Animals and treatment

8- to 10-week-old mice were used for all experiments with males and females in roughly equal proportions. Experimenters were not blinded to mouse age or sexes. Ai14-tdTomato (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze),30 dsRed (Tg(CAG-DsRed*MST)1Nagy),31 UBI-GFP (Tg(UBC-GFP)30Scha),32 R26R-eYFP (Gt(ROSA)26Sortm1(EYFP)Cos),33 SPC-GFP (Tg(Sftpc,-EGFP)1Dobb),34 SPC-CreERT2 (Sftpctm1(cre/ERT2,rtTA)Hap),20 Pdgfrα-GFP (Pdgfratm11(EGFP)Sor),35 and Wnt2-CreERT236 mice have been previously described. All studies were approved by the University of Pennsylvania’s Institutional Animal Care and Use Committees, protocol 806262, and followed all NIH Office of Laboratory Animal Welfare regulations.

Influenza infection

Control and infected mice were first anesthetized using 3.5% isoflurane in 100% O 2 via an anesthesia vaporizer system. Mice were intranasally administered 40–70 U TCID50 of influenza A/H1N1/Puerto Rico/8/34 (PR8) by pipetting 30 μL of virus dissolved in phosphate-buffered saline (PBS) onto the nostrils of anesthetized mice in visually confirmed agonal breathing. Control mice were administered 30 μL PBS using the same method. Only infected mice that lost ≥15% of their starting body weight by 11 DPI were considered to be adequately infected and were used for all experiments involving influenza infection. Mouse weights were tracked by measuring at days 0, 3, 6, 9, 14, 17, 20, and 23 post infection. Cells were transplanted at day 11 post infection.

Acid injury

Mice were anesthetized by intraperitoneal injections of 100 μL ketamine/xylazine (100 mg/kg). Sedated mice were intubated with a 20 G angiocatheter (BD) as previously described.37 Mice were then placed in the right lateral recumbent position and a polyethylene 10 (PE10) catheter (Clay Adams) was directed into the right main stem bronchus, while pressure was applied to the left lung. Injury was induced by instilling 2 μL/g of osmotically balanced 0.1 N HCl into the right lung (maximum dose = 50 μL) through the PE10 catheter as previously described.38 Cells were transplanted at day 1 post acid administration.

Bleomycin administration

For bleomycin-induced lung injury, we utilized intranasal administration as described for influenza infection with the following modifications. Mice received between 1.5 and 2.25 mg/g body weight bleomycin sulfate (13877-10, Cayman Chemicals) in a total volume of 50 μL PBS. Given the inherent variability of bleomycin injury, this range of doses was used to ensure a cohort of mice that was sufficiently injured (lost ≥10% body weight) but survived the injury. Cells were transplanted at day 10 post bleomycin administration.

S. pneumonia infection

Sp strain TIGR4 (serotype 4) was grown in tryptic soy broth for 2 hours as previously described.39 Mice to be infected were anesthetized by intraperitoneal injections of 100 μL ketamine/xylazine (100 mg/kg) and inoculated intranasally with 30 μL of 5.3 × 107 CFU Sp suspension. Cells were transplanted 2 days post Sp administration.

Organoid culture

CD45− EPCAM+ β4− AT2 cells used for organoid culture experiments were fluorescence-activated cell (FACS) sorted from C57BL6, SPC-GFP, UBI-GFP, or dsRed mice. For each growth condition, 80,000 cells were seeded onto 90 μL of Matrigel (BD) in a Corning® 96-well clear polystyrene flat-bottom microplate (Millipore Sigma). Organoid culture reagents used are as follows and each culture condition combination in addition to final in-solution concentrations of growth factors can be found in Table 1: 5% charcoal-treated fetal bovine serum (FBS) (Life Technologies), penicillin–streptomycin (P/S) solution (ATCC), SABMTM Basal Medium (Lonza) + SAGMTM SingleQuotsTM Supplement Pack (Lonza) excluding hydrocortisone, gentamycin solution (Millipore Sigma), A83-01 (Millipore Sigma), recombinant murine Wnt3a (Peprotech), recombinant murine Noggin (Peprotech), recombinant human FGF10 (Peprotech), recombinant human KGF (FGF7, Peprotech), recombinant murine EGF (Peprotech), recombinant human Rspondin-1 (Peprotech), Y-27632 dihydrochloride (Stemcell Technologies), B27TM supplement (50 × , Thermo Fisher Scientific), GlutaMAXTM supplement (100 × , Thermo Fisher Scientific), HEPES buffer solution (1 M, Thermo Fisher Scientific), and N2 supplement (100 × , Thermo Fisher Scientific). Organoids cultured in all conditions were grown in a 37 °C incubator for 13 days without passaging and with media changes on day 5 and day 9 before collecting for RNA or transplant.

Organoid diameter quantification

Organoids grown from all mice listed in “Organoid culture” were used for diameter quantification. Tilescans of organoid-containing wells were imaged using the bright-field setting on a Leica DMi8 Microscope using a Leica DFC9000 sCMOS camera and Leica Application Suite X (LASX) software. Organoids were always grown in at least technical duplicates and all technical replicate wells were imaged and quantified. Diameter measurement was performed by using the built-in “Draw scalebar” function on LASX. Ten to 50 organoids were measured and averaged in each technical replicate, and technical replicates were averaged for a final biological replicate, consisting of >30 organoids measured across all technical replicates. The resultant diameter from each biological replicate was then normalized to the average diameter of C1 organoids, which were grown as a control in at least one technical replicate for all experiments. Reported average diameter for each condition comes from at least n = 3 biological replicates.

Organoid and primary cell transplants

dsRed mice bred to a C57BL6 background were used to grow donor organoids for organoid transplant experiments. Organoid transplant recipients received 20,000– 150,000 AT2 organoid cells grown in C2 and C12 for 13 days with media changes on days 5 and 9. UBI-GFP, SPC-GFP, or SPC-CreERT2(Ai14-tdTomato) mice bred to a C57BL6 background were used as donors for transplant experiments. SPC-CreERT2(Ai14-tdTomato) mice were given three doses of 0.25 mg/g body weight tamoxifen dissolved in 50 μL corn oil every other day for 6 days. Mice given tamoxifen were killed for FACS 1 week following the final dose. Primary AT2 recipients received 900,000 CD45− EPCAM+ β4− AT2 cells. All recipients of both organoid and primary cell transplants were C57BL6. Recipient mice anesthetized with 3.5% isoflurane in 100% O 2 via an anesthesia vaporizer system were intranasally administered cells by pipetting single-cell suspension in PBS + 1% P/S onto the nostrils of anesthetized mice in visually confirmed agonal breathing.

Collecting organoids for RNA and transplants

Organoids were treated with 15 U/mL dispase II (Roche) in Hank’s balanced salt solution (HBSS) for 30 minutes, rinsed with 2 mM EDTA (Thermo Fisher Scientific), incubated in 2 mM EDTA for 5 min at 37 °C, mechanically dissociated by pipetting 50–100 times with a p200, and pelleted at 550 × g for 5 minutes at 4 °C. Cell pellets to be used for RNA were frozen by first aspirating supernatant followed by direct placement in a −80 °C freezer. Cell pellets to be used for organoid transplant had their supernatant aspirated and were resuspended in 30 μL PBS + 1% P/S per number of cells to be transplanted. Resuspended organoid transplant cells were kept on ice between final resuspension and transplantation.

Preparation of primary AT2 cells for transplant

Directly after sorting, primary AT2 cells were pelleted at 550 × g for 5 minutes at 4 °C. After aspirating the supernatant, the cell pellet was resuspended in 30 μL PBS + 1% P/S per 900,000 cells to be transplanted. Resuspended primary AT2 cells were kept on ice between final resuspension and transplantation.

Pulse oximetry

Repeated measurements of peripheral oxygen saturation (SpO 2 ) were taken using a MouseOx Plus Rat & Mouse Pulse Oximeter and a MouseOx small collar sensor (Starr Life Sciences Corp.). Mice were shaven around the neck and shoulders where the collar sensor sits. Recordings were taken using MouseOx Premium Software (Starr Life Sciences Corp., Oakmont, PA, USA). Measurements were taken continuously for > 3 minutes at a measurement rate of 15 Hz. Measurements were imported into Microsoft Excel and all readings with a non-zero Error Code were filtered out. The average of these error-free readings was used to calculate the SpO 2 reading for each mouse for each given time point. SpO 2 measurements were normalized to pretreatment SpO 2 values at day 0.

Lung tissue preparation for immunostaining

Following sacrifice via isoflurane overdose, lungs were inflated at a constant pressure of 25 cm H 2 O with 3.2% paraformaldehyde (PFA) for 30 minutes followed by incubation in 3.2% PFA for another 30 minutes at room temperature. Fixed lungs were then washed in multiple PBS washes over the course of 1 hour at room temperature, followed by an overnight incubation in 30% sucrose shaking at 4 °C, and then a 2 hour incubation in 15% sucrose 50% OCT compound (Fisher HealthCare) at room temperature. Finally, fixed lungs were embedded in OCT by flash freezing with dry ice and ethanol.

Organoid preparation for cytospins

Organoids were collected by first washing each organoid well with PBS, then scraping the Matrigel plug and organoids into a 15 mL tube using a p20 pipet tip. Each well was washed once more with PBS and all washes were collected in their respective tubes. Each tube was topped off with 10 mL cold PBS and the organoids in suspension were gently pipetted up to ten times with a p1000 to break up the Matrigel. Organoids were pelleted at 200 × g for 5 minutes at 4 °C, resuspended in 10 mL cold PBS, and shaken at room temperature on ice for 30 min. Organoids were once more gently pipetted up to ten times to break up the remaining Matrigel before pelleting organoids again at 200 × g for 5 minutes at 4 °C. Organoids were resuspended in 10 mL cold 3.2% PFA and shaken at room temperature on ice for 30 min. Organoids were pelleted once more at 200 × g for 5 minutes at 4 °C.

Cytospins

Sorted cells were pelleted at 550 × g and organoids at 200 × g for 5 minutes at 4 °C before resuspension in 200 μL 3.2% PFA per number of slides to be cytospun. 200 μL of resuspended cells or organoids was loaded into cytospin chambers. Cells were spun at 75 r.p.m. for 4 minutes and organoids were spun at 200 rpm for 5 minutes on a Cytospin 2 (Shandon).

Immunostaining

Seven-micrometer sections were cut on a Leica CM3050 S Research Cryostat (Leica Biosystems). Tissue sections and cytospins were further fixed for 5 minutes in 3.2% PFA, rinsed three times with PBS, and blocked in blocking solution (PBS + 1% bovine serum albumin (Affymetrix) + 5% normal donkey serum (Jackson Immuno Research) + 0.1% Triton X-100 (Millipore Sigma)+ 0.02% sodium azide (Millipore Sigma)) for > 30 minutes. Slides were incubated in primary antibodies (listed below) in blocking solution overnight at 4 °C. Slides were then washed three times with PBS + 0.1% Tween-20 (Millipore Sigma) and subsequently incubated with secondary antibodies (listed below) for > 2 h at room temperature. Slides were then washed once more with PBS + 0.1% Tween-20 prior to incubation in 1 μM DAPI (Life Technologies) for 5 minutes, rinsed with PBS, and mounted with either Prolong Gold (Life Sciences) or Fluoroshield (Millipore Sigma). The following primary antibodies were used: rabbit anti-SPC (1:2000, Millipore), rat anti-Lamp3 (1:500, Novus, clone 1010E1.01), rat anti-RAGE (1:500, R&D, clone 175410), rabbit anti-Aqp5 (1:100, Abcam, clone EPR3747), Syrian hamster anti-Pdpn (1:100, DSHB, clone 8.1.1), rabbit anti-Krt5 (1:1000, BioLegend, clone Poly19055), chicken anti-Krt5 (1:500, BioLegend, clone Poly9059), goat anti-Scgb3a2 (1:200, R&D, clone AF3465), and sheep anti-eGFP (1:500, Invitrogen, 10396164). The following secondary antibodies were used: Alexa Fluor™ 488-conjugated donkey anti-sheep (1:1000, Thermo Fisher Scientific), Alexa Fluor™ 488-conjugated donkey anti-rabbit (1:1000, Thermo Fisher Scientific), Alexa Fluor™ 488 donkey anti-rat (1:1000, Thermo Fisher Scientific), donkey anti-chicken Alexa Fluor™ 488 (1:500, Jackson Immuno Research), fluorescein isothiocyanate-conjugated goat anti-chicken (1:1000, BioLegend, Poly24108), Alexa Fluor™ 568-conjugated donkey anti-rabbit (1:1000, Thermo Fisher Scientific), Alexa Fluor™ 568-conjugated donkey anti-goat (1:1000, Thermo Fisher Scientific), Alexa Fluor™ 647-conjugated donkey anti-rabbit (1:1000, Thermo Fisher Scientific), Alexa Fluor™ 647-conjugated donkey anti-goat (1:1000, Thermo Fisher Scientific), Alexa Fluor™ 647-conjugated chicken anti-rat (1:1000, Thermo Fisher Scientific).

Fluorescence-activated cell sorting

Lung cells were isolated by first inflating lungs with 15 U/mL dispase II in HBSS (Thermo Fisher Scientific), tying off the trachea, and cutting lobes away from the main stem bronchi. Lobes were then incubated in dispase for 45 minutes shaking at room temperature and mechanically dissociated by pipetting in sort buffer (SB; Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) + 2% cosmic calf serum (CC; Thermo Fisher Scientific) + 1% P/S). After pelleting at 550 × g for 5 minutes at 4 °C, whole-lung suspension was treated with Red Blood Cell Lysis Buffer (Millipore Sigma) for 5 minutes, pelleted, and resuspended in SB + 1:1000 DNase I (Millipore Sigma) for a 45 minutes recovery period shaking at 37 °C. Whole-lung suspension was then repelleted and resuspended in SB + 1:50 TruStain FcX™ (anti-mouse CD16/32) Antibody (BioLegend) for a 10 minutes blocking period at 37 °C. For sorts from C57BL6 mice, AT2 cells were collected using allophycocyanin (APC)/Cy7-conjugated rat anti-mouse CD45 antibody (1:200, BioLegend, 30-F11), Alexa Fluor® 488-conjugated rat anti-mouse CD326 (EpCAM) antibody (1:200, BioLegend, G8.8), and phycoerythrin (PE)-conjugated rat anti-mouse CD104 (integrin β4) antibody (1:100, BioLegend, 346-11A). For sorts from mice expressing a GFP fluorophore, AT2 cells were collected using the C57BL6 sort scheme above but with PE-conjugated rat anti-mouse EpCAM (1:200, BioLegend, G8.8) and APC-conjugated rat anti-mouse integrin β4 antibody (1:100, BioLegend, 346-11A) instead of the respective EpCAM and integrin β4 antibodies listed above. For sorts from mice expressing a RFP fluorophore, AT2 cells were collected using the C57BL6 sort scheme above but with APC-conjugated rat anti-mouse integrin β4 antibody instead of the respective integrin β4 antibody listed above. Lung mesenchyme collected from all aforementioned mice was collected by adding APC/Fire™ 750-conjugated rat anti-mouse CD31 (PECAM) (1:200, BioLegend, MEC13.3) to the sort scheme to purify CD45- CD31- EpCAM- mesenchymal cells. Staining was performed for 45 minutes at 4 °C, followed by a final spin-down at 550 × g for 5 minutes at 4 °C. Stained cells and fluorescence minus one controls were then resuspended in SB + 1:1000 DNase + 1:1000 Draq7 (Beckman Coulter) as a live/dead stain. All FACS sorting was done on a BD FACSJazz (BD Biosciences) and cells were collected in Falcon™ round-bottom polystyrene tubes (Thermo Fisher Scientific) in 300 μL DMEM + 20% CC + 2% P/S.

Quantitative reverse transcriptase PCR

RNA was isolated from both organoids and sorted cells using a ReliaPrep™ RNA Cell Miniprep kit (Promega). The amount of RNA input for cDNA synthesis was standardized within each experiment to the RNA isolate with the lowest concentration as measured by Nanodrop (Thermo Fisher Scientific). cDNA was synthesized using iScript™ Reverse Transcription Supermix (BioRad). Primers used are as follows: SPC, forward 5′-ATGGACATGAGTAGCAAAGAGGT-3′, reverse 5′-CACGATGAGAAGGCGTTTGAG-3′; Krt5, forward 5′-TCCAGTGTGTCCTTCCGAAGT-3′, reverse 5′-TGCCTCCGCCAGAACTGTA-3′; Scgb3a2, forward 5′-CCACTGCCCTTCTCATCAACC-3′, reverse 5′-TGTCGTCCAAAGGTACAGGTA-3′; L19, forward 5′-ATGTATCACAGCCTGTACCTG-3′, reverse 5′-TTCTTGGTCTCTTCCTCCTTG-3′. Expression of each gene is relative to expression of L19 within that sample. qPCR was run on an Applied Biosystems QuantStudio 6 Real-Time PCR System (Thermo Fisher Scientific).

Statistics

All statistical tests were performed using GraphPad Prism 7. P-values were calculated using unpaired two-tailed t-tests with Welch’s correction or ordinary one-way or two-way analysis of variance for comparisons involving multiple variables. Comparison of slopes from linear regression was calculated using an equivalent test to analysis of covariance in Chapter 18 of ref. 40

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.