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

Characterization of the Human AOs. Based on the recently reported protocol (14) , we established several lines of AOs from small pieces of normal lung tissue adjacent to the diseased tissue from patients undergoing surgical resection for clinical conditions. These AOs, 3D cysts lined by polarized epithelium, accommodated the four major types of airway epithelial cells, i.e., ciliated cells (ACCTUB + or FOXJ1 + ), goblet cells (MUC5AC + ), basal cells (P63 + ), and club cells (CC10 + ) (Fig. 1A) . Apical ACCTUB clearly indicated the orientation of polarization. Most organoids are orientated towards the lumen (Fig. 1A , ACCTUB Left), while a small proportion of the organoids are inverted (Fig. 1A , ACCTUB Right). Beating cilia were visible (Movie S1). No type I and type II alveolar epithelial cells were present. Thus, these organoids resembled the pseudostratified ciliated airway epithelium. The AOs were inoculated with human IAV H1N1pdm, the low-pathogenic avian virus H7N9/Ah, and the highly pathogenic avian virus H5N1. The intracellular (cell lysate) viral loads of all three virus strains increased over 2 log 10 units (Fig. 1B) . The extracellular (supernatant) viral loads, especially the viral titers, were elevated by 2-3 log 10 units.