先天淋巴细胞 (ILC) 在粘膜屏障部位富集，在发育和疾病中发挥着关键作用。粘膜类器官提供了一个强大的平台，可以同时分化和扩增来自共享外周血前体的成熟 ILC 的所有子集。至关重要的是，类器官的身份驱动了培养物衍生的成熟先天淋巴细胞的组织特异性印记，从而可以研究肠道类器官与肠道特异性比例和 ILC 群体之间的双向相互作用。该方案减少了对饲养细胞系和用于成熟和维持 ILC 的复杂细胞因子混合物的需求，而是依赖于粘膜上皮细胞提供的蛋白质信号的天然生态位。该协议详细介绍了从人诱导多能干细胞 (hiPSC) 生成人肠道类器官 (HIO)，以及随后在 HIO 和 ILC 前体之间建立共培养物以进行扩展和成熟。这种方法在基本生物过程的机制研究中具有广泛的应用，并可作为未来细胞疗法中 ILC 的潜在 GMP 兼容来源。© 2024。Springer Science Business Media, LLC。

Innate lymphoid cells (ILC) are enriched at mucosal barrier sites where they play critical roles in development and disease. Mucosal organoids offer a robust platform for the simultaneous differentiation and expansion of all subsets of mature ILC from a shared peripheral blood precursor. Critically, organoid identity drives tissue-specific imprinting of the culture-derived mature innate lymphoid cells, allowing for the study of bidirectional interactions between, e.g., intestinal organoids and intestine-specific ratios and populations of ILC. This protocol reduces the need for feeder cell lines and complex cytokine cocktails used to mature and maintain ILC, instead relying on a native niche of protein signals provided by mucosal epithelial cells. This protocol details the generation of human intestinal organoids (HIO) from human-induced pluripotent stem cells (hiPSC), and the subsequent establishment of co-cultures between HIO and ILC precursors for expansion and maturation. This approach has extensive applications for mechanistic studies of fundamental biological processes and as a potential GMP-compatible source of ILC for future cell therapies.© 2024. Springer Science+Business Media, LLC.