2D culture: For the AECs, a custom culture medium was blended and named AEC basal medium (AECBM). The media and protocols are listed in Supplementary Figure 1. Primary AECs were used in most analyses. In the culture experiments, however, AECs were precultured with AECBM for 5-7 d then used for differentiation. In the present study, the multistep hepatic differentiation protocol of Nie et al[17 (link)] was applied. In that study, iPSCs were differentiated into hepatocytes. The AEC culture medium was changed step by step within 3 wk.
Another simple differentiation protocol from AECs to hepatocytes was reported by Maymó et al[14 (link)]. In brief, the cells were cultured for 1 wk in Iscove’s modified Dulbecco’s medium containing epidermal growth factor (EGF) then cultured with EGF and dexamethasone. This method was named the two-step protocol.
3D culture: A primary 2D culture was prepared to select a subpopulation of adherent AECs. Unattached cells were removed after 72 h. A 24-well 3D micropattern culture plate (Kuraray, Tokyo, Japan) was used for the next step. After coating the plate with lipidure (NOF Corporation, Tokyo, Japan), the precultured AECs were seeded at a density of 106/well. The AECs formed a sphere within 3-7 d. AECs, HUVECs, and MSCs were used for organoid propagation and they formed spheres within 1 d.