Isolation and Characterization of Intestinal Stem Cells

小肠为研究成体干细胞提供了一个最优雅的系统--成体干细胞具有很强的免疫原性， 对再生医学的承诺遗传研究揭示了几种发挥作用的关键信号通路 肠干细胞（ISCs）的调节。这为理解 ISCs更新和自我修复以及分化为多种肠细胞类型的能力。然而，在这方面， 由于不能分离和移植ISCs用于进一步的研究， 在一些实施方案中，所述方法包括在体外（活组织内）或在体外（活生物体外部的受控环境内）进行。本研究 该项目旨在解决ISC研究的这一障碍，包括开发新颖可靠的 用于ISC分离、体外培养和体内功能表征的方法。 ISC分离将通过使用荧光标记蛋白和其他膜表面蛋白来完成， 通过已知在肠干细胞中表达的基因来分选和验证细胞。体外培养将是 通过将对于支持ISC增殖重要的关键因子添加到基质胶或3-氨基-3-甲基-2-苯并三唑中来实现。 地穴球的立体培养。将通过注射分离的ISCs完成功能表征 然后将嵌合球移植到体内微环境中， 表征ISC存活、自我更新、增殖和谱系定型。 如果这一目标能够实现，它将不仅为推进ISC行为的研究开辟道路，而且也为 治疗ISC驱动的组织再生至关重要的肠道疾病，并筛选靶向药物 癌症干细胞。识别、分离和表征ISCs的能力对于治疗性免疫缺陷至关重要。 研究进展，特别是组织替代，以及了解癌症的发展，预防和治疗。

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 charactize ISCs is critical for therapeutic advancements, especially tissue replacement, and for understanding cancer development, prevention and cure.