Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here, we describe a strategy for directed differentiation of hPSCs into mature lung and airway epithelial cells obtained through maturation of NKX2.1⁺ hPSC-derived lung progenitors in a 3D matrix of collagen I in the absence of glycogen synthase kinase 3 inhibition. This protocol is an extension of our previously published protocol on the directed differentiation of lung and airway epithelium from hPSCs that modifies the technique and offers additional applications. This protocol is conducted in defined media conditions, has a duration of 50–80 d, does not require reporter lines and results in cultures containing mature alveolar type II and I cells as well as airway basal, ciliated, club and neuroendocrine cells. We also present a flow cytometry strategy to assess maturation in the cultures. Several of these populations, including mature NGFR⁺ basal cells, can be prospectively isolated by cell sorting and expanded for further investigation.

从人类多能干细胞 (hPSC) 体外产生的肺和气道上皮细胞在再生医学、肺部疾病建模、药物筛选和人类肺部发育研究中具有应用。在这里，我们描述了一种将 hPSC 定向分化为通过 NKX2.1 ^{+成熟获得的成熟肺和气道上皮细胞的策略}在没有糖原合酶激酶 3 抑制的情况下，hPSC 衍生的肺祖细胞在胶原蛋白 I 的 3D 矩阵中。该协议是我们之前发布的关于肺和气道上皮与 hPSC 的定向分化协议的扩展，它修改了技术并提供了额外的应用。该协议在定义的媒体条件下进行，持续时间为 50-80 天，不需要报告线，结果培养物中含有成熟的肺泡 II 型和 I 型细胞以及气道基底细胞、纤毛细胞、俱乐部细胞和神经内分泌细胞。我们还提出了一种流式细胞术策略来评估培养物的成熟度。这些群体中的一些，包括成熟的 NGFR ⁺基底细胞，可以通过细胞分选前瞻性地分离并扩展以供进一步研究。