Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

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