GRAMD2+ alveolar type I cell plasticity facilitates cell state transitions in organoid culture

Alveolar epithelial regeneration is critical for normal lung function and becomes dysregulated in disease. While alveolar type 2 (AT2) and club cells are known distal lung epithelial progenitors, determining if alveolar epithelial type 1 (AT1) cells also contribute to alveolar regeneration has been hampered by lack of highly specific mouse models labeling AT1 cells. To address this, the Gramd2CreERT2 transgenic strain was generated and crossed to ROSAmTmG mice. Extensive cellular characterization, including distal lung immunofluorescence and cytospin staining, confirmed that GRAMD2+ AT1 cells are highly enriched for green fluoresecent protein (GFP). Interestingly, Gramd2CreERT2 GFP+ cells were able to form colonies in organoid co-culture with Mlg fibroblasts. Temporal scRNAseq revealed that Gramd2+ AT1 cells transition through numerous intermediate lung epithelial cell states including basal, secretory and AT2 cell in organoids while acquiring proliferative capacity. Our results indicate that Gramd2+ AT1 cells are highly plastic suggesting they may contribute to alveolar regeneration. Overall design: Primary isolation of FACS sorted GFP+ cells were defined as day 0. On days 8, 13, and 20 of 3D culture, to obtain single cells, colonies were dissociated with dispase (#354235, BD Gibco) and TrypLE express (#12604013, Gibco). Biotinylated EpCAM Ab (#130-096-419, Miltenyi Biotec, San Diego, California), anti-biotin microbeads (#130-900-485, Miltenyi Biotec,) and MS columns (#130-042-201, Miltenyi Biotec) were used to enrich for epithelial cells. For each time point, cells were counted, and viability was measured using an automated cell counter (Countess 3, Thermo Fisher Scientific). Single cell suspensions were processed using Chromium Single Cell 3' Reagent Kits v3.1 (10x Genomics, PN-1000128, PN-1000127 and PN-1000213). Briefly, cells resuspended in the reaction mix were loaded into the Chromium Single Cell Chip G, together with the 10x Barcoded Gel Beads and partitioning oil. The Chip G was placed into the 10x Chromium controller for cell partitioning, targeting a final number of 10,000 cells per sample. After partitioning, the GEMs (Gel Bead-In EMulsions) were transferred to the C1000 Touch Thermal Cycler for the first phase of reverse transcription followed by library preparation following the manufacturer's instructions. Libraries were deep sequenced using the Illumina NovaSeq platform.