Fig. 6: In vitro Nkx2-1 KO of AEP-derived alveolar organoids drives irreversible transition to a Krt8 stressed transitional /PATS/DAPT-like state. | Nature Communications

Fig. 6: In vitro Nkx2-1 KO of AEP-derived alveolar organoids drives irreversible transition to a Krt8 stressed transitional /PATS/DAPT-like state.

From: Alveolar epithelial progenitor cells require Nkx2-1 to maintain progenitor-specific epigenomic state during lung homeostasis and regeneration

Fig. 6

A AAV6.2FF-Cre experimental set-up. Live/CD31−/CD45−/CD326+(EpCAM+)/TdTomato+(Axin2+) cells (AEPs) sorted from Axin2creERT2-tdT; R26REYFP mice and Axin2creERT2-tdT; R26REYFP; Nkx2-1fl/fl mice were treated with AAV6.2FF-Cre and plated with wild-type fibroblasts. B Comparison of brightfield and GFP whole-well images of organoids grown from control (AAV6.2FF-Cre-treated sorted R26REYFP AEPs) and Nkx2-1 KO AEPs (AAV6.2FF-Cre-treated sorted R26REYFP; Nkx2-1fl/fl AEPs) at day 28 of culture. Control (non-GFP) organoids with normal morphology are marked with a white asterisk. C–J H&E and immunofluorescence images of R26REYFP; Nkx2-1fl/fl AEP-derived organoids that did (F–J) or did not (C–E) undergo recombination via AAV6.2FF-Cre. C–E Non-recombined organoids (D) express SPC (red) and Nkx2-1 (white) but do not express the YFP lineage label (green), whereas G recombined organoids do not express SPC or Nkx2-1 but do express the YFP lineage label. Non-recombined (E) and recombined (H) organoids maintain epithelial identity expressing CDH1. Nkx2-1KO organoids express KRT8 and many proliferate and express Ki67 as late as day 40 of culture (J-J”). Data (C–J) represents 4 biological replicates with 3 technical replicates per experiment. K–R Integrated scRNAseq comparing epithelial cells from day 28 control organoids (Uninfected), AAV6.2FF-Cre-treated control organoids (AAV control), and AAV6.2FF-Cre-treated Nkx2-1 KO organoids (NkxKO). NkxKO cells cluster separately from Uninfected and AAV control cells near Krt8+ cells (K, L), which make up a majority of cells in the NkxKO condition (M). Marker genes for normal alveolar epithelium are lost and different markers gained (N) in NkxKO. O–R Module scoring using published gene sets for AEPs (O), Krt8/PATS/DATP/ADI cells (P), lung cancer cells (Q), and foregut endoderm (R). Compare to Supplementary Fig. S9 for marker gene analysis. [Scale bars = 50 µm]. Schematics created with Biorender.com.

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