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[Preprint]. 2023 Jan 20:2023.01.19.524655. [Version 1] doi: 10.1101/2023.01.19.524655

Figure 7: iAT1s cultured at air-liquid interface (ALI) express AT1-like molecular and functional phenotypes.

Figure 7:

A) Experimental schematic indicating BU3 NGAT iAT1s were cultured in 3D in L+DCI medium for 8–11 days before single cell passaging and replating onto transwell inserts in L+DCI medium. Upper chamber media was aspirated after 3 days (airlift) to form an air-liquid interface (ALI). B) Live cell imaging showing retention of AGERtdTomato in iAT1s after ALI culture. scale bar = 100um. C) Flow cytometric analysis of NKX2-1GFP and AGERtdTomato reporter expression in 3D or ALI cultures of iAT1s (6 days). D) Transepithelial electrical resistance (TEER) measurements of BU3 NGAT iAT1s over 10 days of ALI cultures (Air lifted at day 3; N=3). E) Profiling by scRNA-seq of BU3 NGAT iAT2s in 3D CK+DCI, iAT1s in 3D L+DCI, or iAT1s in ALI cultures. iAT1s in ALI cultures were plated either from iAT2s into L+DCI (iAT1 ALI P0) or were plated from 3D iAT1s after 9 days of pre-culturing in 3D L+DCI prior to transfer to ALI culture (iAT1 ALI P1). UMAP projection shows the 3D cultures cluster separately while the two iAT1 ALI cultures cluster closer together. F) UMAP overlays of YAP/TAZ 22 gene signature61 and primary human AT1 50-gene signature (Table S1). G) Cell cycle phase distribution across all samples. H) Expression of transcripts encoding extracellular matrix (ECM) components. I) Expression of transcripts encoding secreted ligands, comparing the samples from (E). J) Analysis of secreted VEGFA protein in conditioned media at day 10 of culture of each indicated sample. K) TEER of iAT1s after plating at High, Medium, and Low densities and outgrowth in ALI cultures in L+DCI. One-way ANOVA, N=3 per condition. L) Tight junction protein ZO-1 staining (magenta) at high, medium, and low plating density outgrowths at day 10 Scale bar = 50um. M) Average surface area of cells calculated using ZO-1 cell outlines at three different iAT1 ALI plating densities. N=3 per condition, averaged from ~150 cells per sample. N) Cross sectional imaging of SPC2-ST-B2 iAT2s at ALI as previously published,68 and SPC2-ST-B2 iAT1s plated at low densities in L+DCI. (Toluidine Blue stain, scale bar = 10um). O) Frequency distribution of cell surface areas of iAT2, iAT1 high density, and iAT1 low density. N=149, 139, and 79 respectively). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.001 for all panels.