Extended Data Fig. 8: Dual reporter system demonstrates SFTPC expression in iAT2 cells and highlights dynamics of cell transitions in vitro.

A) Schematic of vectors for SFTPC-eGFP targeting in the dual reporter hES cell line. B) Brightfield microscopy showing mCherry and eGFP expression in 3D cultures of sorted iRAS cells grown in either airway (top) or alveolar (bottom) media for 14 days. Scale bar = 100 µm. N = 3. C) Flow cytometry showing endogenous SCGB3A2-mCherry and SFTPC-eGFP expression in iRAS cells grown in airway (top) or alveolar (bottom) media compared to NKX2.1 progenitor controls. D) Gating strategy for all hES cell flow cytometry and FACS experiments shown in Extended Data Figs. 6c, 8b, and 9h, i and n. E) Flow cytometry corresponding to experiment in Fig. 2a, of SCGB3A2-mCherry and SFTPC-eGFP expression over time as iRASC were propagated in Alveolar media. N = 3. F) UMAP of scRNA-seq analysis of all populations derived from iRAS cells at day 14 of differentiation in alveolar media reveals several clusters. G) The resulting culture was heterogenous and included both iAT2 lung endoderm progenitors as well as a small number of other foregut endoderm cell types. Feature plots showing expression of canonical AT1 and AT2 cell alveolar epithelial markers, Airway cell markers, Lung endoderm progenitor markers, neuroendocrine and tuft cell markers, and gastric fate markers, allowing putative identification of all observed clusters.