Isolation and Characterization of Intestinal Stem Cells

小肠为成体干细胞的研究提供了一个最好的系统？？ 对再生医学的承诺。遗传学研究揭示了几个关键的信号通路在其中发挥作用 在肠道干细胞(ISCs)的调节中。这为理解 ISCs的更新、自我修复和分化为多种肠道细胞类型的能力。然而， 由于无法在体内分离和移植ISCs进行高级研究，进一步的进展受到阻碍 (在活组织内)或体外(在活有机体外的受控环境中)。这项研究 该项目建议解决ISC研究的这一障碍，包括开发新颖和可靠的 ISC分离、体外培养和体内功能鉴定的方法。 ISC的分离将通过使用荧光标记蛋白和其他膜表面蛋白来完成 根据肠道干细胞中已知的表达基因对细胞进行分类和验证。体外培养将是 通过添加对支持ISC扩散到Matrigel或3- 隐窝球体的立体培养。功能表征将通过注入隔离的ISCs来完成 然后将嵌合球体移植到体内微环境中 描述ISC的生存、自我更新、增殖和世系承诺。 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，也将为 治疗肠道疾病，其中ISC驱动的组织再生是必不可少的，并用于筛选靶向药物 癌症干细胞的研究。识别、分离和鉴定干细胞的能力对治疗至关重要 进展，特别是组织置换，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.