Department of Cell and Molecular Biology, Karolinska Institutet.
Department of Cell and Molecular Biology, Karolinska Institutet;
J Vis Exp. 2023 Jan 6(191). doi: 10.3791/64905.
Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescence-activated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively high-throughput method for identifying and understanding esophageal stem cell niche components in vitro.
上皮干细胞和祖细胞有助于在整个生命周期中形成和维持上皮屏障。大多数干细胞和祖细胞群体都藏在解剖学上不同的位置，使其能够与维持干细胞特性的小生境信号进行独特的相互作用。虽然上皮类器官培养的发展为理解干细胞和祖细胞在体内平衡和疾病中的作用提供了有力的工具，但小生境环境内的相互作用在很大程度上被忽略了，从而阻碍了识别影响干细胞行为的因素。成纤维细胞在指导上皮干细胞和祖细胞命运方面发挥着关键作用。在这里，我们提出了一个全面的类器官-成纤维细胞共培养方案，该方案能够阐明食管祖细胞更新和分化过程中成纤维细胞亚群的作用。在本方案中，描述了一种从食管中平行分离上皮细胞和成纤维细胞的方法。概述了用于从转基因报告小鼠或野生型小鼠中分离食管祖细胞以及成纤维细胞亚群的独特荧光激活细胞分选策略。该方案提供了一种通用的方法，可以适应特定成纤维细胞亚群的分离。本方案还包括食管上皮类器官单培养的建立和传代，以便与共培养系统进行直接比较。此外，还描述了一种 3D 清除方法，可用于对上皮-成纤维细胞相互作用进行详细的图像分析。总之，该方案描述了一种用于体外鉴定和理解食管干细胞小生境成分的比较和相对高通量的方法。
