Stem Cell

Alveolar epithelial progenitor cells require Nkx2-1 to maintain progenitor-specific epigenomic state during lung homeostasis and regeneration – Nature Communications

Time Stamp: December 18, 2023 7:00 PM
Source Node: 396634

To optimize important aspects of the AEP-O culture, we undertook extensive reagent testing and standardization. The primary components of epithelial 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 (i.e., Transwell filter). We tested each of these components iteratively. As previously reported, AEPs form more and larger organoids than unselected AT2 cells³, so we focused on using FACS-sorted AEPs derived from Axin2^CreERT2-tdT mice as the epithelial starting fraction. To control for observed variability in organoid morphology and composition from different types of supportive mesenchymal cells, we used large, consistent preparations of primary lung fibroblasts obtained by selective adhesion from P28 wild-type C57BL/6 mice at passage 3-4. We then lot tested 4 available commercial matrices, all with compositions similar to Matrigel (Corning), and found that the best lots of Matrigel support organoid growth with 2-3-fold increased CFE compared to other Matrigel lots and competing products; we therefore confined our studies to a single lot of Matrigel and all data herein uses this standardized reagent. We cultured 5000 FACS-purified AEPs and 50,000 lung fibroblasts in each well on a Transwell filter in a 1:1 ratio of Matrigel and media (SAGM with 5% FBS and limited additives); we took this approach to minimize exogenous signaling modulators in the media and allow evaluation of the supportive capacity of the mesenchymal fraction. To evaluate the growth of these organoids more fully, we adapted methods from iPSC culture for whole-mount immunohistochemistry of organoids²⁷.

Ethical compliance and animals

All animal studies were conducted under the guidance and supervision of the Cincinnati Children’s Hospital Medical Center (CCHMC) Institutional Animal Care and Use Committee (IACUC) in accordance with CCHMC regulatory and biosafety protocols. Mouse lines used included: C57BL/6J mice (Jackson Strain #000664), PDGFRα^EGFP (B6.129S4-PDGFRa^{tm11(EGFP)Sor}/J; Jackson Strain #007669) Axin2^creERT2-TdT (a gift from Edward Morrisey), Tfcp2l1^CreERT2 (B6;129S-Tfcp2l1^{tm1.1(cre/ERT2)Ovi}/J; Jackson Strain #028732), Nkx2-1^fl/fl (a gift from Shioko Kimura), and R26R^EYFP (B6.129×1-Gt(ROSA)26Sor^tm1(EYFP)Cos/J; Jackson Strain #006148). All experiments for both organoids and in vivo lineage tracing included both male and female mice. For Cre recombinase induction in mouse models, 8–12-week-old mice were treated intraperitoneally (IP) with Tamoxifen (Sigma, T5648; dissolved in ethanol and resuspended in corn oil) at a dose of 50 mg/kg, one or three times (every other day), at the experimental time points indicated previously.

Mouse lung harvest

Mice were anesthetized via IP 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. For tissue dissociation for organoids, the lungs were removed and placed in cold PBS on ice. For tissue fixation for histology and immunofluorescence, the trachea was cannulated and lungs were inflated to a pressure of 30 cm H₂O using 4% paraformaldehyde (PFA). Inflated lungs were immersed in a conical of 4% PFA and then left on a rocker at 4 °C overnight.

Processing fixed lung tissue for histology and immunofluorescence

The day following inflation, fixed lung tissue was trimmed and placed in cassettes. The cassettes were washed (15 min 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 µm. Paraffin sections were incubated at 65 °C for 2 h, deparaffinized in xylene (3x for 10 min), rehydrated through an ethanol gradient, and standard H&E staining was performed. Slides were mounted with Permount Mounting Medium (Electron Microscopy Sciences, 17986-05) and cover slipped with #1.5 Gold Seal 3419 Cover Glass (Electron Microscopy Sciences, 63790-01).

Immunofluorescence on paraffin sections was performed as previously described³. Briefly, following deparaffinization, rehydration, and sodium citrate antigen retrieval (10 mM, pH 6.0), and blocking, immunofluorescence was performed on paraffin sections using antibodies in Supplementary Table 1b and the following reagents: ImmPRESS® HRP Horse Anti-Rabbit IgG Polymer Detection Kit (Vector Labs, MP-7401-50), ImmPRESS® HRP Horse Anti-Goat IgG Polymer Detection Kit (Vector Labs, MP-7405-50), and ImmPRESS® HRP Goat Anti-Rat IgG, Mouse adsorbed Polymer Detection Kit (Vector Labs, MP-7444-15). Following the application of TSA fluorophores (listed in Supplementary Table 1b; 1:100), sections were stained with DAPI (Invitrogen, D1306; 1:1000) and mounted using Prolong Gold antifade mounting medium (Invitrogen, P36930).”

Mouse lung digestion and single cell suspension

Clonal mouse alveolar epithelial progenitor (AEP)-based alveolar organoids were generated as previously described³ with minor modifications. Briefly, following harvest, 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 µL of DNase (stock: 5 mg/mL; final concentration: 0.025 mg/mL or 50 U/ml, GoldBio, D-301), and 100 µL of Collagenase Type I (stock: 48,000 U/mL; final concentration of 480 U/mL, Gibco, 17100-017)]. C tubes were placed on a gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, 130-096-427), and the following protocols were run: “m_lung_01_02” (36 s) twice, “37C_m_LIDK_1” (36 min 12 s) once, and “m_lung_01_02” (36 s) once. Samples were passed through a 70 µm filter (Greiner Bio-One, 542070) and centrifuged at 500 × g for 5 min at 4 °C. Following removal of the supernatant, 5 mL of RBC Lysis Buffer (Invitrogen, 00-4333-57) was added and incubated for 5 min. All centrifugation steps with this single-cell suspension were performed at 500×g for 5 min at 4 °C for the following procedures.

Fibroblast stock preparation and maintenance

For generation of fibroblast stocks, 4-week C57BL/6J mice and/or 4-week PDGFRα^EGFP mouse 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]). After removing supernatant from final wash, 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 h post-plating.

Cells were passaged at 80% confluency to P3. For passaging, media was removed from each plate and cells were washed with 5 mL of DPBS (Gibco, 14190-094). Then, 3 mL of 0.25% Trypsin-EDTA (Gibco, 25200-056) was added and plates were incubated at 37 °C for 7 min. Next, 5 mL of fibroblast medium was added to each plate, pipetted to dissociate cells, and transferred to a 15 mL conical tube. Cells were centrifuged at 500 × g for 5 min at 4 °C, the supernatant was removed, and the cell pellet was resuspended in 2 mL fibroblast medium/per plate (split 1:2 or 1:3) and transferred to plates containing 6 mL fibroblast medium. Once confluent at P3, cells were washed, trypsinized, centrifuged as above, and resuspended in 1 mL of freezing medium (90% FBS, 10% DMSO) (one plate per cryovial) and transferred to Mr. Frosty Cryogenic Freezing Container (Nalgene, 5100-0001) filled with isopropyl alcohol, which was placed in a −80 °C freezer overnight, before samples were moved to long-term liquid nitrogen storage.

For the use of frozen fibroblast stocks in organoids, 48 h prior to use in organoids, cells were rapidly thawed and resuspended in a 10 mL fibroblast medium in a 15 mL conical. Cells were centrifuged at 500 × g for 5 min 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.

Processing for organoids, FACS, or cell sorting

Single-cell suspensions were obtained as above, and cells were resuspended in 5 mL MACS Buffer (autoMACS Rinsing Solution [Miltenyi Biotec, 130-091-222] with MACS BSA Stock Solution [Miltenyi Biotec, 130-091-376]) and passed through a 40 µm filter (Greiner Bio-One, 542040). Cells were centrifuged, the supernatant was removed, and the cell pellet was resuspended in Fc Receptor Binding Inhibitor Polyclonal Antibody (Invitrogen, 14-9161-73) diluted 1:100 in MACS buffer and incubated for 10 min at room temperature. Following centrifugation, cells were resuspended in a mixture of the following antibodies diluted 1:100 in MACS buffer and incubated for 10 min protected from light: CD31 (PECAM-1; Monoclonal Antibody [390], eFluor 450) (Invitrogen, 48-0311-82), CD45 (Monoclonal Antibody [30-F11], eFluor 450) (Invitrogen, 48-0451-82), CD326 (EpCAM; Monoclonal Antibody [G8.8], APC) (Invitrogen, 17-5791-82). Cells were washed 1x with 1–5 mL of MACS buffer and resuspended in Fixable Viability Dye eFluor 780 (Invitrogen, 65-0865-14) diluted 1:1000 in MACS buffer and incubated for 15 min protected from light. Cells were centrifuged and washed in 1–5 mL MACS buffer 3x. After the final wash/centrifugation, the cell pellet was resuspended in MACS buffer (volume adjusted for cell count) and passed through a 35 µm filter lid (Corning, 352235) into a FACS tube for sorting.

Using single-stain controls from experimental animals and wild-type littermates (TdTomato⁻) for compensation and adjusting gating to remove debris/doublets, 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 µm nozzle. Yield is approximately 10⁵ AEPs per mouse using this protocol. For generation of organoids from Tfcp2l1⁺ epithelial cells, Tfcp2l1-Cre^ERT2; R26R^EYFP mice were treated with tamoxifen as described above, followed by sorting of Live/CD31⁻/CD45⁻/CD326⁺[EpCAM⁺]/EYFP⁺ cells using the same sorter settings. Organoid initiation and maintenance with cells generated with both lines were performed identically.

Organoid growth medium

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).

Standard organoid plating and maintenance

AEPs (live/CD31⁻/CD45⁻/CD326⁺[EpCAM⁺]/TdT⁺ cells) sorted from Axin2^creERT2-tdT mice were counted using a hemocytometer and resuspended in ‘spiked’ SAGM at a concentration 500 cells/µL. Fibroblasts were prepared (as described above), counted, and resuspended in ‘spiked’ SAGM at a concentration of 5000 cells/µL. For the remaining steps, it was extremely important that all reagents were kept cold/on ice and that bubbles were not introduced to mixtures when pipetting. It is recommended to prepare the plate (Falcon 24-well companion plates [Corning, 353504]), insert Transwells (Falcon Transwell Insert/Permeable Support with 0.4 µm membrane [Corning, 353095]), and place them on ice before use.

For each well of organoids to be plated, 10 µL AEPs (5000 total cells), 10 µL fibroblasts (50,000 total cells), and 25 µL ‘spiked’ SAGM were combined (create one master mix of cells and medium for all wells before adding Matrigel) and placed on ice. Corning Matrigel GFR Membrane Matrix (Corning, 356231) was added to the cell mixture (45 µL per well, 1:1 ratio of SAGM to Matrigel) and carefully mixed, then placed back on ice.

For plating, 90 µL 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 min, then 500 µL of ‘spiked’ SAGM supplemented with ROCK Inhibitor/Y-27632 Dihydrochloride (Sigma, Y0503, final concentration 0.01 mM) was added beneath the Transwell insert. After 48 h (and for subsequent media changes), media was replaced every 2 days with ‘spiked’ SAGM without ROCK inhibitor, and plates were maintained at 5% CO₂ and 37 °C.

AAV6.2FF-Cre organoids plating and maintenance

AAV6.2FF-Cre (titer of 2.779 × 10¹⁰ viral genomes [vg]/µL) was generated and characterized in vivo as previously described^69,70. Working dilutions (2.779 × 10⁹ vg/µL, 2.779 × 10⁸ vg/µL, and 2.779 × 10⁷ vg/µL) 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/µL. The total cells needed for the desired number of wells were transferred to a new 1.5 mL tube (i.e., 10 wells → 50,000 cells → 50 µL cells [1000 cells/µL]). Total cell number per tube, desired MOI (i.e., 1000, 10000, 20000), and known viral titers were used to calculate the volume of virus needed from viral stocks. For each MOI, the calculated volume of the virus was added to each cell mixture, mixed, and incubated on ice for 60 min. Following viral incubation, ‘spiked’ SAGM and fibroblasts (5000 cells/µL) 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 + 50,000 fibroblasts in 45 µL ‘spiked’ SAGM). The cell mixture was mixed with Matrigel (45 µL/well) and plated/maintained as described above for standard organoids.

Organoid plating with fibroblasts on the basolateral side of transwell

One day prior to organoid plating, fibroblasts were prepared (as described above), counted, and resuspended at a concentration of 50,000 cells in 100 µL in fibroblast medium. Transwells were placed in the wells of the companion plate, and 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. Then, 100 µL 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 h. After incubation, the 100 µL of the 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 µL of DPBS (beneath the Transwell insert) and then moved to a fresh well/plate with 500 µL fibroblast medium beneath the Transwell insert. After standard isolation of epithelial cells (AEPs) for organoid plating, the Transwells were again washed with 500 µL of DPBS (beneath the Transwell insert), then the epithelial cell/Matrigel mixture (5000 AEPs in 45 µL ‘spiked’ SAGM + 45 µL Matrigel) was added to the apical side of the Transwell filter. Organoids were maintained as described above for standard organoids.

Fixation and processing for sections, histology, and H&E

Organoids were washed with 500 µL PBS, above and below Transwells. After removing PBS, 500 µL of 4% PFA was added above and below the Transwell for fixation overnight at 4 °C. 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 liquid consistency) was added on top of the Matrigel plug/organoids until covered on all sides. Once solidified (~15–30 min), the sample was transferred to a tissue processing cassette (Fisher, 15-182-702 A). Once in cassettes, samples were processed as described for whole-lung processing above for paraffin embedding and sectioning, H&E, and immunofluorescence of paraffin sections.

Isolation of organoids for whole-mount immunofluorescence

Previously established whole-mount organoid staining protocols from Dekkers et al.²⁷ were adapted for mouse lung alveolar organoids using the following modifications. All steps used cut or wide-bore pipette tips. All steps after first wash and prior to fixation were performed on ice/with chilled reagents and used cut/wide-bore pipette tips coated in 1% BSA in PBS.

Briefly, Transwells were washed (above and below) with 500 µL of room-temperature PBS. Then, 500 µL of Cell Recovery Solution (Corning, 354253) was added to each Transwell, and a cut pipette tip was used to mechanically disrupt the Matrigel—the mixture was pipetted up and down and transferred to a new 24-well plate. Each Transwell was washed with an additional 250 µL of cell recovery solution and added to the new plate. The plate was incubated on ice (gel ice packs were optimal) on an orbital/horizontal shaker for 60 min. The organoid mixture was transferred to a 15 mL conical pre-coated in 1% PBS-BSA. Each well was washed with 500 µL of 1% PBS-BSA and added to the 15 mL conical. Wells from the same experimental condition (up to 4 wells) were combined in one conical. Conicals were filled to 10 mL with ice-cold PBS and centrifuged at 70×g for 5 min 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 matrixl]. If Matrigel was still visible, the organoid pellet was gently resuspended in 1 mL of ice-cold 1% PBS-BSA and centrifuged again at 70×g for 5 min at 4 °C. After careful removal of the supernatant, the organoid pellet was resuspended in 1 mL of 4% PFA and incubated at 4 °C for 45 min (resuspending once halfway through incubation). For permeabilization, 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 min at room temperature).

Whole-mount blocking and immunofluorescence

After isolation, fixation, and permeabilization, organoids were centrifuged at 70×g for 5 min at 4 °C, resuspended in 500 µL of 5% Normal Donkey Serum (Jackson ImmunoResearch, 017-000-121) in 0.1% PBS-Triton X-100, and transferred to a 24-well plate for blocking. Organoids were incubated at room temperature on an orbital shaker for 1–2 h.

After blocking, the supernatant was removed from each well without disturbing organoids [note: supernatant was removed more easily when the plate was placed at a 45° angle for 5–10 min to allow organoids to settle to the bottom edge of the well]. Primary antibodies (see Supplementary Table 1) were diluted to a final concentration of 1:100 in 5% Normal Donkey Serum in 0.1% PBS-Triton X-100 (approximately 200–250 µL total) and incubated overnight at 4 °C on an orbital shaker. For this protocol, a ‘quick wash’ was defined as adding 1 mL of organoid wash buffer (0.2% BSA, 0.1% Triton X-100 in PBS)²⁷ and immediately allowing organoids to settle/removing the wash, and a ‘long wash’ was defined as adding 1 mL of organoid wash buffer placing the plate on an orbital shaker for 1–2 h before allowing organoids to settle/removing the wash. After primary antibody staining, one ‘quick wash’ and three ‘long washes’ were performed. Then, secondary antibodies (see Supplementary Table 1) were diluted to a final concentration of 1:200 in 5% Normal Donkey Serum in 0.1% PBS-Triton X-100 (approximately 200–250 µL total) and incubated overnight at 4 °C on an orbital shaker. For this step and all subsequent steps, samples were covered/protected from light to prevent photobleaching. After secondary antibody staining, one ‘quick wash’ was performed, then organoids were incubated in DAPI (Invitrogen, D1306, final concentration of 1:1000) in 5% Normal Donkey Serum in 0.1% PBS-Triton X-100 (approximately 200–250 µL total) for 15 min. After removing the supernatant, one ‘quick wash’ and three ‘long washes’ were performed.

Whole-mount clearing and mounting

Following the final wash after immunostaining, as much wash buffer as possible was removed from each well, and organoids were transferred to a 1.5 mL tube. Organoids were centrifuged at 70 × g for 5 min at 4 °C and as much supernatant as possible was removed without disturbing the organoids. Using a cut or wide-bore pipette tip, organoids were gently resuspended in room temperature fructose-glycerol clearing solution (60% vol/vol glycerol + 2.5 M fructose)²⁷. Depending on organoid volume, ~50–200 µL 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. Prior to preparing slides, cleared organoids were allowed to equilibrate to room temperature. Organoids were mounted as described previously²⁷—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). Using a PAP pen (Abcam, ab2601), a square was drawn between the two pieces of tape. Using a cut P200 pipette tip, approximately 20 µL of organoids in clearing solution was placed in the middle of the drawn square, avoiding bubbles. A #1.5 Gold Seal 3419 Cover Glass (Electron Microscopy Sciences, 63790-01) was applied over the organoids, bridging the two pieces of tape. Slides were imaged immediately or stored at 4 °C.

Whole-mount Click-iT EdU staining

For whole-mount Click-iT EdU staining, the standardized commercial protocol for Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye (Invitrogen, C10337) was combined with our optimized whole-mount immunofluorescence protocol. Kit reagents were prepared as directed in commercial protocols. Briefly, 48 h prior to harvest/fixation, the organoid medium was replaced with ‘spiked’ SAGM supplemented with EdU (final concentration of 10 µM) from the commercial kit. After EdU incubation, all steps described in “Isolation of organoids for whole-mount immunofluorescence” were performed. Next, steps for EdU detection from the commercial kit’s standardized protocol with kit reagents were followed (i.e., 30-min incubation of “Click-iT Reaction Cocktail” at room temperature, followed by 1 mL wash with 3% PBS-BSA). Following EdU detection, whole-mount immunofluorescence was performed as described in the “Whole-mount blocking and immunofluorescence” and “Whole-mount clearing and mounting” sections above.

Whole-mount Click-iT TUNEL staining

For whole-mount Click-iT TUNEL staining, the standardized commercial protocol for Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor 488 dye (Invitrogen, C10617) was combined with our optimized whole-mount immunofluorescence protocol. Kit reagents were prepared as directed in commercial protocols. All steps described in “Isolation of organoids for whole-mount immunofluorescence” were performed. Organoids were washed twice with DI H₂O. Next, steps for ‘TdT Reaction’ and ‘Click-iT Plus Reaction’ from the commercial kit’s standardized protocol with kit reagents were followed (i.e., 60-min incubation of “TdT Reaction Mixture” at 37 °C, followed by 2 washes with 3% PBS-BSA, and a 30-min incubation of “Click-iT Plus TUNEL reaction cocktail” at 37 °C). Organoids were washed twice with 3% PBS-BSA, and then whole-mount immunofluorescence was performed as described in the “Whole-mount blocking and immunofluorescence” and “Whole-mount clearing and mounting” sections above.

Hoechst and live imaging preparation

For live imaging of organoids grown with PDGFRα^EGFP fibroblasts, Hoechst 33342 (Invitrogen, H3570) was diluted 1:10000 in ‘spiked’ SAGM, and 500 µL was added above and below the Transwell and incubated at 37 °C for 30–45 min. 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. Samples were covered in ‘spiked’ SAGM and cover slipped (MatTek, PCS-1.5-18) prior to imaging on an inverted confocal microscope.

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 λ 10x, Plan Apo λ 20x, Apo LWD 20x WI λS (water immersion), Apo LWD 40x WI λS DIC N2 (water immersion), and SR HP Plan Apo λ S 100xC Sil (silicone immersion). Second harmonic generation was performed to visualize fibrillar collagens I and II 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 plate imaging/cytation imager

For whole-well imaging, plates were loaded into a Cytation 5 Imager (BioTek, CYT5PV) configured with a CO₂ gas controller (BioTek, 1210012). Plates were maintained at 5% CO₂ and 37 °C during imaging using Cytation Gen5 Microplate Reader and Imager Software (BioTek, version 3.08.01). Protocols specific to Falcon 24-well companion plates (Corning, 353504) and Falcon Transwell Insert/Permeable Support with 0.4 µm membrane (Corning, 353095) were established and used to take brightfield and fluorescent (GFP) 4x tile scans at 10 z-steps (~50 µm per step). 4x tile scans were used to generate z-projections. Individual tile scans and z-projections were used for further quantification.

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 the 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. T-tests were used for comparison of 2 groups, and ANOVA with prespecified multiple comparisons was used to compare 3 or more groups.

Electron microscopy

Fixation, sectioning, and acquisition of electron micrographs of alveolar cells were performed as previously described⁹¹.

Organoid dissociation and preparation of single-cell suspension for scRNAseq and scATACseq

Transwells were washed (above and below) with 1 mL of PBS. Then, 60 µL of 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 a digest buffer for 30 min 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 the same experimental condition combined into each tube). Then, 500 µL of cold PBS was added to each tube and incubated on ice for 5–10 min. Samples were centrifuged at 500×g for 5 min at 4 °C. Supernatant was removed carefully and the sample was washed with 1 mL of cold DPBS (Gibco, 14190-094). Following centrifugation at 500×g for 5 min at 4 °C and removal of supernatant, samples were resuspended in 60 µL of 0.25% Trypsin-EDTA (Gibco, 25200-056) and incubated for 30 min at 37 °C. Then, 1 mL of ice-cold PBS was added to each tube, samples were centrifuged at 500×g for 5 min at 4 °C, and the supernatant was carefully removed. Samples were washed 2x in 1 mL of cold 0.04% PBS-BSA and centrifuged at 500×g for 5 min at 4 °C. Following the removal of the supernatant, the samples were resuspended in 100 µL of 0.04% PBS-BSA. Prior to filtering cells, 40 µm Flowmi Cell Strainers (Bel-Art, H13680-0040) were equilibrated by passing 100 µL of 0.04% PBS-BSA through the strainer using a P1000 pipette tip. The 100 µL cell suspension was then pipetted through the 40 µm Flowmi Cell Strainer. Cells were counted manually using a hemocytometer and resuspended at a concentration of 1000 cells/µL prior to processing for scRNAseq.

Nuclei isolation from organoids for scATACseq

Using the same filtered cell suspension generated for scRNAseq, 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 500×g for 5 min at 4 °C. Following removal of the supernatant, 100 µL of ATAC lysis buffer (from standard 10x Genomics protocol, CG000212 Revision B) was added, gently mixed and incubated on ice for 4–4.5 min. Immediately following incubation, 1 mL of chilled ATAC wash buffer (from standard 10x Genomics protocol) was added and gently mixed. Nuclei were centrifuged at 500×g for 5 min at 4 °C, supernatant was removed, and nuclei were resuspended in 100 µL of 1x nuclei buffer (diluted from 20x nuclei buffer [10x Genomics, 2000153/2000207]). Prior to filtering nuclei, 40 µm Flowmi Cell Strainers were equilibrated by passing 100 µL of nuclei buffer through the strainer using a P1000 pipette tip. The 100 µL of nuclei suspension was then pipetted through the 40 µm Flowmi Cell Strainer. Nuclei were counted manually using a hemocytometer and resuspended at a concentration of 5000 nuclei/µL prior to processing for scATACseq.

Mouse lung preparation for single-cell suspension for scRNAseq and scATACseq

For scRNAseq and scATACseq of adult mouse lungs, lungs were harvested, digested, and processed as described above in “Mouse lung digestion and single cell suspension.” Samples were then passed through a 40 µm filter (Greiner Bio-One, 542040) and centrifuged at 500×g for 5 min at 4 °C. Following removal of the supernatant, cells were resuspended in DMEM-F12 (Gibco, 11320-033), counted, and resuspended at a final concentration of 1000 cells/μL (50,000 cells total in 50 μL) for downstream processing for scRNAseq.

Cells for scATACseq were further processed as described in the “Nuclei isolation from organoids for scATACseq” section, with the following modifications: cells were left in lysis buffer for 120 to 150 s total before the addition of wash buffer. All other steps were completed as described for organoids. Finally, the nuclear count was adjusted to 5000 nuclei/μL for sequencing preparation.

Sequencing/library preparation

From each single cell or single nuclear preparation described above, a maximum of 16,000 cells or nuclei were loaded into a 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. To detect YFP expressing cells following Cre-mediated activation, 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. Briefly, 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.

scRNAseq analysis and visualization

For RNAseq analysis, output data from CellRanger was partitioned into spliced and unspliced reads using Velocyto⁹². 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 DoubletFinder⁹³, and libraries from individual time points and treatments were integrated using SelectIntegrationFeatures and IntegrateData commands in Seurat. Following integration, cells were re-clustered, UMAP project generated, and samples identified based on expression similarity to published data as described in the “Results”. Module scoring was performed using AddModuleScore function in Seurat for gene sets indicated in the figures. For lineage inference, these Seurat objects were directly used for Slingshot⁴⁷ pseudotime inference and were converted to a h5ad file using the SaveH5Seurat command. These h5ad file were used as input to scVelo⁴⁸ and CellRank⁹⁴ in Python 3.9.12 in Spyder following the standard pipeline (scVelo.readthedocs.io) to generate RNA velocity mapped to the Seurat UMAP and cell populations. For ligand-receptor analysis, we used CellChat³⁴ (https://github.com/sqjin/CellChat) v1.1 using the SecretedSignaling subset of the Mouse CellChatDB, with default parameters. Visualizations were generated with these tools and ggplot2.

scATACseq analysis and visualization

For ATACseq analysis, CellRanger output was loaded into ArchR⁵⁰, and Arrow files were generated per package defaults. Clusters were generated based on ATACseq parameters and named based on the evaluation of integrated gene expression from the paired Seurat RNA object. Peak calls for regions of open chromatin were generated from pseudobulk analysis of each cell state, followed by peak calling in MACS2. Differential open chromatin peaks were identified based on FDR < 0.01 and Log2FC ≥ 1 between cell states. Visualizations were generated using standard ArchR commands.

Transcriptional regulatory networks and visualization

For TRN inference, scRNAseq gene expression data and scATACseq chromatin accessibility data from each epithelial cell population were used as input for the Inferelator 3.0 (https://github.com/flatironinstitute/inferelator) with minor modifications in Python 3.9.22. Enriched transcriptional regulators were identified per cell type by performing Fisher’s exact test to compare the observed vs expected number of genes regulated in a cell type based on the TRN model, and visualizations were generated in Illustrator with details as noted in the figure legends.