Extended Data Fig. 6: Human ES cell model of RAS cells demonstrates capacity to differentiate into AT2-like cells in vitro.

A) General experimental schematic of development of iRAS cells and propagation in airway or transition to alveolar media. B) Brightfield microscopy showing organoid formation and fluorescence after sorted iRAS cells were grown in airway (top) or alveolar (bottom) media. Scale bar = 100 µm. C) Corresponding flow cytometry analysis of endogenous mCherry reporter. D) Organoids from 3D culture with EPCAM or SCGB3A2 and SFTPC staining demonstrating SFTPC expression in alveolar organoids (bottom) and retention of SCGB3A2 expression in airways organoids (top). Scale bar = 100 µm, 20 µM for enlarged regions. N = 3 for panels B–D. E) Schematic of SFTPC protein processing in AT2 cells and F) western blot of primary translation product and processing intermediates (top, bands 1 and 2) and mature S-PC protein (band 3) in NKX2.1 progenitors, iRAS cells propagated in airway media, iRAS cells grown in alveolar media, and primary human AT2 cells (HT2-280+ cells). Molecular weights indicated on left. N = 3. G) SFTPC staining in individual iAT2 cells (top) and primary human AT2 cells (bottom) showing punctate nature of SFTPC staining. Scale bar 10 µm. N = 4. H) q-RT-PCR from iRASC grown in airway (red) or alveolar (green) media demonstrates differential gene expression of known RAS cell and AT2 cell marker genes. Data presented as box with median (bar) and upper and lower quartiles (box bounds) and whiskers for min and max values (n = 5). Unpaired Student’s test performed *p < 0.05, **p < 0.01. I) Gene expression based on scRNA sequencing of iRAS cells, and iRAS cells after 14 days in alveolar media showing downregulation of primary RAS cell defining genes and upregulation of AT2 defining genes. All N refer to biological replicates

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