Journal: Nature Communications

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

doi: 10.1038/s41467-023-44184-0

Figure Lengend Snippet: A Schematic of experimental design and overview. Live/CD31 − /CD45 − /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) were mixed with mouse lung fibroblasts from P28 mice and cultured for up to 35 days, followed by analysis via high content imaging. B H&E of 5 µm sections of FFPE day 35 Axin2 + organoids, showing cellular morphologies typical of both AT1 and AT2 cells. C – G Whole-mount immunofluorescence time course of Axin2 + organoids showing expansion of SFTPC + AT2 cells (red), increased differentiation into RAGE + AT1 cells (green) and increased structural complexity. H Imaris 3D reconstruction of day 35 Axin2 + organoid (z-depth = 174.13 µm) showing cellular arrangement/organization within mature organoids. I Click-iT EdU (green) whole-mount day 25 Axin2 + organoids, with proliferating cells primarily on outer edges or ‘buds’ growing outward from the organoid. J , K Electron microscopy of day 28 organoids. J Image of properly polarized AT2 cell with apical microvilli (black arrowhead) secreting surfactant (blue arrowhead) into a lumen. K Image of AT2 cell with lamellar bodies (black arrowhead) adjacent to an AT1 cell (green arrowhead, right). L , M Comparison of in vivo mouse lung (9-month C57BL/6J mouse) and in vitro day 25 Axin2 + organoids. Data throughout the figure represents at least 5 biological replicates with 3 technical replicates per experiment. [Scale bars = 50 µm, except for electron microscopy ( J , K ) scale bars = 2.5 µm]. (RAGE Receptor for Advanced Glycation End-products [AT1 cell marker], SFTPC Surfactant Protein C [AT2 cell marker], EdU 5-ethynyl-2’-deoxyuridine, FFPE formalin-fixed, paraffin-embedded). Schematics created with Biorender.com.

Article Snippet: To generate ‘spiked’ SAGM medium for mouse lung alveolar organoids, SABM Small Airway Epithelial Cell Growth Basal Medium (Lonza, CC-3119) was combined with the following additives: SAGM Small Airway Epithelial Cell Growth Medium SingleQuots Supplements and Growth Factors (using only the BPE [2 mL], Insulin [0.5 mL], Retinoic Acid [0.5 mL], Transferrin [0.5 mL], and hEGF [0.5 mL] aliquots) (Lonza, CC-4124), Heat Inactivated Fetal Bovine Serum (Corning, 35-011-CV, final concentration 5%), Antibiotic-Antimycotic (Gibco, 15240-062, final concentration 1x), Cholera Toxin from Vibrio cholerae (Sigma, C8052, final concentration 25 ng/mL).

Techniques: Cell Culture, Imaging, Immunofluorescence, Electron Microscopy, Comparison, In Vivo, In Vitro, Marker, Formalin-fixed Paraffin-Embedded

Journal: Nature Communications

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

doi: 10.1038/s41467-023-44184-0

Figure Lengend Snippet: A AAV6.2FF-Cre experimental set-up. Live/CD31 − /CD45 − /CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) sorted from Axin2 creERT2-tdT ; R26R EYFP mice and Axin2 creERT2-tdT ; R26R EYFP ; Nkx2-1 fl/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 R26R EYFP AEPs) and Nkx2-1 KO AEPs (AAV6.2FF-Cre-treated sorted R26R EYFP ; Nkx2-1 fl/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 R26R EYFP ; Nkx2-1 fl/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. Nkx2-1 KO 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 (Nkx KO ). Nkx KO cells cluster separately from Uninfected and AAV control cells near Krt8 + cells ( K , L ), which make up a majority of cells in the Nkx KO condition ( M ). Marker genes for normal alveolar epithelium are lost and different markers gained ( N ) in Nkx KO . 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. for marker gene analysis. [Scale bars = 50 µm]. Schematics created with Biorender.com.

Article Snippet: To generate ‘spiked’ SAGM medium for mouse lung alveolar organoids, SABM Small Airway Epithelial Cell Growth Basal Medium (Lonza, CC-3119) was combined with the following additives: SAGM Small Airway Epithelial Cell Growth Medium SingleQuots Supplements and Growth Factors (using only the BPE [2 mL], Insulin [0.5 mL], Retinoic Acid [0.5 mL], Transferrin [0.5 mL], and hEGF [0.5 mL] aliquots) (Lonza, CC-4124), Heat Inactivated Fetal Bovine Serum (Corning, 35-011-CV, final concentration 5%), Antibiotic-Antimycotic (Gibco, 15240-062, final concentration 1x), Cholera Toxin from Vibrio cholerae (Sigma, C8052, final concentration 25 ng/mL).

Techniques: Comparison, Control, Immunofluorescence, Derivative Assay, Expressing, Marker