Extended Data Fig. 1: Purified hAEC2s transdifferentiate into KRT5+ basal cells when co-cultured with AHLM.
From: Human alveolar type 2 epithelium transdifferentiates into metaplastic KRT5+ basal cells
(a) FACS plot shows the strategy for isolating hAEC2s. After selecting live/CD45-/CD31-/CD11b- cells, hAEC2s were further isolated by gating EpCAM + /HTII-280+ cells. (b) Quantification of SFTPC + and KRT5 + cells in freshly sorted HTII-280+ cells by cytospins. Data are mean ± SD, representative of n = 4 biologically independent human lung cytospins analyzed. (c) confirmation of the purity of HTII-280+ cells by scRNA-seq showed no appreciable detection of non-AEC2 markers. (d) scRNA-seq of AHLM isolated as live/CD31-/CD45-/CD11b-/EpCAM- cells that express PDGFRA. (e) Colony forming efficiency of hAEC2/MRC5 and hAEC2/AHLM co-culture at D14. Data are mean ± SD, representative of n = 3 biologically independent for AHLM and hAEC2 and representative of n = 3 independent experiments for MRC5. (f) and (g) IF analysis of p63 and KRT17, and KRT5 in the hAEC2s + MRC5 and hAEC2s+AHLM organoids at D14. Images are representative of n = 2 (in f) and n = 3 (in g) biological replicates. (h) Quantification of KRT17 + and p63+ cells in total KRT5 + basal cells in the hAEC2s + MRC5 and hAEC2s+AHLM organoids at D14. n = 2 (for MRC5) and n = 3 (for AHLM) biologically independent samples were analyzed. Data are mean ± SD. (i) IF analysis of KRT8 in the hAEC2s+AHLM organoids at D14. Representative of n = 2 independent experiments. (j) IF analysis and quantification of KRT5 + and SFTPC + cells in the hAEC2-derived organoids co-cultured with freshly sorted AHLM (Passage 0) and cultured AHLM (Passage 2) at D14. Each dot represents a technical replicate. Representative of n = 2 independent experiments. (k) Enrichment of hAEC2s using a mesenchyme-free system, confirmed by IF analysis and quantification of SFTPC + and KRT5 + cells (left), and co-culture of the enriched hAEC2s with AHLM or MRC5, the organoids are analyzed at D14 (right). Data are expressed as the mean ± SD, representative of n = 3 biologically independent experiments.
