Differentiated human airway organoids to assess infectivity of emerging influenza virus

Zhou, Jie; Li, Cun; Sachs, Norman; Chiu, Man Chun; Wong, Bosco Ho-Yin; Chu, Hin; Poon, Vincent Kwok-Man; Wang, Dong; Zhao, Xiaoyu; Wen, Lei; Song, Wenjun; Yuan, Shuofeng; Wong, Kenneth Kak-Yuen; Chan, Jasper Fuk-Woo; To, Kelvin Kai-Wang; Chen, Honglin; Clevers, Hans; Yuen, Kwok-Yung

Consistently, the transcriptional levels of the ciliated cell markers FOXJ1 and SNTN were strongly up-regulated in the PD organoids compared with the organoids in AO medium. The expression levels of the basal cell markers P63 and CK5 and the goblet cell marker MUC5AC also increased, whereas the levels of club cell markers CC10 and SCGB3A2 were substantially down-regulated in the PD organoids (Fig. 3A) . Importantly, we observed globally elevated expression of serine proteases including TMPRSS2, TMPRSS4, TMPRSS11D (HAT), and matriptase, which are essential for the activation and propagation of human IAVs and low-pathogenic avian IAVs (8) . We also performed flow cytometry analysis to measure the percentages of the four cell types in the organoids cultured in two distinct media at day 16. It was shown that the percentage of ciliated cells increased remarkably, by about threefold, after PD, to over 40% in the PD organoids, while the ciliated cells invariably constituted a minority of the cells in the organoids in AO medium (Fig. 3 B and C). Goblet cells also increased marginally, while club cells consistently decreased after PD (Fig. 3 B and C) . Collectively, we successfully induced mucociliary differentiation and developed PD AOs which morphologically and functionally simulate human airway epithelium.