Transplantation of Testis Stem Cells in Large Animals

大型动物睾丸干细胞移植

• 批准号: 10358575
• 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/10358575
• 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 function; 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系干细胞(GSC)的基因编辑，以 建立人类疾病和再生医学的大型动物模型(PAR-16-093)，并提供 可利用的系统来研究非啮齿动物的GSC生态位。睾丸干细胞是独一无二的；它是 成年男性的一种细胞类型，它将基因分裂并贡献给后代，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，因为它们的系统发育和 在生理上比啮齿动物更接近人类。通过GSCs而不是胚胎进行基因编辑将会 缩短生产生殖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。这个 本次更新项目的目标是：1)建立促进猪生殖细胞增殖的培养体系 在体外；2)探讨睾丸体细胞在形成有功能的GSC生态位中的作用；3) 开发一种在猪GSC中进行靶向基因编辑的精确方法。而生殖系的传播 通过移植原代pGSCs进行基因改造是可能的，该方法的效率将受益 来自一个强大的体外系统，用于移植前编辑细胞的扩增。为了实现这一目标，我们 将定义pGSCs的代谢表型，以便为设计合适的培养条件提供信息。嫁接到 小鼠宿主和移植到受体猪将监测培养细胞的支持能力 精子发生作为功能终点。生殖细胞的功能受与生态位相互作用的控制 微环境。我们新颖的睾丸器官系统和睾丸细胞的异种移植将使 对特定细胞类型的修饰，并描述干细胞-利基相互作用的特征。我们将检验这一假设 睾丸体细胞上的初生纤毛对于生态位的形成是必不可少的，体细胞环境 猪诱导多能干细胞对生殖细胞分化的调控及对生殖系分化的支持 细胞(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 人类疾病等位基因。由于HDR仅限于微小的DNA改变(~1-50个碱基)，我们还将探索精确的 整合到目标染色体(Pitch)以进行靶向基因编辑，从而能够引入大的转基因 和/或使猪基因组的某些部分人性化以用于再生医学。一种突变猪胰岛素的引进 与永久性新生儿糖尿病中存在的人类INSC96Y突变匹配的基因(INSC94Y)将 提供原则证明，证明我们的新策略是可行和高效的。总体而言，这项工作将确立 精确定位猪的生殖系基因编辑，并进一步定义干细胞与生态位的相互作用。