A Novel Large Animal Model for Studying the Developmental Potential and Function of LGR5 Stem Cells in Vivo and in Vitro

项目摘要 Lgr5是成体和胎儿干细胞在不同组织和器官中的标志，已被广泛研究 在过去几十年里。表达LGR5的细胞在器官/组织发育、动态平衡、再生、 和疾病，包括癌症。因此，拥有在体外识别、跟踪和操纵这些细胞的能力 而在体内，将使它们在正常和病理器官功能中发挥详细的作用。到目前为止，大多数研究 在LGR5细胞上进行了实验，小鼠具有显著的解剖学、生理学和分子生物学特征 与人类的不同。利用新育成的LGR5-H_2B-GFP猪品系，我们展示了其广泛的 用于研究这些独特细胞的作用，现在已经在各种 组织/器官。然而，我们也无法解决一些关键方面的问题，因为 现有模型分析LGR5细胞在发育过程中的分化后代(谱系跟踪)，在 移植，或在损伤和修复过程中。此外，虽然我们已经能够停用这两个 LGR5基因的等位基因，我们没有能力删除细胞本身。能够删除LGR5 表达细胞将增加我们对这种独特细胞(相对于LGR5基因本身)的重要性的理解 在感兴趣的表型中。因此，要克服这两个不足，进一步增加价值和 在原有LGR5-H_2B-GFP动物模型的影响下，我们建议建立一个高度改良的LGR5猪系 这将允许标记LGR5细胞，在受伤后跟踪其后代，并在需要时将其删除。高潮 到目前为止，人和猪在LGR5表达和功能方面的结果一致，不仅是 增加了这条线路对基础研究的价值，也增加了将临床结果快速转化为人类的价值， 进一步增加了这种独特的动物模型的影响。成功完成拟议的目标将 为研究肠道、肺、皮肤的LGR5干细胞的翻译方面建立强大的动物模型， 肝脏、耳蜗骨、肾脏等。

Project Summary LGR5, a marker of adult and fetal stem cells in various tissues and organs, has been studied extensively for the past decades. Cells expressing LGR5 play key roles in organ/tissue development, homeostasis, regeneration, and disease, including cancer. Therefore, having the ability to identify, track, and manipulate these cells in vitro and in vivo, will allow their detailed role in normal and pathological organ function. To date, most of the research on LGR5 cells has been performed using mice, which have significant anatomical, physiological, and molecular differences from the human. Utilizing a newly developed LGR5-H2B-GFP pig line we have demonstrated its wide utility for studying the role of these unique cells and have now made seminal observations in a variety of tissues/organs. However, we have also been unable to address some key aspects due to the inability of the existing model to analyze the differentiated progeny (lineage tracking) of LGR5 cells during development, after transplantation, or during the process of injury and repair. In addition, while we have been able to inactivate both alleles of the LGR5 gene, we do not have the ability to delete the cell itself. Being able to delete the LGR5 expressing cell will increase our understanding of the importance of this unique cell (versus the LGR5 gene itself) in the phenotypes of interest. Thus, to overcome these two deficiencies, and further increase the value and impact of the original LGR5-H2B-GFP animal model, we propose to develop a highly improved LGR5 pig line that will allow labeling of the LGR5 cell, tracking of its progeny after injury, and delete it when desired. The high concordance between human and pig results to date with respect to LGR5 expression and function, not only increases the value of this line for basic research, but also for rapidly translating clinical findings to humans, further increasing the impact of this unique animal model. Successful completion of the proposed aims will generate a powerful animal model for study of translational aspects of LGR5 stem cells of the gut, lung, skin, liver, cochlea, and kidney among others.