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

PD of the AOs. To improve proximal differentiation (PD), we tried various protocols and variations thereof and settled on a medium described by Konishi et al. (18) for inducing ciliary differentiation in human PSC-derived airway epithelial spheroids. The ASCderived organoids in the original AO medium gradually enlarged, while those in PD medium became more compact ( Fig. 2A) . After 16 d of culture, the organoids in AO medium grew approximately two times larger, while the organoids in PD medium remained basically unchanged in size (Fig. 2B) . From day 7, the number of ciliated cells increased markedly in PD medium. At day 16, beating cilia were observed in a minority (<10%) of the organoids in AO medium, while abundant beating cilia were present in every PD organoid ( Fig. 2C and Movies S2 and S3). The synchronously beating cilia drove the cell debris within the organoid lumens to swirl unidirectionally (Movie S3). The dramatically increased abundance of ciliated cells in the PD organoids was verified by immunofluorescence staining (Fig. 2D) .