Fasciano Alyssa C, Blutt Sarah E, Estes Mary K, Mecsas Joan
Program in Immunology, Sackler School of Graduate Biomedical Sciences;
Department of Molecular Virology and Microbiology, Baylor College of Medicine.
J Vis Exp. 2019 Jul 26(149). doi: 10.3791/59894.
M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer's patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-α to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.
肠道的M（微褶）细胞的功能是将抗原从顶端管腔转运至下方有免疫细胞驻留的派尔集合淋巴结和固有层，因此有助于肠道的黏膜免疫。目前尚缺乏对M细胞在肠道中如何分化以及M细胞摄取抗原的分子机制的全面了解。这是因为M细胞在肠道中是稀有细胞群体，且M细胞的体外模型不够完善。一种名为肠类器官的肠道自我更新干细胞培养系统的发现，为培养M细胞提供了新的可能性。肠类器官比标准培养细胞系更具优势，因为它们可以分化为肠道中发现的几种主要细胞类型，包括杯状细胞、潘氏细胞、肠内分泌细胞和肠上皮细胞。细胞因子RANKL在M细胞发育中至关重要，向培养基中添加RANKL和TNF-α可促进回肠肠类器官中的一部分细胞分化为M细胞。以下方案描述了一种使用人回肠肠类器官在肠道的Transwell上皮极化单层系统中分化M细胞的方法。该方法可应用于M细胞发育和功能的研究。
