WO2024049773A1 - Organoïdes épithéliaux alvéolaires avancés - Google Patents

Organoïdes épithéliaux alvéolaires avancés Download PDF

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WO2024049773A1
WO2024049773A1 PCT/US2023/031312 US2023031312W WO2024049773A1 WO 2024049773 A1 WO2024049773 A1 WO 2024049773A1 US 2023031312 W US2023031312 W US 2023031312W WO 2024049773 A1 WO2024049773 A1 WO 2024049773A1
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aep
cells
cell
optionally
nkx2
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William ZACHARIAS
Andrea Toth
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Children's Hospital Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/42—Respiratory system, e.g. lungs, bronchi or lung cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0688—Cells from the lungs or the respiratory tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/27—Lung cells, respiratory tract cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Definitions

  • lung organoid approaches have been reported in recent years, derived both from primary lung epithelium and induced-pluripotent stem cells (iPSC)-.
  • iPSC induced-pluripotent stem cells
  • iPSC-derived alveolar cells have advanced understanding of human alveolar type 2 (AT2) differentiation and biology, it is difficult to model complex adult lung epithelial phenotypes and pathologies using human iPSC cultures.
  • lung regeneration involves complex in vivo morphogenesis occurring in tandem with cellular differentiation – a major barrier to building an “alveolus in a dish” is the lack of morphological similarity between in vitro and in vivo models.
  • Lung epithelial regeneration after acute injury requires coordination of extensive cellular and molecular processes controlling proliferation and differentiation of specialized alveolar cells to pattern the morphologically complex alveolar gas exchange surface.
  • a refined primary murine alveolar organoid assay which recapitulates important aspects of in vivo lung epithelial regeneration, providing a tractable model to dissect regenerative processes. Clonal expansion of single AEPs generated complex alveolar organoids with extensive structural maturation and organization.
  • AEP to AT1 intermediate states a widely reported transitional state defined by cell stress markers (Krt8 + /PATS/DATP/ADI cells, also referred to as a “Krt8+ stressed transitional state”) and a second state defined by differential receptivity to cellular signaling pathways important in AT1 cell differentiation.
  • Transcriptional regulatory network (TRN) analysis demonstrates that these AT1 transition states are driven by distinct regulatory networks controlled in part by differential activity of the lung master regulatory factor Nkx2-1, which was absent in the TRN for Krt8+ cells.
  • Nkx2-1 in AEP-derived organoids causes irreversible transition to a proliferative stressed Krt8 + state (Krt8 + /PATS/DATP/ADI-like state) by disorganized, uncontrolled growth.
  • AEP-specific deletion of Nkx2-1 in adult mice using a Tfcp2l1 CreERT2 mouse line leads to rapid, irreversible loss of AEP state, clonal expansion, and disorganization of alveolar structure, (optionally with loss of proliferation).
  • Tfcp2l1 CreERT2 mouse line leads to rapid, irreversible loss of AEP state, clonal expansion, and disorganization of alveolar structure, (optionally with loss of proliferation).
  • Embodiments of the present disclosure include the following numbered embodiments: 1. A method of making an alveolar epithelial progenitor cell (AEP)-derived organoid (AEP-O), the method comprising coculturing Wnt-responsive alveolar type 2 cells (AEP cells) and mesenchyme cells. 2. The method of embodiment 1, wherein the mesenchyme cells are fibroblast cells. 3. The method of any one of the preceding embodiments, wherein the mesenchyme cells are alveolar fibroblasts. 4. The method of any one of the preceding embodiments, wherein the mesenchyme cells are from P28 wild type C57BL/6 mice, optionally at passage 3-4. 5.
  • AEP alveolar epithelial progenitor cell
  • AEP-O alveolar epithelial progenitor cell
  • AEP cells are FACS sorted to select CD31-/CD45-/CD326 + (EpCAM + ) cells, wherein optionally the AEP cells are TdTomato + . 6. The method of any one of the preceding embodiments, wherein the AEP cells are from Axin2 creERT2-tDT mice. 7.
  • AEP and mesenchyme cells are cocultured in a ratio of AEP to mesenchyme that is, or is about, 2:1, 1:1, 1:2, 1:5, 1:10, 1:15, 1:20, 1:50, or a range defined by any two of the preceding values, optionally 2:1-1:50, 1:2-1:20, 1:5-1:15, or 1:10. 8.
  • any one of the preceding embodiments wherein about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000, or a range defined by any two of the preceding values, of sorted AEPs are cocultured with about 10K, 20K, 30K, 40K, 50K, 60, 70K, 80K, 90K, or 100K, or a range defined by any two of the preceding values, mesenchymal cells.
  • the coculturing is in a small airway epithelial cell growth basal medium (Lonza, CC-3119) or equivalent media. 10.
  • the coculturing is in a media supplemented with BPE, Insulin, Retinoic Acid, Transferrin, and hEGF. 11. The method of any one of the preceding embodiments, wherein the coculturing is in a media supplemented with heat inactivated fetal bovine serum, optionally at a final concentration of about 1-10%, 2-8%, 3-7%, or 5%. 12. The method of any one of the preceding embodiments, wherein the coculturing is in a media comprising an extracellular membrane matrix. 13. The method of any one of the preceding embodiments, wherein the coculturing is in a media comprising Matrigel. 14.
  • the coculturing is in a media comprising an extracellular membrane matrix, wherein the media and the extracellular membrane matrix are combined in a ratio of 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, or 1:10, or a range defined by any two of the preceding values.
  • the coculturing is in a transwell.
  • the coculturing is in a transwell, wherein media supplemented with a ROCK inhibitor is added beneath the transwell. 17.
  • any one of the preceding embodiments wherein the coculturing is in a transwell, wherein media supplemented with ROCK Inhibitor Y-27632 dihydrochloride is added beneath the transwell, optionally at a final concentration of about 0.001-0.1, 0.005- 0.05, or 0.01 mM. 18. The method of any one of the preceding embodiments, wherein the coculturing is in a transwell, wherein media supplemented with a ROCK inhibitor is added beneath the transwell, and wherein the coculture is incubated in the presence of the ROCK inhibitor for about 36-60, or 48 hours. 19.
  • the coculturing is in a transwell, wherein media supplemented with a ROCK inhibitor is added beneath the transwell, and wherein the coculture is incubated in the presence of the ROCK inhibitor for about 36-60, or 48 hours, and wherein thereafter the media does not contain a ROCK inhibitor.
  • the coculturing is for a period of, or of at least, about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or a range defined by any two of the preceding values, optionally 1-8, 2-8, 3-8, 2-6, 2-5, or 3-5 weeks. 21.
  • the AEP-O comprises AEP cells, AT1 cells, and AT2 cells, optionally wherein the AEP, AT1 and AT2 cells express AEP, AT1, and AT2 cell markers, respectively. 22. The method of any one of the preceding embodiments, wherein the AEP-O comprises RAGE + AT1 cells. 23. The method of any one of the preceding embodiments, wherein the AEP-O comprises cavities. 24. The method of any one of the preceding embodiments, wherein the AEP-O comprises cavities forming alveolar-like structures. 25.
  • the AEP-O comprises mature and/or polarized AT1 cells within the central portion of the organoid, optionally wherein AT2 cells are intermixed with the mature and/or polarized AT1 cells. 26. The method of any one of the preceding embodiments, wherein the AEP-O comprises cavities with epithelial lining that comprises AT2 cells. 27. The method of any one of the preceding embodiments, wherein the AEP-O comprises cavities with epithelial lining that comprises AT2 cells containing lamellar bodies with the apical surface directed towards the internal lumen. 28. The method of any one of the preceding embodiments, wherein the AEP-O comprises active surfactant secretion. 29.
  • the AEP-O comprises mesenchymal cells, optionally fibroblasts.
  • the AEP-O does not comprise mesenchymal cells, optionally fibroblasts.
  • the AEP-O does not comprise fibillar collagen type I and/or type II.
  • mesenchymal cells, optionally fibroblasts are near and/or adjacent to the AEP-O. 33.
  • the AEP-O is cultured in a container comprising a monolayer of mesenchymal cells, optionally fibroblasts. 34. The method of any one of the preceding embodiments, wherein the AEP-O comprises immune cells. 35. The method of any one of the preceding embodiments, wherein the AEP-O does not comprise immune cells. 36. The method of any one of the preceding embodiments, wherein immune cells are near and/or adjacent to the AEP-O. 37. The method of any one of the preceding embodiments, wherein the AEP-O are cultured in a container comprising a monolayer of fibroblasts comprising immune cells. 38.
  • the AEP-O comprises AT1 cells expressing WNT ligands and/or PDGF ligands. 39. The method of any one of the preceding embodiments, wherein the AEP-O comprises AT2 cells that are WNT-responsive. 40. The method of any one of the preceding embodiments, wherein the mesenchymal cells are WNT-responsive, PDGFR ⁇ + , express HGF, express non-canonical WNT, and/or express FGF ligands. 41.
  • the AEP-O comprises AEP cells expressing one or more of the AEP-enriched markers Id2, Ctnnb1, Lrp5, Lrp2, Napsa, Bex2, Hdc, and Fgfr2. 42. The method of any one of the preceding embodiments, wherein the AEP-O comprises AEP cells expressing high levels of cycle genes (pAEPs). 43. The method of any one of the preceding embodiments, wherein the AEP-O comprises AT2tr cells, optionally comprising high level expression of glutathione pathway genes and a shift towards lipid metabolism. 44.
  • the AEP-O comprises mature AT2 cells expressing one or more markers selected from Sftpa1, Lys2, Sftpc and Sftpb. 45. The method of any one of the preceding embodiments, wherein the AEP-O comprises Krt8 + transition cells (Krt8 + ) expressing one or more markers selected from Krt8, Lgals3, Tp53, Nupr1, Ddit3, and Cldn4, or optionally one or more markers selected from Krt8, Lgals3, Tp53, and Cldn4. 46.
  • the AEP-O comprises AT1 transition (AT1tr) cells expressing one or more markers selected from Hes1 and Igfbp7. 47. The method of any one of the preceding embodiments, wherein the AEP-O comprises a modification reducing or eliminating expression of Nkx2-1. 48. The method of any one of the preceding embodiments, wherein the AEP-O comprises AEPs harboring a R26R-lox-stop-lox-EYFP allele. 49. The method of any one of the preceding embodiments, wherein the method comprises infecting AEPs with AAV6.2FF-Cre, optionally after FACS sorting and prior to coculturing with mesenchyme cells. 50.
  • AEPs are from Axin2 CreERT2-Tdt x Rosa-EYFP x Nkx2-1 flox/flox x animals. 51. The method of any one of the preceding embodiments, wherein the AEPs are from Tfcp2l1 CreERT2 x R26R EYFP x Nkx2-1 flox/flox animals. 52. The method of any one of the preceding embodiments, wherein the AEPs are from Nkx2-1 knockout animals. 53.
  • the AEP-O comprises at least one alveolar-like cavity filled with debris, a pseudostratrified epithelial lining, and/or a glandular-like appearance.
  • the AEP-O does not comprise substantial expression of one or more foregut endoderm markers selected from Sox2, Sox9, Cdx2, Gata4, and Pdx1.
  • An AEP-O made by the method of any one of the preceding embodiments.
  • An AEP-O comprising AEP cells, AT1 cells, and AT2 cells, optionally wherein the AEP, AT1 and AT2 cells express AEP, AT1, and AT2 cell markers, respectively. 57.
  • the AEP-O of any one of the preceding embodiments, wherein the AEP-O comprises cavities with epithelial lining that comprises AT2 cells. 62. The AEP-O of any one of the preceding embodiments, wherein the AEP-O comprises cavities with epithelial lining that comprises AT2 cells containing lamellar bodies with the apical surface directed towards the internal lumen. 63. The AEP-O of any one of the preceding embodiments, wherein the AEP-O comprises active surfactant secretion. 64. The AEP-O of any one of the preceding embodiments, wherein the AEP-O comprises mesenchymal cells, optionally fibroblasts. 65.
  • the AEP-O of any one of the preceding embodiments, wherein the AEP-O comprises immune cells. 70. The AEP-O of any one of the preceding embodiments, wherein the AEP-O does not comprise immune cells. 71. The AEP-O of any one of the preceding embodiments, wherein immune cells are near and/or adjacent to the AEP-O. 72. The AEP-O of any one of the preceding embodiments, wherein the AEP-O are cultured in a container comprising a monolayer of fibroblasts comprising immune cells. 73. The AEP-O of any one of the preceding embodiments, wherein the AEP-O comprises AT1 cells expressing WNT ligands and/or PDGF ligands. 74.
  • the AEP-O of any one of the preceding embodiments, wherein the AEP-O comprises AT2 cells that are WNT-responsive.
  • the AEP-O of any one of the preceding embodiments, wherein the AEP-O comprises AEP cells expressing one or more of the AEP-enriched markers Id2, Ctnnb1, Lrp5, Lrp2, Napsa, Bex2, Hdc, and Fgfr2. 77.
  • Krt8 + transition cells
  • the AEP-O comprises AT1 transition (AT1tr) cells expressing one or more markers selected from Hes1 and Igfbp7.
  • the AEP-O of any one of the preceding embodiments, wherein the AEP cells are from Axin2 creERT2-tDT mice.
  • AEP-O of any one of the preceding embodiments, wherein the AEP-O is a model for a disease state.
  • a method comprising exposing an AEP-O of any one of the preceding embodiments to a compound.
  • the compound is selected from a therapeutic compound, a candidate therapeutic compound, a toxin, mutagen, and/or a compound that induces a disease-like state in the AEP-O.
  • 97. The method of embodiment 95 or 96, wherein the method comprises screening multiple compounds and/or multiple AEP-Os.
  • FIG. 1A-1M An embodiment of AEP-derived alveolar organoids clonally expand and pattern complex, polarized alveolar-like cavities.
  • FIG.1A 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.
  • FIG. 1A 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.
  • FIG. 1B H&E of 5 ⁇ m sections of FFPE day 35 Axin2 + organoids, showing cellular morphologies typical of both AT1 and AT2 cells.
  • FIGs. 1C-1G 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.
  • FIG. 1H Imaris 3D reconstruction of day 35 Axin2 + organoid showing cellular arrangement/organization within mature organoids.
  • FIG. 1I Click-iT EdU (green) whole- mount day 25 Axin2 + organoids, with proliferating cells primarily on outer edges or ‘buds’ growing outward from the organoid.
  • FIGs.1J-1K Electron microscopy of day 28 organoids.
  • FIG. 1J Image of properly polarized AT2 cell with apical microvilli (black arrowhead) secreting surfactant (blue arrowhead) into a lumen.
  • FIG.1K Image of AT2 cell with lamellar bodies (black arrowhead) adjacent to an AT1 cell (green arrowhead, right).
  • FIG. 2A-2H An embodiment of single cell composition and epithelial- mesenchymal interactions in alveolar organoids over time course of differentiation.
  • FIG.2A UMAP of all cell populations combining d14, d21 and d28 AEP-derived organoid of scRNA- seq datasets.
  • FIG. 2B Cell population proportions at each time point with increasing proportion of epithelial cells as organoids grow.
  • FIG.2C Heat map showing expression of top 10 most differentially expressed genes in each population.
  • FIGs. 2D-2E Ligand-receptor analysis of organoid culture demonstrating extensive mesenchymal-epithelial communication in organoids.
  • FIG.2F Schematic of experimental set-up of live imaging and 3D reconstruction of live day 20 organoids generated using PDGFR ⁇ eGFP fibroblasts stained with Hoechst, with data shown in FIGs. 2F-2H.
  • FIG. 2F 3D reconstruction of confocal z-stacks of whole wells including transwell filter, showing the majority of GFP + fibroblasts are growing on the filter; F’-F’’’) Whole-mount immunofluorescence showing lack of PDGFR ⁇ + cells within day 20 organoids, with scattered cells found throughout the surrounding matrigel.
  • FIG. 2G Whole mount IHC showing few GFP + fibroblasts inside of organoids.
  • FIG. 2H CD45 staining of organoids; see also Figure 12I.
  • AEP alveolar epithelial progenitor
  • pAEP proliferative AEP
  • AT2tr AT2 transitional cell
  • AT2 alveolar type 2 cell
  • AT1tr AT1 transitional cell
  • AT1 alveolar type 1 cell
  • Krt8 Krt8/DATP/PATS-like transitional cell
  • pMes proliferative mesenchymal cell
  • AlvFB1 alveolar fibroblast type1
  • AlvFB2 alveolar fibroblast type 2
  • SM smooth-muscle like mesenchyme.
  • FIG. 3A An embodiment of AEP-derived organoids elucidate dynamics of alveolar epithelial differentiation.
  • FIG. 3A scVelo RNA velocity UMAP showing differentiation dynamics (A) and pseudotime inferred from RNA velocity (A”) in AEP organoids.
  • FIG. 3B Slingshot trajectory analysis and pseudotime inference of AEP organoids demonstrates similar lineage relationships to RNA velocity.
  • FIGs.3C-3E Lineage drivers defined by CellRank for differentiation of pAEP/AEP to AT2 cells (C), AT1 cells via AT1tr (D), and Krt8 cells (E).
  • FIG.3F Heatmap showing major cell markers differentiating cell states in alveolar epithelium.
  • FIG. 3A scVelo RNA velocity UMAP showing differentiation dynamics (A) and pseudotime inferred from RNA velocity (A”) in AEP organoids.
  • FIG. 3B Slingshot trajectory analysis and pseudotime inference of AEP organoids demonstrates similar lineage relationships to
  • FIG. 4A UMAP of cellular populations within organoids, named according to RNA integration.
  • FIGs. 4B-4C Volcano plots showing differential chromatin accessibility regions between AEP and AT2 cells (B) and AT1tr and Krt8 + transitional cells (C).
  • FIG. 4A UMAP of cellular populations within organoids, named according to RNA integration.
  • FIGs. 4B-4C Volcano plots showing differential chromatin accessibility regions between AEP and AT2 cells (B) and AT1tr and Krt8 + transitional cells (C).
  • FIG. 4D Paired heatmap of differentially accessible genomic loci in ATAC (left) and RNA expression of nearest-neighbor gene production (right) showing overview of regulators of AT1 cell differentiation (AT1 path), AT2 cell differentiation (AT2 path), and AEP state (AEP path) derived from integrated analysis. Cell populations shown along top bar, with colors the same as in (A).
  • FIGs. 4E-4F Pseudotime prediction of separate AT1 differentiation trajectories from AEPs to AT1 cells through AT1tr path (E) and PATS/DAPT path (F).
  • Figures 5A-5F An embodiment of in vitro gene editing of AEP-derived alveolar organoids via AAV6.2FF-Cre.
  • FIG 5A AAV6.2FF-Cre experimental set-up. Live/CD31-/CD45-/CD326 + (EpCAM + )/TdTomato + (Axin2 + ) cells (AEPs) sorted from mice with the R26R EYFP allele (Axin2 creERT2-tDT ; R26R EYFP ) were treated with AAV6.2FF-Cre and plated with wild-type fibroblasts.
  • FIG 5B H&E of 5 ⁇ m sections of FFPE day 29 AAV6.2FF- Cre-treated organoids, exhibiting morphology and structural complexity similar to untreated/control organoids ( Figure 1B).
  • FIG 5C Whole-well brightfield and GFP images of day 29 organoids (untreated vs.
  • FIG 5F Whole-mount immunofluorescence of day 32 AAV6.2FF-Cre-treated AEP-derived organoids (same experimental set-up as Figure 2).
  • FIG. 6A 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.
  • FIG. 6A 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
  • FIGs. 6C-6J 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.
  • 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 identify expressing CDH1.
  • Nkx2-1 KO organoids express KRT8 and many proliferate and and express Ki67 expression (J-J’’), as late as day 40 of culture.
  • 6K-6R Integrated scRNA- sequencing datasets 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 novel markers gained (N) in Nkx KO .
  • FIGs. 7A-7N An embodiment of genetic deletion of Nkx2-1 in vivo leads to loss of distal lung fate and acquisition of PATS/Krt8 + state.
  • FIGs. 7A-7B Experimental design of in vivo genetic ablation of Nkx2-1 in AEPs. Mouse genetic construct (A) and experimental treatment plan and schematic (B).
  • FIGs.7C-7N 8-12-week Tfcp2l1-CreERT2; R26R EYFP (C-F) and Tfcp2l1-CreERT2; R26R EYFP ; Nkx2-1 fl/fl (G-N) were treated with three doses of IP tamoxifen (50 mg/kg) and harvested at 2 to 4 weeks post-treatment; control is from 2-week timepoint.
  • C Control (Tfcp2l1-CreERT2; R26R EYFP ) mice exhibited YFP induction in a subset of AT2 cells (SPC + [red]/Nkx2-1 + [white]) with normal histological characteristics.
  • FIG. 8A Experimental design of in vivo genetic ablation of Nkx2-1 in AEPs prior to scRNAseq.
  • FIGs.8B-8C Common UMAP of whole lung scRNAseq from WT and Nxk2-1 KO animals. (FIG. 8C) shows cell identities using LungMAP labels.
  • FIGs.8D-8F Distal epithelial cell populations in WT (red) and Nkx2- 1KO (blue) animals; a Nkx2-1 KO specific population is present which expresses markers of Krt8+ cells but not AT1 or AT2 cells.
  • FIG. 8G-8I Overlap and label transfer of AEP-O identities to in vivo epithelial cells from FIGs. 8D-8F.
  • FIG. 8G shows reference UMAP for comparison, reproduced from Figure 6.
  • FIG. 8H shows clustering of cells from in vivo on reference UMAP, with colors per population as in (E).
  • Proportions of cells from WT and Nkx2- 1KO are show in (FIG.8I).
  • FIG.8J-8K scATACseq of AEP-O [0015]
  • Figure 9. An embodiment of a model of Nkx2-1 activity in controlling progenitor and transitional cell state.
  • AEP can differentiate to AT1 cells via either the AT1tr or Krt8 + states during homeostasis, with Nkx2-1 release from AT2 genes during transition through a Krt8 + state.
  • Nkx2-1 activity and expression are lowest in Krt8 + cells, and Nkx2-1 must re-engage chromatin to complete AT1 transition from the Krt8 + state.
  • Permanent Nkx2- 1 loss in AEPs causes transition to proliferative, stressed, Krt8+-like state characterized by unconstrained growth in vitro and in vivo. Nkx2-1 is therefore important for the progenitor activity of AEPs and supports the transition to AT1 cells by Krt8 + cells.
  • FIG. 11A-11D An embodiment of sorting gates used to isolate Axin2-positive AT2 cells (AEPs) for use in organoids.
  • AEPs Axin2-positive AT2 cells
  • cells were gated away from debris based on size (1), then single cells were identified by SSC and FSC gating (2,3).
  • Live cells were identified by Live/Dead staining (4), followed by removal of CD31- or CD45-positive cells in a dump channel (5).
  • Epithelial cells were identified by Epcam expression (6), and TdTomato-positive epithelium were sorted into complete SAGM media and used immediately for organoids or single cell RNA sequencing (Figure 13).
  • Figures 11A-11D An embodiment of TUNEL + cells largely confined to cell clumps and debris outside organoids.
  • FIG. 12A-12C UMAP projections (left) demonstrating relative detected cells for each epithelial cell state at day 14 (FIG.12A), day 21 (FIG.12B), and 28 (FIG.12C).
  • FIG.12D Quantification of cell population abundance at each time point.
  • Figures 13A-13I An embodiment of localization of mesenchymal and immune cells in wells surrounding AEP-O.
  • FIG. 13A Generation of fibroblast stocks from control (C57BL/6J) and PDGFRr ⁇ eGFP mice.
  • FIG.13B Imaging of control and GFP fibroblast stocks at P2 (second passage).
  • FIG.13C Whole-well scans comparing day 35 AEP-derived organoids grown from control and PDGFR ⁇ eGFP fibroblasts.
  • FIG. 13D Quantification comparing counts and size of day 35 AEP-derived organoids grown from control and PDGFR ⁇ eGFP fibroblasts.
  • FIG. 13E Schematic of experimental set-up of live imaging; 3D reconstruction of live day 20 organoids and PDGFR ⁇ eGFP fibroblasts stained with Hoechst; (E’) 3D reconstruction of confocal z-stacks of Matrigel/organoids with the transwell filter, showing the majority of GFP + fibroblasts are growing on the filter and not within organoids.
  • FIG.13F Whole-mount immunofluorescence of organoids grown with PDGFR ⁇ eGFP fibroblasts showing lack of PDGFR ⁇ + cells within day 35 organoids.
  • FIG. 13G Cultures with fibroblasts on the basolateral side of the filter and AEPs on the apical side of the filter do not grow organoids (G’ – 31-day culture), although fibroblasts do persist on the basolateral side of the filter after 31 days (G’’).
  • FIG.13H Confocal imaging utilizing second harmonic generation to show lack of fibrillar collagen in paraffin sections of organoids from long term culture (H’’) (injured adult mouse lung section as positive control [H’]).
  • FIG.13I Immunofluorescence staining showing presence of contaminating immune (CD45 + ) population (green) in fibroblast stocks (I).
  • (ns p > 0.05)
  • Scale bars 50 ⁇ m].
  • Figures 14A-14B An embodiment of the sorted AEP fraction contains both stressed and unstressed cells at baseline, but organoids arise from AEP-like AT2 by day 7 of organoid culture.
  • FIG. 14A scRNAseq analysis of freshly sorted AEPs. Two epithelial populations are apparent with a small mesenchymal contaminant. Right panel shows marker genes for each cell population.
  • FIG.14B scATACseq of d7 AEP-O. The majority of cells at this stage are mesenchymal, with a small epithelial population which appears most similar to the pAEP state seen in late scRNAseq. Imputed gene expression from ATAC data is shown in right panel for marker gene identification. Low level expression of Krt8 markers is present, with higher level expression of AT2 genes such as Abca3 and AEP-enriched genes in the Wnt signaling pathway. [0021] Figures 15A-15C.
  • FIG. 15A UMAP of AEP organoids used as basis of integration and labels.
  • FIGs. 15B-15C Integrated data from all three organoid datasets, labeled by cell type (FIG.15B) or dataset of origin (FIG.15C). Composition of each organoid dataset is shown in FIGs.15A’-15C’.
  • Figure 16. An embodiment of transition of SMAD-regulated gene expression in AT2 to AT1 transitions in AEP-O. Top row shows gene activity of SMAD target genes overlayed on scATACseq UMAP (compare to Figure 4A).
  • FIG.17A Time series of Nkx2-1 KO organoids in culture from day 12 to day 28.
  • FIG.17B Immunofluorescence of paraffin section from Nkx2- 1 organoid transwells showing non-recombined (Nkx2-1 + ) organoids adjacent to recombined (Nkx2-1-) organoids with atypical morphology.
  • FIGs.17C and 17J H&E of paraffin sections of Nkx2-1 KO organoids (FIG.17C) and E12.5 mouse embryos (FIG.17J).
  • FIGs.17K’-17P’ used as positive controls for protein expression. Immunofluorescence of paraffin section from Nkx2-1 KO organoids (FIGs.17D-17I) and E12.5 mouse embryos (positive controls; FIGs.17K- 17P) stained for canonical endodermal markers – Sox2 (FIGs. 17D, 17K), Sox9 (FIGs.
  • FIG. 18A An embodiment of RNA Expression of Canonical Markers in Control (Uninfected), AAV Control, and Nkx2-1 KO AEP-derived Organoids.
  • FIG. 18A Overview of cell input used to generate AEP-O and subsequent timepoints for scRNAseq.
  • FIGs. 18B-18D Repeated data from Figure 6K-M; Source (FIG. 18B), cell clusters (FIG.
  • FIG.18E-18T RNA expression of common markers of alveolar epithelial cell identity.
  • FIG.19A Experimental design to generate Nkx2-1 null AEPs using Axin2 CreERT2 .
  • FIG.19B IHC at 2 weeks post tamoxifen injection demonstrating lineage labeled AT2 cells expressing Nkx2-1 protein, suggesting incomplete knockout in the AEP lineage.
  • FIG.19A Experimental design to generate Nkx2-1 null AEPs using Axin2 CreERT2 .
  • FIG.19B IHC at 2 weeks post tamoxifen injection demonstrating lineage labeled AT2 cells expressing Nkx2-1 protein, suggesting incomplete knockout in the AEP lineage.
  • FIG.19A Experimental design to generate Nkx2-1 null AEPs using Axin2 CreERT2 .
  • FIG.19B IHC at 2 weeks post tamoxifen injection demonstrating lineage labeled AT2 cells expressing Nkx2-1 protein, suggesting incomplete knockout in the AEP lineage.
  • FIG. 19C Experimental design to evaluate efficiency of Nkx2-1 knockout in lineage labeled cells.
  • FIG. 19D Nkx2-1 expression was reduced by approximately 50% in lineage labeled cells, confirming inefficient recombination despite high dose tamoxifen via the Axin2Cre ERT2 .
  • Figures 20A-20L An embodiment of Tfcp2l1Cre ERT2 functions as an epithelial-specific method to target the AEP lineage.
  • FIG. 20A Comparison of expression level of Axin2 and Tfcp2l1 in published LungMAP data shows significant epithelial enrichment.
  • FIG. 20B-20D Comparison of lineage labeling in homeostatic lung using Axin2Cre ERT2 and Tfcp2l1Cre ERT2 .
  • FIG.20E Experimental design to compare the molecular state of Tfcp2l1-lineage cells with Axin2Cre ERT2-Tdtomato sorted AEPs.
  • FIG.20F scRNAseq of sorted AEPs, reproduced from Figure 14 for comparison.
  • FIGs. 20G-20I UMAP project of Tfcp2l1 CreERT2 x R26R EYFP whole lung scRNAseq confirms Tfcp2l1-lineage cells comprise a subpopulation of AT2 cells in adult homeostatic lung.
  • FIG. 20J-20L Comparison of molecular state of freshly sorted Axin2+ AT2 cells (from FIG. 20F) and Tfcp2l1-lineage labeled AT2 cells (from FIGs. 20G-20H). Integration of these cells leads to clustering in a single cell population, and label transfer from AEP-O scRNAseq identifies >80% of cells as in the AEP state.
  • Figures 21A-21D An embodiment of Tfcp2l1-lineage organoids form complex organoids in AEP-O culture conditions.
  • FIG. 21A Experimental design for generation of Tfcp2l1-lineage organoids.
  • FIG.21B Whole mount image showing virtually all organoids in culture derive from Tfcp2l1-lineage labeled EYFP+ cells.
  • FIGs. 21C-21D Tfcp2l1-derived organoids form complex organoids which develop complex cellular differentiation and 3D organization indistinguishable from Axin2-lineage organoids.
  • DETAILED DESCRIPTION [0028] In embodiments disclosed herein, we refined and standardized the culture conditions and inputs of co-culture of murine AT2 cells and alveolar fibroblasts. Recent data demonstrates that Wnt-responsive AT2 cells, also called alveolar epithelial progenitors (AEPs), harbor extensive progenitor capacity.
  • AEPs alveolar epithelial progenitors
  • AEPs expand rapidly, differentiate into new AT1 and AT2 cells, and repair regions of alveolar injury following epithelial loss or infectious stress.
  • AEP-derived organoids or AEP- O
  • AEP- O develop from clonal expansion of single progenitor cells, undergo progressive cellular differentiation and spontaneous cavity formation in vitro, giving rise to complex alveolar-like structures with properly polarized epithelial cells, recapitulating key aspects of the alveolar regenerative process in a flexible in vitro assay.
  • multistage single cell transcriptomics and epigenomics we defined the organoid cellular milieu, identified separable progenitor, AT2, AT1, and transitional states in organoids.
  • the primary components of organoid co-culture assays are 1) epithelial cells; 2) supportive cells, if any; 3) matrix for three-dimensional suspension and growth; 4) media and media additives; and 5) growth surface (e.g., transwell filter).
  • the AEPs form more and larger organoids than unselected AT2 cells, so in some embodiments, we used FACS-sorted AEPs (Figure 10).
  • the AEPs are derived from Axin2 CreERT2-Tdt mice as the epithelial starting fraction.
  • the fibroblasts are obtained by selective adhesion from P28 wild type C57BL/6 mice at passage 3-4.
  • SAGM Matrigel and small airway growth media
  • mesenchymal cells e.g., lung fibroblasts
  • mesenchymal cells are added to each well on a transwell filter.
  • mesenchymal cells e.g., lung fibroblasts
  • Matrigel and media optionally small airway growth media (SAGM) with about 1-10%, optionally about 5%, FBS and optionally with limited additives as disclosed herein, in a ratio of about 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, or 1:10, or a range defined by any two of the preceding values.
  • SAGM small airway growth media
  • FBS optionally with limited additives as disclosed herein, in a ratio of about 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, or 1:10, or a range defined by any two of the preceding values.
  • AEPs expand into small clusters of SFTPC + cells during the first week of culture ( Figure 1C).
  • Figure 1D By day 14 of culture, differentiation of RAGE + AT1 cells was observed within the central portion of the organoids ( Figure 1D), consistent with previous reports from murine organoids.
  • Figure 1E During the third week of culture, these developing AT1 cells begin to elongate and polarize (Figure 1E), and by day 14 of culture cavities are present within the organoids ( Figure 1B, F).
  • the AEP-O comprise AEP cells, AT1 cells, and AT2 cells, optionally wherein the AEP, AT1 and AT2 cells express markers disclosed herein.
  • the AEP-O comprises RAGE + AT1 cells.
  • the AEP-O comprise cavities, optionally forming alveolar-like structures.
  • the AEP-O comprises mature and/or polarized AT1 cells within the central portion of the organoid, optionally wherein AT2 cells are intermixed with the mature and/or polarized AT1 cells.
  • the AEP-O comprise cavities with epithelial lining that comprises AT2 cells, optionally containing lamellar bodies with the apical surface directed towards the internal lumen.
  • the AEP-O comprises active surfactant secretion.
  • AEP-O maturation is driven by mesenchymal paracrine signaling without direct mechanical contribution of mesenchymal cells. [0031] To better characterize the progressive cellular maturation occurring during paired cell differentiation and cavity formation in AEP-O, we performed single cell RNA sequencing at 14, 21, and 28 days after culture initiation. We identified clear epithelial and mesenchymal fractions, as well as an unexpected immune fraction (Figure 2A-C).
  • PDGFR ⁇ eGFP + fibroblasts localize predominantly in two locations – with the minority of cells surrounding the epithelial organoids suspended in matrigel, and the majority growing on the transwell filter in a monolayer ( Figures 2F-2H, Figures 13E-13F). Few PDGFR ⁇ eGFP + cells were detected within organoids ( Figure 2F-2G) and no clear deposition of fibrillar collagen (types I and II) was seen within organoids ( Figure 13H), suggesting that the morphological maturation and complex structural organization of AEP-O did not require direct mesenchymal cell localization within the organoid itself.
  • the AEP-O comprises mesenchymal cells, e.g., fibroblasts. In some embodiments, the AEP-O does not comprise mesenchymal cells, e.g., fibroblasts. In some embodiments, the AEP-O does not comprise fibillar collagen type I and/or type II. In some embodiments, mesenchymal cells, e.g., fibroblasts, are near and/or adjacent to the AEP-O.
  • the AEP- O is cultured in a container comprising a monolayer of mesenchymal cells, e.g., fibroblasts.
  • the AEP-O comprises immune cells.
  • the AEP-O does not comprise immune cells.
  • immune cells are near and/or adjacent to the AEP-O.
  • the AEP-O are cultured in a container comprising a monolayer of fibroblasts comprising immune cells.
  • AT1 cells expressed extensive WNT ligands with predicted receptivity in both WNT-responsive AT2 cells and multiple mesenchymal populations. They also had high level expression of AT1 also produced PDGF ligands predicted to signal to the PDGFR ⁇ + mesenchyme.
  • Mesenchymal cells expressed HGF, non-canonical WNT, and FGF ligands, consistent with published data describing roles of these pathways in alveolar regeneration. Together, these data suggest that the AEP-O signaling milieu recapitulates key aspects of the in vivo regenerative niche, and that the mesenchymal cells provide primarily a supportive paracrine signaling niche important for alveolar cavity formation.
  • mesenchymal cells cultured with the AEP-O are WNT-responsive, PDGFR ⁇ + , express HGF, express non-canonical WNT, and/or express FGF ligands.
  • scRNAseq defines separable epithelial maturation trajectories of AEPs toward AT1 and AT2 cells within alveolar organoids.
  • the AEP-O comprises AEP cells expressing one or more of the AEP-enriched markers Id2, Ctnnb1, Lrp5, Lrp2, Napsa, Bex2, Hdc, and Fgfr2.
  • the AEP-O comprises AEP cells expressing high levels of cycle genes (pAEPs).
  • the larger epithelial fraction corresponded to AT2 cells bearing the AEP signature and a second smaller state contained cells in the Krt8+ stressed transitional state (also referred to herein as “Krt8 + /PATS/DATP/ADI” cells) ( Figure 14A), suggesting that process of digestion and sorting of Axin2+ AT2 cells causes cell stress.
  • Krt8 + /PATS/DATP/ADI Krt8 + /PATS/DATP/ADI
  • AT2tr AT2 transitional intermediate state
  • Figure 3C The AT2tr state is defined by high level expression of glutathione pathway genes and shift towards lipid metabolism, with the AT2 state expressing high levels of mature AT2 markers including Sftpa1 and Lys2 in addition to Sftpc and Sftpb, which are expressed broadly within multiple differentiating AEP states ( Figure 2C, 3F).
  • the AEP-O comprises AT2tr cells, optionally comprising high level expression of glutathione pathway genes and a shift towards lipid metabolism.
  • the AEP-O comprises mature AT2 cells expressing one or more markers selected from Sftpa1, Lys2, Sftpc and Sftpb. These predictions correspond to the lineage trajectories implied by the progression of organoid composition shown in the time course data underlying the combined set ( Figures 12A-12D). [0037] We next examined AT1 differentiation in AEP-O.
  • RNA markers of the AT1tr state included Hes1 and Igfbp7, and RNA velocity analysis showed decreasing AT2 gene expression and intermediate expression of AT1 markers within this population ( Figure 3D-F). Label transfer and integration with published organoid data sets showed AT1tr and Krt8+ cell states were detectable in published data ( Figures 14A-C).
  • the AEP-O comprises Krt8 + transition cells (Krt8 + ) expressing one or more markers selected from Krt8, Lgals3, Tp53, Nupr1, Ddit3, and Cldn4, or optionally expressing one or more markers selected from Krt8, Lgals3, Tp53, and Cldn4.
  • the AEP-O comprises AT1 transition (AT1tr) cells expressing one or more markers selected from Hes1 and Igfbp7.
  • AEP chromatin state is defined by a progenitor-enriched transcriptional regulator network.
  • TRN transcriptional regulatory network
  • RNA expression and chromatin accessibility can improve TRN predictions by reducing both false positive and false negative regulatory predictions; application to single cell techniques have extended the power of these approaches to estimate TF regulators of individual cell states. Therefore, we performed TRN inference comparing regulatory networks of various cell states within AEP-O to identify differential TF activity and predict regulators of state transitions (Figure 4G-K). Focusing on the core TFs specific to each TRN, we found that AEP regulators included Nkx2-1 and Tfcp2l1, both enriched in expression in bulk RNAseq from AEPs, as well as TFs modulating Wnt (Tcf4) and BMP (Smad1/4) activity concordant with known AT2 progenitor signaling response (Figure 4H).
  • the top regulators in Krt8 + cells are Atf4 and its target Ddit3, the gene encoding C/EBP homologous protein (CHOP) ( Figure 4J); together these factors are activated by the multiple inputs of the integrated stress response (ISR), with CHOP implicated in regulation of checkpoints in apoptosis vs cellular differentiation in other systems.
  • ISR integrated stress response
  • CHOP integrated stress response
  • the ISR is activated in alveolar epithelium following ventilator-induced lung injury, and ISR activation contributes to lung fibrosis; Atf4 and CHOP activation underlies Krt8+ cell accumulation in fibrosis.
  • the AT1tr TRN showed multiple distinct factors which are shown for comparison ( Figure 4K and Figure 16); given the overlap of these factors with both AT2 and AT1 cells, testable regulators of the AT1tr state were not clearly identifiable in our dataset.
  • Loss of Nkx2-1 activity defines the Krt8 + transitional cell TRN.
  • Recent epigenomic profiling of the activity of Nkx2-1 during AT2 to AT1 transitions demonstrated Nkx2-1 occupancy at different genomic regions in each cell state, suggesting a role for Nkx2- 1 dis-engagement and re-engagement in the genome during differentiation.
  • Nkx2-1 expression is lowest in stressed transitional epithelial cells at the time when Krt8 expression is highest during in vivo lung regeneration. Together, these observations supported the hypothesis that lack of Nkx2-1 activity promotes transition to the stressed Krt8 + transitional state. [0042] To validate this observation, we developed an approach to genetically manipulate AEPs during development of AEP-O (Figure 5). Using an AAV6.2FF-Cre, which has recently been described as a high-fidelity reagent for genetic manipulation of AT2 cells in vivo, we infect AEPs harboring a R26R-lox-stop-lox-EYFP allele (from R26R EYFP mice) immediately after FACS sorting (Figure 5A).
  • AAV6.2FF-Cre efficiently targeted AEPs, which produced morphologically complex organoids (Figure 5B) expressing the EYFP lineage label ( Figure 5C).
  • MOI 1000 led to targeting of approximately 60% of organoids with no change in colony formation efficiency or size of organoids (Figure 5E); whole mount IHC confirmed EYFP expression with no reduction in internal complexity of AEP-O ( Figure 5F).
  • AAV6.2FF-Cre was capable of efficiently targeting AEPs in vitro for genetic manipulation without perturbing the AEP-O system, and confirmed the clonal nature of AEP-derived organoids.
  • Nkx2-1 deficient AEPs transition to the Krt8+ stressed transitional cell state.
  • AAV6.2FF-Cre was applied to AEPs from Axin2 CreERT2-Tdt x R26R EYFP x Nkx2- 1 flox/flox animals to generate Nkx2-1 knockout AEPs which were used to initiate organoid 6A).
  • Nkx2-1 KO AEPs lost expression of AT2 markers, including Sftpc, with associated increased expression of E-cadherin (Cdh1) and change in cell shape and organoid morphology (Figure 6F-H).
  • Diverse morphological types were visible in Nkx2-1 KO AEP-O, with loss of alveolar-like cavities, prominence of one or a small number of large cavities full of debris, and pseudostratified epithelial lining with some organoids exhibiting a glandular appearance (Figure 6F,I).
  • the AEP-O comprises at least one alveolar-like cavity filled with debris, a pseudostratrified epithelial lining, and/or a glandular-like appearance. In some embodiments, the AEP-O does not comprise substantial expression of one or more foregut endoderm markers selected from Sox2, Sox9, Cdx2, Gata4, and Pdx1.
  • Sox2, Sox9, Cdx2, Gata4, and Pdx1 selected from Sox2, Sox9, Cdx2, Gata4, and Pdx1.
  • EYFP + Nkx2-1 KO cells form multiple distinct clusters separated from Nkx2-1 expressing cells in both EYFP- cells of the same wells and the two control libraries (wild-type/uninfected and AAV control) (Figure 6K).
  • Nkx2-1 KO cells clustered near WT Krt8 + transitional epithelial cells than other control clusters ( Figure 6L) and expressed high levels of markers of Krt8 + /PATS/DATP/ADI state including Cldn4 and Tff2, while expressing intermediate levels of Lgals3 ( Figures 6N, 18Q-18T).
  • a distinct proliferative cluster is present by scRNAseq among Cldn4-high Nkx2-1 KO cells ( Figure 6K).
  • Ki67 expression in Krt8 + Nkx2-1 KO AEP-O confirms ongoing proliferation at d40 of culture despite their large size (Figure 6J).
  • Seurat module scoring Given the similarities of Nkx2-1 KO epithelial cells to the Krt8 + state in Nkx2-1 WT AEP-O, we used Seurat module scoring to compare Nkx2-1KO and WT cells.. Cells in Nkx2-1 KO AEP-O lost AEP-associated gene expression ( Figure 6O) while activating cell stress markers associated Krt8 + cells in WT AEP-O.
  • Nkx2-1 KO AEP-O were highly enriched for gene sets associated with human lung adenocarcinoma, concordant with previous findings implicating Nkx2-1 loss in the pathogenesis of lung cancer. While we did not detect non-lung foregut endoderm markers by IHC, Nkx2-1 KO epithelial cells did express low levels of non-lung endodermal genes at the RNA level,consistent with loss of instructive activity of Nkx2-1 in constraining lung epithelial cell fate. Taken together, these data supported the conclusion that Nkx2-1 loss in AEPs led to acquisition of the Krt8 + /PATS/DATP/ADI cell state, validating the prediction of the Krt8 + cell state TRN analysis.
  • Nkx2-1 loss is sufficient to cause acquisition of the Krt8 + /PATS/DATP/ADI cell state in AEPs.
  • Tfcp2l1-lineage AT2 cells efficiently form complex organoids indistinguishable from those generated by sorted Axin2-positive AEPs.
  • Tfcp2l1 CreERT2 constituted an AEP-enriched inducible Cre line suitable for epithelial specific knockout of Nkx2-1.
  • Nkx2-1 knockout in AEPs we detected multi-cell clones of lineage labeled EYFP + , Nkx2-1 KO epithelial cells throughout the lung (Figure 7F-I).
  • Nkx2-1 KO Tfcp2l1- lineage cells in vivo lost expression of AT2 markers including Sftpc underwent change in cell shape and morphology, and acquired an E-cadherin high, Krt8-expressing, proliferative state (Figure 7J-M), consistent with changes seen in Nkx2-1 KO organoids.
  • Krt8 + clones have grown substantially, with persistent shape change and high-level proliferation (Figure 7N-U).
  • the AEPs are from Tfcp2l1 CreERT2 x R26R EYFP x Nkx2-1 flox/flox animals.
  • Combined scRNAseq and scATACseq of AEP-derived alveolar organoids allowed definition of regulatory networks along multiple differentiation trajectories of lung epithelial progenitor cells toward differentiated alveolar epithelium.
  • Nkx2-1 is important for maintaining the AEP state, and loss of Nkx2-1 activity is sufficient for AEPs to enter the Krt8+ stressed transitional state. Nkxk2-1 therefore plays an importnat, previously unrecognized role in the maintenance of progenitor function in the adult lung.
  • Nkx2-1 loss is sufficient to cause transition of AEPs to a proliferative stressed transitional state, a finding which emphasizes the need for active maintenance of adult AT2 alveolar progenitor capacity.
  • Prior reports have demonstrated a requirement for Nkx2-1 expression in maintenance of lung epithelial fate in adult differentiated cells. Loss of one or more alleles of Nkx2-1 is a common mutation found in lung adenocarcinoma.
  • Nkx family factors are known to function as pioneer transcription factors in diverse contexts, and pioneer factors catalyze changes in chromatin structure that maintain epigenetic stability. Evaluation of epigenomic state enriches single cell RNA evaluation of multiple similar cell states.
  • Nkx2-1 as a regulator of AEP progenitor state relied on the ability to distinguish similar cell states in a dynamic system.
  • Our data from AEP-O provided two useful adjuncts to traditional single cell analysis: known input/initial cell state in a clonal culture(obtained by sorting pure AEPs), and scATACseq to add epigenomic state data for comparison to matched RNA transcriptomes. These additions provided multiple benefits.
  • the known initial cell state and clonal nature of AEP-O provided a clear ability to identify the starting point of differentiation. Identification of initial root cell in trajectory, pseudotime, and RNA velocity analyses can be challenging and the shortfalls can be mitigated by controlling input.
  • Krt8 + stressed transitional cells have been described in diverse models, are found in mouse and human, are increased in several disease states, and are readily identifiable based on high level expression of enriched markers.
  • our data shows that permanent acquisition of a stressed transitional state, such as seen in Nkx2-1 KO organoids and Nkx2-1 KO AEPs in vivo, drives aberrant proliferation, expression of lung cancer programs, and loss of lung identity. Progressive acquisition of this cell state is deleterious; even if most cells pass through or ‘recover’ from this stressed transitional state, accumulation of ‘stuck’ transitional cells represents a risk factor for development of lung disease.
  • AEP-Os are a high-fidelity model of alveolar epithelial regeneration in a dish.
  • Development of new therapeutics to promote functional lung regeneration will benefit from high fidelity in vitro models of the regenerative process. Restoration of a gas exchange surface through repair or replacement of injured alveoli is the central process needed to promote therapeutic regeneration.
  • AEP-derived lung organoids recapitulate the key aspects of the epithelial portion of the alveolar regenerative process, modeling progenitor cell expansion, alveolar epithelial differentiation, and formation of alveolar-like cavities with properly polarized and functional epithelium.
  • Nkx2-1 deletion caused concordant changes both in organoids and in vivo, providing proof of principle that AEP-O model key aspects of adult alveolar biology and alveolar regeneration.
  • the cultures described are generated from a clearly defined epithelial cell input, are clonal, and show high level reproducibility after optimization of matrix, mesenchymal components, and media conditions.
  • Single cell analysis demonstrated that AEP-O recapitulate key aspects of in vivo epithelial differentiation and epithelial/mesenchymal interactions.
  • TRN inference from these data implied a key role for Nkx2-1 in detected AT1 differentiation states, which was validated using both in vitro and in vivo lineage tracing and genetic targeting.
  • AEP- O will allow screening, molecular testing, and genetic manipulation via AAV6.2FF-Cre in a system which provides a balance between fidelity and reproducibility. While we characterized AEP-O only from mice in this study, in some embodiments the cells (AEP and/or mesenchyme cells) are human, resulting in human AEP-Os. [0053] Controversy exists regarding the similarity of alveolar regeneration and alveolar development; some have argued that regeneration is fundamentally different due to the altered milieu of the injured alveolus, or even suggested that pathological remodeling rather than functional regeneration is the end state of a significant portion of lung injury .
  • AEPs contain the required information to undergo progenitor self-renewal, multilineage differentiation, and complex morphogenesis in the presence of a minimal signaling niche. This challenge bears striking resemblance to the injured lung, where epithelial, mesenchymal, endothelial, immune lineages and the underlying matrix environment are all altered by pathogens.
  • AEP-driven cavity formation occurs in the absence of mechanical contribution from myofibroblasts, quite different from during alveologenesis when the mechanical activity of myofibroblasts is required for formation of alveoli.
  • mice All experiments for both organoids and in vivo lineage tracing included both male and female mice.
  • IP intraperitoneally
  • Tamoxifen Sigma, T5648; dissolved in ethanol and resuspended in corn oil
  • Mouse Lung Harvest Mice were anesthetized via intraperitoneal Ketamine + Xylazine followed by euthanasia via cervical dislocation and thoracotomy. The chest cavity was opened to expose the heart and lungs.
  • the right ventricle was perfused with 5-10 mL of cold PBS (Gibco, 10010-023) to clear blood from the lungs.
  • cold PBS cold PBS
  • lungs were removed and placed in cold PBS on ice.
  • tissue fixation for histology and immunofluorescence the trachea was cannulated and lungs were inflated to a pressure of 30 cm H 2 O using 4% paraformaldehyde (PFA). Inflated lungs were immersed in a conical of 4% PFA, then left on a rocker at 4°C overnight.
  • PFA paraformaldehyde
  • the cassettes were washed (15 minutes each) 3x in DEPC-treated PBS, 1x in DEPC-treated 30% ethanol, 1x in DEPC-treated 50% ethanol, and 3x in DEPC-treated 70% ethanol. Following a standardized overnight automated processing protocol (Thermo Scientific, Excelsior ES), the samples were embedded in paraffin. Samples were sectioned at a thickness of 5 ⁇ m. Paraffin sections were incubated at 65°C for two hours, deparaffinized in xylene (3x for 10 minutes), rehydrated through an ethanol gradient, and standard H&E staining was performed.
  • lungs were removed from ice cold PBS and non-pulmonary tissue and gross airways were removed via manual dissection, and lung tissue was finely chopped and transferred to a GentleMACS C tube (Miltenyi Biotec, 130-093-237) (tissue from one mouse per C tube) containing 5 mL of digestion buffer [composed of 9 mL of phosphate-buffered saline (PBS; Gibco, 10010-023) combined with 1 mL of Dispase (stock: 50 U/mL; final concentration: 5 U/mL, Corning, 354235), 50 ⁇ L of DNase (stock: 5 mg/mL; final concentration: 0.025 mg/mL or 50 U/ml, GoldBio, D-301), and 100 ⁇ L of Collagenase Type I (stock: 48,000 U/mL; final concentration of 480 U/mL, Gibco, 17100-017)].
  • digestion buffer composed of 9 mL of phosphate-buffered saline (PBS; Gibco,
  • Fibroblast Stock Preparation (with PDGFR ⁇ eGFP fibroblasts)/Media: For generation of fibroblast stocks, 4-week C57BL/6J mice and 4-week PDGFR ⁇ eGFP mice lungs were harvested, digested, and processed as described above. Following centrifugation, cells were washed 3x with MACS Buffer (autoMACS Rinsing Solution [Miltenyi Biotec, 130-091- 222] with MACS BSA Stock Solution [Miltenyi Biotec, 130-091-376]).
  • MACS Buffer autoMACS Rinsing Solution
  • MACS BSA Stock Solution [Miltenyi Biotec, 130-091-376]
  • the cell pellet was resuspended in 10 mL fibroblast medium (DMEM/F-12 [Gibco, 11320-033], Antibiotic-Antimycotic [Gibco, 15240-062, final concentration 1x], and Heat Inactivated Fetal Bovine Serum [Corning, 35-011-CV, final concentration 10%]) and plated on a 10 cm tissue culture plate (approximately 1 mouse per plate). Non-adherent cells were removed via media change 2-12 hours post-plating. [0060] Cells were passaged at 80% confluency to P3.
  • Cells were centrifuged at 500g for 5 minutes at 4°C, supernatant was removed, and cell pellet was resuspended in 2 mL fibroblast medium and transferred to a 10 cm tissue culture plate containing 6 mL of fibroblast medium. Fibroblasts used for organoids were washed, trypsinized, and resuspended (as described for passaging) before counting.
  • CD31 PECAM-1; Monoclonal Antibody [390], eFluor 450) (Invitrogen, 48-0311-82)
  • CD45 Monitoringoclonal Antibody [30-F11], eFluor 450) (Invitrogen, 48-0451-82)
  • CD326 EpCAM; Monoclonal Antibody [G8.8], APC) (Invitrogen, 17-5791-82).
  • the live/CD31-/CD45-/CD326 + (EpCAM + )/TdTomato + (AEP) population was sorted into a tube containing ‘spiked’ SAGM organoid medium (see ‘Organoid Medium’ section below) at 4°C, using a BD FACSAria Fusion cell sorter with a 100 ⁇ m nozzle. Approximately 10 5 AEPs have been sorted from one mouse using this protocol.
  • AEPs live/CD31-/CD45- /CD326 + [EpCAM + ]/TdT + cells
  • Axin2 creERT2-tDT mice were counted using a hemocytometer and resuspended in ‘spiked’ SAGM at a concentration 500 cells/ ⁇ L.
  • Fibroblasts were prepared (as described above), counted, and resuspended in ‘spiked’ SAGM at a concentration of 5000 cells/ ⁇ L. For the remaining steps, it was extremely important that all reagents are kept cold/on ice and that bubbles were not introduced to mixtures when pipetting.
  • Corning Matrigel GFR Membrane Matrix (Corning, 356231) was added to the cell mixture (45 ⁇ L per well, 1:1 ratio of SAGM to Matrigel) and carefully mixed, then placed back on ice.
  • 90 ⁇ L of the combined cell/Matrigel mixture was pipetted carefully directly into the center of the transwell (placed in the companion plate) without introducing bubbles.
  • Organoid plates were incubated at 37°C for 15 minutes, then 500 ⁇ L of ‘spiked’ SAGM supplemented with ROCK Inhibitor/Y-27632 Dihydrochloride (Sigma, Y0503, final concentration 0.01 mM) was added beneath the transwell insert.
  • AAV6.2FF-Cre Organoids Plating/Maintenance AAV6.2FF-Cre (titer of 2.779x10 10 viral genomes [vg]/ ⁇ L) was generated and characterized in vivo as previously described. Working dilutions (2.779x10 9 vg/ ⁇ L, 2.779x10 8 vg/ ⁇ L, and 2.779x10 7 vg/ ⁇ L) were generated via serial dilution of viral stocks in ‘spiked’ SAGM and frozen -80°C in single use aliquots.
  • AEPs live/CD31-/CD45-/CD326 + [EpCAM + ]/TdT + cells) sorted from Axin2 creERT2-tDT ; R26R EYFP mice were counted using a hemocytometer and resuspended in ‘spiked’ SAGM at a concentration 1000 cells/ ⁇ L.
  • the total cells needed for the desired number of wells were transferred to a new 1.5 mL tube (i.e., 10 wells ⁇ 50000 cells ⁇ 50 ⁇ L cells [1000 cells/ ⁇ L]).
  • Total cell number per tube, desired MOI i.e., 1000, 10000, 20000
  • known viral titers were used to calculate the volume of needed viral stocks.
  • the calculated volume of virus was added to each cell mixture, mixed, and incubated on ice for 60 minutes. Following viral incubation, ‘spiked’ SAGM and fibroblasts (5000 cells/ ⁇ L) were added to create a mixture with the same proportions of cells as described above for standard plating of organoids (i.e., for each well – 5000 AEPs + 50000 fibroblasts in 45 ⁇ L ‘spiked’ SAGM).
  • the cell mixture was mixed with Matrigel (45 ⁇ L/well) and plated/maintained as described above for standard organoids.
  • Organoid Plating with Fibroblasts on Basolateral Side of Transwell One day prior to organoid plating, fibroblasts were prepared (as described above), counted, and resuspended at a concentration of 50000 cells in 100 ⁇ L in fibroblast medium. Transwells were placed in the wells of the companion plate, then the plate was flipped so the transwells rested on the inside of the lid. The companion plate was removed exposing the basolateral side of the transwells/filters.100 ⁇ L of the resuspended fibroblast mixture was added to the basolateral side of each transwell filter.
  • the plate base was placed back on top of the transwells, and was incubated (basolateral side up) at 37°C and 5% CO2 for 4 hours. After incubation, the 100 ⁇ L of medium was removed via gentle pipetting (without disturbing the filter) and the plate was flipped to the standard orientation.
  • the transwells were washed with 500 ⁇ L of DPBS (beneath the transwell insert) then moved to a fresh well/plate with 500 ⁇ L fibroblast medium beneath the transwell insert.
  • Transwells were washed 5x (above and below) with PBS. Using a small knife or scalpel, the transwell filter and Matrigel plug/organoids were cut out of the transwell and placed on parafilm. Using forceps, the transwell filter was carefully removed from the Matrigel plug/organoids [note: older organoid cultures are more likely to adhere to the filter]. Using a transfer pipet, HistoGel (Epredia, HG4000012) (pre-heated to a liquid consistency) was added on top of the Matrigel plug/organoids until covered on all sides. Once solidified ( ⁇ 15-30 minutes), the sample was transferred to a tissue processing cassette (Fisher, 15-182-702A).
  • Conicals were filled to 10 mL with ice cold PBS and centrifuged at 70g for 5 minutes at 4°C. The supernatant was removed very carefully [note: if the organoid pellet is not compact/tight, the entire pellet may be lost with suction due to loose matrix]. If Matrigel was still visible, the organoid pellet was gently resuspended in 1 mL of ice cold 1% PBS-BSA and centrifuged again at 70g for 5 min at 4 °C.
  • the organoid pellet was resuspended in 1 mL of 4% PFA and incubated at 4°C for 45 minutes (resuspending once halfway through incubation).
  • conicals were filled to 10 mL with 0.1% PBS-Tween and incubated overnight at 4°C (alternate permeabilization option: for Click-iT protocols or shorter permeabilization, remove PFA and incubate in 0.25% Triton X-100 for 20 minutes at room temperature).
  • Primary antibodies were diluted to a final concentration of 1:100 in 5% Normal Donkey Serum in 0.1% PBS-Triton X-100 (approximately 200-250 ⁇ L total) and incubated overnight at 4°C on an orbital shaker.
  • a ‘quick wash’ was defined as adding 1 mL of organoid wash buffer (0.2% BSA, 0.1% Triton X-100 in PBS) and immediately allowing organoids to settle/removing the wash
  • a ‘long wash’ was defined as adding 1 mL of organoid wash buffer placing the plate on an orbital shaker for 1-2 hours before allowing organoids to settle/removing the wash.
  • organoids were gently resuspended in room temperature fructose- glycerol clearing solution (60% vol/vol glycerol + 2.5 M fructose). Depending on organoid volume, ⁇ 50-200 ⁇ L of clearing solution was used. Organoids were left to clear for at least 1 day (and as long as several months) at 4°C before mounting. [0078] Prior to preparing slides, cleared organoids were allowed to equilibrate to room temperature. Organoids were mounted as described previously – briefly, two pieces of double-sided tape were applied to a microscope slide approximately 25-30 mm apart, perpendicular to the length of the slide (for larger organoids, additional layers of tape can be used).
  • Hoechst and Live Imaging Preparation For live imaging of organoids grown with PDGFR ⁇ eGFP fibroblasts, Hoechst 33342 (Invitrogen, H3570) was diluted 1:10000 in ‘spiked’ SAGM and 500 ⁇ L was added above and below the transwell and incubated at 37°C for 30-45 minutes. Using a small knife or scalpel, the transwell filters and Matrigel plug/organoids were cut out of the transwells and placed into a coverslip bottom dish (MatTek, P35G-1.5-20-C). For some samples, the entire Matrigel plug/filter was imaged, and for others the Matrigel plug and filter were separated and imaged independently.
  • Imaging Brightfield H&E images were acquired on a Nikon Eclipse NiE Upright Widefield Microscope (Nikon DS-Fi3 Camera – with a Plan Apo VC 20x DIC N2 objective).
  • Fluorescent images were acquired on Nikon A1 inverted LUNV and Nikon A1R inverted LUNV confocal microscopes using the following objectives: Plan Apo ⁇ 10x, Plan Apo ⁇ 20x, Apo LWD 20x WI ⁇ S (water immersion), Apo LWD 40x WI ⁇ S DIC N2 (water immersion), and SR HP Plan Apo ⁇ S 100xC Sil (silicone immersion).
  • Second harmonics images were obtained using a Nikon FN1 Upright Multiphoton microscope using the following objectives: Plan Apo VC 20x DIC N2 and Apo LWD 25x 1.10W DIC N2. Images were processed in Nikon Elements with minimal, global adjustment of LUTs for acquired channels.
  • Organoid Quantification Z-projections of stitched 4x images from each well were loaded into a custom FIJI-macro (run in FIJI/ImageJ v1.53) to count organoids per well, GFP + organoids per well, and organoid area.
  • This macro allowed for batch analysis of each experiment, reducing subjectivity of counts. Briefly, given specific input parameters, the macro contained commands to: set the scale based on the diameter of each transwell, subtract background, adjust image threshold, convert to mask, analyze particles/count objects meeting a specific threshold, and export data. Data was imported into GraphPad Prism 9.0 for analysis.
  • Electron Microscopy Fixation, sectioning, and acquisition of electron micrographs of alveolar cells was performed as previously described.
  • Organoid Dissociation and Preparation of Single Cell Suspension for scRNA-seq and scATAC-seq Transwells were washed (above and below) with 1 mL of PBS.
  • organoid digest buffer (Dispase [Corning, 354235, undiluted, 50 U/mL], DNase I [GoldBio, D-301, final concentration 5 U/mL], Collagenase Type I [Gibco, 17100017, final concentration 4800 U/mL)]) was added and Matrigel plugs were gently disrupted and pipetted using a cut or wide-bore pipette tip. Organoids were incubated in digest buffer for 30 minutes at 37°C. Following incubation, the digested organoid mixture was pipetted several times and transferred to a low-binding 1.5 mL tube (3 wells of same experimental condition combined into each tube).
  • Samples were washed 2x in 1 mL of cold 0.04% PBS-BSA and centrifuged at 500g for 5 minutes at 4°C. Following removal of the supernatant, the samples were resuspended in 100 ⁇ L of 0.04% PBS-BSA. Prior to filtering cells, 40 ⁇ m Flowmi Cell Strainers (Bel-Art, H13680-0040) were equilibrated by passing 100 ⁇ L of 0.04% PBS-BSA through the strainer using a P1000 pipette tip. The 100 ⁇ L cell suspension was then pipetted through the 40 ⁇ m Flowmi Cell Strainer.
  • Bel-Art, H13680-0040 Prior to filtering cells, 40 ⁇ m Flowmi Cell Strainers (Bel-Art, H13680-0040) were equilibrated by passing 100 ⁇ L of 0.04% PBS-BSA through the strainer using a P1000 pipette tip. The 100 ⁇ L cell suspension was then
  • Nuclei Isolation from Organoids for scATAC-seq Using the same filtered cell suspension generated for scRNA-seq, the standard 10x Genomics protocol for ‘Nuclei Isolation for Single Cell ATAC Sequencing’ (CG000212 Revision B) was followed. Briefly, the single cell suspension was centrifuged at 500g for 5 minutes at 4°C.
  • ATAC lysis buffer from standard 10x Genomics protocol, CG000212 Revision B
  • ATAC wash buffer from standard 10x Genomics protocol
  • Flowmi Cell Strainers Prior to filtering nuclei, 40 ⁇ m Flowmi Cell Strainers were equilibrated by passing 100 ⁇ L of nuclei buffer through the strainer using a P1000 pipette tip. The 100 ⁇ L of nuclei suspension was then pipetted through the 40 ⁇ m Flowmi Cell Strainer. Nuclei were counted manually using a hemocytometer and resuspended at a concentration of 5000 nuclei/ ⁇ L prior to processing for scATAC-seq.
  • Sequencing/Library Preparation From each single cell or single nuclear preparation described above, a maximum of 16,000 cells or nuclei were loaded into on channel of a 10x Genomics Chromium system by the Cincinnati Children’s Hospital Medical Center Single Cell Sequencing Core. Libraries for RNA (v3) and ATACseq (v2) were generated following the manufacturer’s protocol. Sequencing was performed by the Cincinnati Children’s Hospital DNA Sequencing Core using Illumina reagents. Raw Sequencing data was aligned to the mouse reference genome mm10 with CellRanger 3.0.2 to generate expression count matrix files.
  • a YFP contig was added to the mm10 genome following 10x Genomics “Build a Custom Reference” instructions(https://support.10xgenomics.com/single-cell-gene- expression/software/pipelines/latest /using /tutorial_mr) with modifications.
  • a custom EYFP .fasta file was generated using the EYFP segment (682-1389) of the pEYFP-N1 plasmid sequence available through Addgene. This sequence was integrated into the standard mm10 assembly available from Ensembl to create a reference compatible for alignment with the CellRanger pipeline described above.
  • RNAseq Analysis and Visualization For RNAseq analysis, output data from CellRanger was partitioned into spliced and unspliced reads using Velocyto. Velocyto output files were loaded into Seurat 4.0 using SeuratWrappers and SeuratDisk using the ReadVelocity command and spliced transcripts were used as the expression input to SCTransform. Cells with less than 2000 or more than 8000 features were filtered and cells were clustered using the standard Seurat workflow. Putative doublets were identified and removed using DoubletFinder, and libraries from individual time points and treatments were integrated using SelectIntegrationFeatures and IntegrateData commands in Seurat.
  • the Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment. Immunity 49, 764-779.e769, doi:10.1016/j.immuni.2018.09.020 PMID - 30332632 (2016). 71 Tata, P. R. et al. Developmental History Provides a Roadmap for the Emergence of Tumor Plasticity. Dev Cell 44, 679-693.e675, doi:10.1016/j.devcel.2018.02.024 PMID - 29587142 (2018). 72 Laughney, A. M. et al. Regenerative lineages and immune-mediated pruning in lung cancer metastasis.
  • DoubletFinder Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Systems 8, 329-337.e324, doi:10.1016/j.cels.2019.03.003 PMID - 30954475 (2019).
  • Katsura, H. et al. Human lung stem cell-based alveolospheres provide insights into SARS-CoV-2 mediated interferon responses and pneumocyte dysfunction.
  • LungGENS' a web-based tool for mapping single-cell gene expression in the developing lung. Thorax 70, 1092-1094, doi:10.1136/thoraxjnl-2015-207035 (2015). 95 Du, Y. et al. Lung Gene Expression Analysis (LGEA): an integrative web portal for comprehensive gene expression data analysis in lung development. Thorax 72, 481- 484, doi:10.1136/thoraxjnl-2016-209598 (2017). 96 Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell reprogramming. Genes Dev 28, 2679-2692, doi:10.1101/gad.253443.114 (2014). 97 Mayran, A.

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Abstract

L'invention concerne des organoïdes alvéolaires qui récapitulent des aspects importants de régénération épithéliale pulmonaire in vivo, fournissant un modèle tractable pour disséquer des processus régénératifs. L'expansion clonale d'AEP uniques a généré des organoïdes alvéolaires complexes avec une organisation interne étendue. L'immunohistochimie d'échantillon entier de ces organoïdes met en évidence des cellules AT1 et AT2 correctement modelées, polarisées et fonctionnelles entourant de nombreuses cavités de type alvéolaire avec une contribution structurale minimale à partir de cellules mésenchymateuses, impliquant une fonction de régénération autonome cellulaire étendue codée dans des AEP adultes.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20210030811A1 (en) * 2019-07-26 2021-02-04 The Children's Medical Center Corporation Use of alveolar or airway organoids for the treatment of lung diseases and disorders
US20210395695A1 (en) * 2018-12-20 2021-12-23 Korea Research Institute Of Chemical Technology Method for fabrication of three-dimensional lung organoid comprising human stem cell-derived alveolar macrophage
WO2023030158A1 (fr) * 2021-08-30 2023-03-09 Versitech Limited Organoïdes alvéolaires, leurs procédés de fabrication et leurs utilisations

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210395695A1 (en) * 2018-12-20 2021-12-23 Korea Research Institute Of Chemical Technology Method for fabrication of three-dimensional lung organoid comprising human stem cell-derived alveolar macrophage
US20210030811A1 (en) * 2019-07-26 2021-02-04 The Children's Medical Center Corporation Use of alveolar or airway organoids for the treatment of lung diseases and disorders
WO2023030158A1 (fr) * 2021-08-30 2023-03-09 Versitech Limited Organoïdes alvéolaires, leurs procédés de fabrication et leurs utilisations

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Title
HU YAN, NG-BLICHFELDT JOHN-POUL, OTA CHIHARU, CIMINIERI CHIARA, REN WENHUA, HIEMSTRA PIETER S., STOLK JAN, GOSENS REINOUD, KÖNIGSH: "Wnt/β-catenin signaling is critical for regenerative potential of distal lung epithelial progenitor cells in homeostasis and emphysema", STEM CELLS, WILEY, vol. 38, no. 11, 1 November 2020 (2020-11-01), pages 1467 - 1478, XP093147742, ISSN: 1066-5099, DOI: 10.1002/stem.3241 *

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