Isolation and Characterization of Intestinal Stem Cells

描述（由申请人提供）：小肠为成人干细胞的研究提供了一个最优雅的系统，这些细胞在再生医学中具有很大的前景。遗传学研究揭示了在肠干细胞（ISCs）调控中发挥作用的几个关键信号通路。这为理解ISCs的更新、自我修复和分化为多种肠细胞类型的能力提供了重要的见解。然而，由于无法在体内（活组织内）或体外（活生物体外的受控环境内）分离和移植ISCs进行深入研究，进一步的进展受到阻碍。本研究项目旨在解决ISC研究的这一障碍，包括开发新颖可靠的ISC分离、体外培养和体内功能表征方法。

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