Isolation and Characterization of Intestinal Stem Cells

描述（由申请人提供）：小肠为成体干细胞的研究提供了一个最优雅的系统，成体干细胞对再生医学有很大的希望。遗传学研究揭示了几个关键的信号通路，在肠道干细胞（ISCs）的调节中发挥作用。这为理解ISCs的更新和自我修复以及分化为多种肠细胞类型的能力提供了重要的见解。然而，由于不能分离和移植ISCs用于体内（活组织内）或体外（活生物体外的受控环境内）的高级研究，进一步的进展受到阻碍。该研究项目旨在解决ISC研究的这一障碍，包括开发用于ISC分离、体外培养和体内功能表征的新颖可靠的方法。 ISC分离将通过使用荧光标记蛋白和其他膜表面蛋白来分选和验证已知在肠干细胞中表达的基因的细胞来完成。体外培养将通过将对支持ISC增殖重要的关键因子添加到基质胶或隐窝球的三维培养物中来完成。将通过将分离的ISC注射到辐照的隐窝球中，然后将嵌合球移植到体内微环境中以表征ISC存活、自我更新、增殖和谱系定型来完成功能表征。 如果这一目标能够实现，它不仅将为推进ISC行为的研究开辟道路，而且还将为治疗ISC驱动的组织再生至关重要的肠道疾病以及筛选靶向癌症干细胞的药物开辟道路。识别、分离和表征ISCs的能力对于治疗进展，特别是组织替代，以及理解癌症的发展、预防和治愈至关重要。 公共卫生相关性：美国消化系统疾病的直接费用总计超过850亿美元，间接费用超过230亿美元。有证据表明，肠干细胞（ISCs）参与了许多病例-从克罗恩病到结肠炎再到癌症。这项研究将增加ISC生物学的知识，可用于提高治疗效果，改善患者预后，并最终影响消化系统疾病的公共卫生负担

DESCRIPTION (provided by applicant): The small intestine provides a most elegant system for the study of adult stem cells -- cells which hold great promise for regenerative medicine. Genetic studies have revealed several key signaling pathways that play a role in the regulation of intestinal stem cells (ISCs). This has provided important insight into understanding the capacity of ISCs for renewal and self-repair and differentiation into multiple intestinal cell types. However, further progress is impeded by the inability to isolate and transplant ISCs for advanced study either in vivo (within living tissue) or in vitro (within a controlled environment outside of a living organism). This research project proposes to address this barrier to ISC research, including the development of novel and reliable methods for ISC isolation, in vitro culture, and in vivo functional characterization. ISC isolation will be accomplished by using fluorescent label protein and other membrane surface proteins to sort and verify cells by genes known to be expressed in intestinal stem cells. In vitro culture will be accomplished by adding key factors that are important for supporting ISC proliferation into Matrigel or 3-dimensional culture of crypt sphere. Functional characterization will be accomplished by injecting isolated ISCs into the irradiated crypt sphere and then transplanting the chimeric sphere to an in vivo microenvironment to characterize ISC survival, self-renewal, proliferation, and lineage commitment. If this goal can be achieved, it will open avenues not only for advancing the study of ISC behavior, but also for treating intestinal disorders in which ISC-driven tissue regeneration is essential and for screening drugs to target on cancer stem cells. The ability to identify and isolate and characterize ISCs is critical for therapeutic advancements, especially tissue replacement, and for understanding cancer development, prevention and cure. PUBLIC HEALTH RELEVANCE: The cost of digestive diseases in the U.S. collectively exceeds $85 billion in direct and $23 billion in indirect expenses. Evidence indicates intestinal stem cells (ISCs) are involved in many cases -- from Crohn's to colitis to cancer. This research will increase knowledge of ISC biology, which can be applied to enhance treatment efficacies, improve patient outcomes, and ultimately, impact the public health burden of digestive diseases