br Results br Discussion We established

Results
Discussion We established a method of 3D differentiation without feeder 2b3a inhibitors to generate hPSC-derived PAEC spheroids via isolated progenitor cells using CPM as a surface antigen, which is reportedly a biomarker of lung diseases, such as acute pneumonia and lung cancer (Dragović et al., 1995). It is noted that the inhibition of the Notch pathway induced not only MCACs but also PNECs from hPSCs, which is consistent with the studies of genetic murine models (Tsao et al., 2009; Morimoto et al., 2012). PNECs have been proposed to be the origin of small-cell lung cancer (Song et al., 2012), thus suggesting its future application in cancer studies. The ciliary function analyses of hPSC-derived MCACs, as well as induction efficiency, are important aspects of the present study. Previously, the functional analyses of hPSC-derived PAECs mostly focused on CFTR (Wong et al., 2012; Firth et al., 2014), and not on ciliary movement. In addition, the ciliary function was not shown in hPSC-derived lung organoids due to immaturity (Dye et al., 2015). In the ciliary function tests, the CBF of hPSC-derived MCACs in spheroids appeared to be lower than that in the 3D-ALI protocol (Figure 4E) for at least two reasons. First, mucoid secretion was trapped in the closed lumen and its increased viscosity might reduce the CBF in the spheroids (Figure 2C and Movie S1, left). Second, we had to mince the spheroids and place cover slips on the samples during image acquisition, which may have reduced the CBF in the hPSC-derived spheroids, while we could directly observe the samples in the 3D-ALI protocol. The CBF of hPSC-derived MCACs in the 3D-ALI protocol was near the normal CBF of human MCACs, which range from 10 to 14 Hz (Rutland et al., 1982). Next, to quantify mucociliary transport, fluorescent beads were tracked as previously demonstrated in resected murine trachea (Kunimoto et al., 2012). Because synchronized ciliary beating for generating a unidirectional flow appeared to be incomplete in both hPSC- and NHBEC-derived MCACs (Movies S1 and S2; Figure 4F), as was reported for NHBEC-derived MCACs (Matsui et al., 1998), we further focused on the diffusion of the beads, demonstrating the potency of mucociliary clearance in hPSC-derived MCACs (Figures 4F and 4G). The difference between MCACs derived from hPSCs and NHBECs might be partly due to the difference in maturity. In addition, the ideal balance in the number of between MCACs and mucus-producing cells for mucociliary clearance remains to be elucidated. MUC5AC and SPDEF levels on day 56 were lower than on day 42 (Figure S3F), which might be due to differentiation (Chen et al., 2009) and/or apoptosis. In this respect, the regulation of MUC5AC+ cells by modulating factors, such as IL-13 (Atherton et al., 2003), remains to be a future subject. In conclusion, the findings of the present study are thus considered to pave the way for future applications toward modeling airway diseases, such as PCD and CF, or developing methods of airway reconstruction such as an artificial trachea.
Experimental Procedures
Author Contributions
Acknowledgments We are grateful to K. Osafune, K. Okita, K. Takahashi, I. Asaka, and S. Yamanaka (Center for iPS Cell Research and Application, Kyoto University) for providing the iPSC lines, and method of iPSC culture. We thank K. Okamoto-Furuta and H. Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, Kyoto University), Y. Maeda and A. Inazumi for technical assistance. The fluorescence studies were performed at the Medical Research Support Center, Kyoto University. This work was supported by Grant-in-Aid for Scientific Research (KAKENHI), Translational Research Network Program from MEXT of Japan, Kyoto-Funding for Innovation in Health-related R&D Fields of Kyoto City, Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (S.T.), and, in part, Uehara Memorial Foundation.