Immune Compromised Zebrafish for Cell Transplantation

将细胞移植到免疫受损的小鼠中改变了我们对人类疾病的理解， 已广泛用于评估异种移植物的再生、干细胞自我更新和癌症 移植环境尽管它们具有很大的实用性，但小鼠模型不适合大规模研究， 导致高饲养成本，并且不容易促进以单细胞分辨率直接观察移植细胞。 相比之下，斑马鱼价格低廉，可以大量饲养，并且适合大规模养殖。 化学遗传方法，其中化合物可以直接添加到水中。此外，光学清晰 免疫缺陷斑马鱼品系已经允许大规模细胞移植研究动态成像 荧光标记的细胞在单细胞分辨率。尽管取得了这些成功， 免疫受损斑马鱼作为一个强大的和长期的异种移植细胞移植模型。长期 本申请的目的是开发一种通用的斑马鱼移植模型，用于移植广泛的 从斑马鱼、小鼠和人类获得的再生和癌细胞类型。本申请的总体目标 为优化再生组织的同种异体移植物植入提供新的免疫缺陷斑马鱼模型 以及人癌症、ES、iPS和CD 34+脐带血细胞的异种移植物植入。我们的理由是， 研究表明，斑马鱼的血液发育是高度保守的， 模型已经导致再生干细胞过程的独特理解和动态可视化 新的细胞行为来驱动细胞生长。目的1是开发复合突变体和人源化转基因 斑马鱼进行优化细胞移植。我们将开发缺乏所有T、B和NK细胞的新模型， 包括最近鉴定的表达NK-溶素的细胞毒性血细胞中的突变体和功能完全丧失 rag2基因突变，这是成熟T和B细胞功能所必需的。我们还将产生 人源化斑马鱼，其转基因表达支持人细胞生长提高的因子，包括 人"别吃我"信号抑制调节蛋白α（SIRPa）和人细胞因子。目标2将 利用这些模型来评估原位和异种移植物植入，鉴定具有上级， 人癌细胞系、ES和iPS细胞以及CD34+脐带血细胞的长期植入。目标3将 完善一个系统，用于全球分布和快速传播突变系到斑马鱼，干细胞， 再生医学社区我们的工作是重要的，因为它将开发急需的资源， 社区，促进下一代低成本，高通量细胞移植模型， 移植大量再生细胞类型。这项工作预计将产生积极的翻译影响， 开发临床前动物模型，以降低的成本促进移植细胞的直接可视化， 允许化学遗传方法来揭示与再生和干细胞功能相关的途径。 免疫受损斑马鱼的这种广泛应用跨越了许多NIH研究所的使命。

Cell transplantation into immune compromised mice has transformed our understanding of human disease and has been used extensively to assess regeneration, stem cell self-renewal, and cancer in the xenograft transplantation setting. Despite their great utility, mouse models are not amenable to large-scale studies due to high husbandry costs and do not easily facilitate direct visualization of engrafted cells at single cell resolution. By contrast, zebrafish are inexpensive, can be reared in large numbers, and are amenable to large-scale chemical genetic approaches where compounds can be added directly to the water. Moreover, optically-clear immune-deficient zebrafish strains have permitted large-scale cell transplantation studies to dynamically image fluorescent-labeled cells at single cell resolution. Despite these successes, more needs to be done to develop immune compromised zebrafish as a robust and long-term xenograft cell transplantation model. The long-term goal of this application is to develop a universal zebrafish transplantation model for engrafting a wide array of regenerative and cancer cell types from zebrafish, mouse, and human. The overall objective of this application is to provide new immune deficient zebrafish models for optimized allograft engraftment of regenerative tissues and xenograft engraftment of human cancer, ES, iPS, and CD34+ cord blood cells. The rationale for our research is that zebrafish blood development is highly conserved and that developing zebrafish transplantation models has already led to unique understanding of regenerative stem cell processes and dynamic visualization of new cell behaviors that drive cell growth. Aim 1 will develop compound mutant and humanized transgenic zebrafish for optimized cell transplantation. We will develop new models that lack all T, B, and NK cells, including mutants in the recently identified NK-lysin expressing cytotoxic blood cells and full loss-of-function mutations in the rag2 gene, which is required for mature T and B cell function. We will also generate humanized zebrafish that transgenically express factors that support elevated growth of human cells, including the human “don’t eat me” signal inhibitory regulatory protein alpha (SIRPa) and human cytokines. Aim 2 will utilize these models for assessing orthotopic and xenograft engraftment, identifying lines that have superior, long-term engraftment of human cancer cell lines, ES and iPS cells, and CD34+ cord blood cells. Aim 3 will refine a system for global distribution and rapid dissemination of mutant lines to the zebrafish, stem cell, and regenerative medicine community. Our work is significant because it will develop a much-needed resource for the community, facilitating the next generation of low-cost, high throughput cell transplantation models to engraft a wide array of regenerative cell types. This work is expected to have a positive translational impact by developing pre-clinical animal models that facilitate direct visualization of engrafted cells at reduced cost and allow chemical genetic approaches to uncover pathways associated with regeneration and stem cell function. Such broad reaching applications for immune compromised zebrafish spans the mission of many NIH institutes.