丸体细胞上的初级纤毛对于生态位的形成至关重要，并且体细胞环境 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 cells (piPSCs).最后，我们将检验 CRISPR/Cas9 核糖核蛋白 (RNP) 介导的假设 同源定向修复（HDR）将允许在 GSC 中进行高效的靶向基因组编辑，以精确复制 human disease alleles.由于 HDR 仅限于小的 DNA 改变（~1-50 bp），我们还将探索 Precise 整合到目标染色体 (PITCh) 中进行靶向基因编辑，以引入大型转基因 和/或将猪基因组的部分人性化用于再生医学。引入突变型猪胰岛素 与永久性新生儿糖尿病中存在的人类 INSC96Y 突变相匹配的基因 (INSC94Y) 将 提供原理证明，证明我们的新策略是可行且有效的。总体而言，这项工作将建立 对猪进行精确靶向种系基因编辑，并进一步定义干细胞与生态位的相互作用。

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.