irradiated mrc 5 feeder  (ATCC)

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: (A) Treatment of AEOs with IAV in media placed on apical surface of transwell above matrigel plug. Minimal NP expression is evident at 24hpi. (B) Morphology of AEOs in suspension culture after liberation from Matrigel. AT1 and AT2 cell morphology is maintained for 24h post-suspension but has decreased by 36h. (C). Cell number per organoid in suspension culture. We noted a nearly significant loss of cell number by 36h post liberation. (D) Cell state of AEOs with infection of IAV using SAGM base media with TPCK-treated trypsin and lacking serum (Inf. media). Suspension in this medium allowed flu infection by 8h (NP) but led to loss of AT1 cells (AGER) without loss of AT2 cells (SPC). (E) Impact of different media compositions on cell junctions in AEOs. Infection media (top row) led to loss of AGER and associated loss of ZO-1 marked cell surface tight junctions. Removal of TPCK-treated trypsin or addition of 5% serum prevented these changes. Scale bars = 50 μm. (SPC = Surfactant Protein C [AT2 marker]; Ager = Advance Glycosylation End-product Specific Receptor [AT1 marker]; NP = IAV nucleoprotein [infected cell marker], ZO-1 = Zonula Occludens-1 [cell surfact tight junction marker]) .

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques: Expressing, Suspension, Infection, Marker, Glycoproteomics

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: (A) Schematic of method for liberation and treatment of AEOs with IAV. (B-E) AEOs treated with H1N1 develop progressively increased influenza infection as measured by nuclear expression of H1N1 NP across 24hpi. (F) Quantification of NP-positive nuclei at each timepoint of infection. (G) Cell type specific infection in AEOs. Significantly more AT2 are infected than AT1 by 8hpi and thereafter. (H) Expression of the flu binding α-2,6-sialic acid in AEOs marked by expression of sambuccus nigra lectin predominantly in AT2 cells. (I-J). In vivo confirmation of SNA expression (I) and increased infection rate (J) in AT2 cells during early flu. Scale bars = 50 μm. (SPC = Surfactant Protein C [AT2 marker]; Ager = Advance Glycosylation End-product Specific Receptor [AT1 marker]; NP = IAV nucleoprotein [infected cell marker], SNA = Sambuccus nigra lectin [flu binding motif α-2,6-sialic acid]) . P values by ANOVA with multiple comparison (F,G,J) or T test (I): * = <0.05, ** = <0.01, *** = < 0.001, **** = < 0.0001.

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques: Infection, Expressing, Binding Assay, In Vivo, Marker, Glycoproteomics, Comparison

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: (A) R26R EYFP mice were infected with a Pr8-Cre virus and then harvested for flow cytometry 48, 72, and 96hpi. (B) Flow cytometry gating showing the identification of our YFP + /IAV-Infected cells with a predominance of infected AT2 cells relative to other populations

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques: Infection, Virus, Flow Cytometry

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: (A) Schematic color scheme of cell states in AEOs. Unexposed = grey, Uninfected = blue, Early IAV = orange, Late IAV = red. (B-C) UMAP (B) and frequency (C) showing infection states of AT2 cell subset of AEOs. (D) Strip plots showing number of differentially expressed genes (DEGs) and degree of enrichment across AT2 cell states. (E-F) Volcano plot of DEGs (E) and GO Biological Process enrichment (F) of genes in Unexposed (grey) vs Uninfected (blue) AT2. (G-H) Volcano plot of DEGs (G) and GO Biological Process enrichment (H) of genes Uninfected (blue) vs Early Infected (orange) AT2. (I-J) Volcano plot of DEGs (I) and GO Biological Process enrichment (J) of genes in Early Infected (orange) vs Late Infected (red) AT2. Upregulated processes are shown in yellow, and downregulated processes in blue. (K) State dynamics of enrichment of GO terms from F, H, and J. Letter to left of process name matches letters to right of bars. Some terms are enriched at multiple timepoints. Diagrams in K show dynamics of change of associated terms (boxes) across cell states in AEOs.

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques: Infection, Stripping Membranes

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: (A) Overall UMAP and AT2 only UMAP based on ATAC assay showing cell infection state. (B) Strip plot showing number and significance of differential peaks across AT2 cell states. (C-D) Volcano plot (C) and GO Terms (D) showing nearest neighbor genes to differentially open chromatin enriched in Uninfected compared to Unexposed AT2 cells. (E) Volcano plot of nearest neighbor genes to differentially open chromatin in Early infected compared to Uninfected AT2 cells. (F) Bar chart showing the number of differentially open peaks, nearest neighbor genes, and associated enriched GO Terms. No enriched GO Terms are associated with newly open chromatin in IAV-infected AT2 cells, and no differentially open chromatin regions passed thresholding for Early vs Late infection. (G) Motifs enriched in differentially open chromatin in Uninfected vs Unexposed (left) or Early infected vs Uninfected (right) AT2 cells.

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques: Infection, Stripping Membranes

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: ( A) Epithelial-only UMAP (left) and psuedotime trajectory (right) suggesting that STE arise from AT2 in IAV-treated AEOs. (B) Marker gene expression of a series of AT2 and STE markers defining cell state in AEOs. (C-D) GO Biological Process Terms based on DEGs for (C) Uninfected AT2s vs STE1s and (D) STE1s vs STE2s. (E-F) Motif activity enrichment based on differentially open chromatin (E) in Uninfected AT2s vs STE1s and (F) and Motif activity enrichment based on differentially open chromatin (F) in STE2 vs STE1. Major reported regulators of STE transition including reduction of Nkx2-1 activity, IL1b signaling driving FOS:JUN signaling, and TGFb signaling are identified in STE1 vs AT2. (G) Cytokine ELISA demonstrating enrichment of multiple IAV-associated cytokines in the media of IAV-infected AEOs. Compare to .

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques: Marker, Gene Expression, Activity Assay, Enzyme-linked Immunosorbent Assay, Infection

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: (A) Schematic describing the co-culture of freshly sorted human epithelial cells with MRC5 fibroblasts for creation of human AEOs. (B) IHC demonstrating clear punctate SPC expression in human AEOs; we noted relatively low level HT2-280 expression in established AEO culture. (C-D) Dynamics of PR8 IAV infection in AEOs, showing high levels of nuclear NP expression (C) which is quantified in (D). (E-F) UMAP projection (EF) and cell marker gene expression (F) of cell states found in human AEOs. (G-H) UMAP of AT2 cell subset (G) and quantification (H) showing imputed flu infection state as previously described for murine organoids. (I-J) Strip plots showing differentially expressed genes (I) or differentially accessible chromatin regions (J) in Uninfected vs Unexposed, Early Infected vs Uninfected, and Late Infected versus Early Infected human AT2 cells.

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques: Co-Culture Assay, Expressing, Infection, Marker, Gene Expression, Stripping Membranes

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: (A-B) Volcano plot of DEGs (A) and GO Biological Process enrichment (B) of genes in Unexposed (grey) vs Uninfected (blue) human AT2 cells. (C-D) Volcano plot of DEGs (C) and GO Biological Process enrichment (D) of genes in Uninfected vs Early IAV (orange) human AT2 cells. (E-F) Volcano plot of DEGs (E) and GO Biological Process enrichment (F) of genes in Early IAV vs Late IAV (red) human AT2 cells. (G) Temporal dynamics of enrichment of GO terms from B, D, and F. Letter to the left of the process name matches letters to the right of the GO term bars. Some terms are enriched in multiple timepoints. Diagrams in G show the dynamics of change of associated terms (boxes) across cell states in AEOs.

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques:

Journal: bioRxiv

Article Title: Early cell autonomous and niche-mediated alveolar epithelial response to influenza infection in primary lung organoids

doi: 10.1101/2025.10.24.684481

Figure Lengend Snippet: (A) Schematic depicting data analysis pipeline (see Results and Methods) used to identify core conserved regulated pathways and gene sets in IAV-infected AEOs. (B-C) Enriched and downregulated GO Terms (B) in Unexposed vs Uninfected AT2 with leading edge genes per GO terms as in (C). (D-E) Enriched GO Terms (D) in Uninfected vs Early IAV AT2 with leading edge genes per GO term as in (E). (F-G) Enriched and downregulated GO Terms (F) in Early vs Late IAV infection in AT2 with leading edge genes per GO term as in (G).

Article Snippet: Human AT2s were plated as described above for murine AT2 except MRC5s (ATCC CCL-171, tested negative for mycobacterial contamination) at P8 were used as a replacement for the murine fibroblast stock.

Techniques: Infection