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This protocol describes the isolation and long-term culture of Sox9 distal tip lung progenitors from mouse embryonic lung tissue. We have developed a serum-free, feeder-free 3D system to stably expand a ~95% pure Sox9 mouse embryonic lung progenitor population in vitro. These lung progenitors retain their multipotency and can differentiate into the full spectrum of lung epithelial lineages—i.e., airway and alveolar lineages— in vitro as well as in vivo upon transplantation into the injured mouse lung. Sox9 progenitors can be isolated from mouse embryonic lungs within ~4h and maintained for at least 6 months (expanding ~10 fold in number). Cultured Sox9 progenitors can be also cryopreserved and resurrected without losing their properties. The ability to expand multipotent Sox9 lung progenitors should avail multiple future applications including the generation of lung organoids and gene editing, with ramifications for disease modeling, drug screening and regenerative medicine. Introduction The lung is a ramified three-dimensional tree of epithelial tubes comprising two major subdivisions—the proximally-located conducting airways and the distally-located alveoli (where gas exchange occurs). Currently, in the adult lung, only airway-restricted and alveolar-restricted stem cells are known to exist. Access to systems in which multipotent lung stem or progenitor cells with the ability to generate both airway and alveolar cell-types, can be cultured and expanded in vitro would be an advantageous starting point to study lung developmental biology, organ function and lung diseases. Although methods to generate 3D aggregates (“organoids”) from adult lung restrictedstem cells or differentiating pluripotent stem cells have already been described, a Nature Methods: doi:10.1038/nmeth.4498 2 true system that recapitulate lung native architecture and functionalities has yet to be achieved. Establishing lung organoid from cells with full lung multipotency could be an alternative to generate such in vitro model. While only airway-restricted and alveolarrestricted stem cells subsist in the adult lung, multipotent Id2 and Sox9 distal tip lung progenitors that have the ability to generate both airway and alveolar lineages transiently exist during mouse embryonic development (~E11.5-E15.5). Here we describe in detail the steps necessary to isolate and expand multipotent Sox9 lung progenitors from lung tissue at day 12.5 (E12.5) of mouse embryonic development. Despite their transient nature in vivo, we show here that these multipotent lung progenitors can be captured and stably expanded in vitro, while maintaining their undifferentiated, proliferative state and their competence to differentiate into both airway and alveolar cell-types. In these feeder-free and serum-free conditions (named Lung Progenitor Medium-3D; “LPM-3D” hereafter), a ~95% pure Sox9 lung progenitor population can be maintained for at least 6 months, transcriptionally resemble the in vivo native distal tip embryonic lung progenitors and retain a normal karyotype. Moreover, the expanded mouse lung progenitors can differentiate into both alveolar and airway lineages in vitro and in vivo, validating their multipotency. A single investigator with expertise in developmental biology and cell culture can execute this protocol— initial cell isolation from embryonic lungs takes ~4 hours and the isolated progenitors can be long-term expanded in vitro. Procedures for passaging (~2 hours) and the cryopreservation of Sox9 lung multipotent progenitors are also provided. The remit of this protocol is to enable the derivation of a highly-pure multipotent Sox9 lung progenitor population. However, it is important to note that methodologies to enable their rapid and efficient differentiation into specific airway and alveolar cell-types or 3D organoids with complete recapitulation of lung native architecture and functionalities have yet to be developed. In the future, we believe that these in vitroexpanded Sox9 multipotent lung progenitors could be used as building blocks to assemble such sophisticated organoids comprising both airway and alveolar lineages with proper tissue anatomy. Nature Methods: doi:10.1038/nmeth.4498 3 Reagents Isolation of E12.5 mouse embryonic lung progenitors Sox9-IRES-GFP (Sox9GFP; JAX 030137) or Wild-Type (C57BL/6J; JAX 000664) mouse strains Molecular grade Ethanol 100% Sterile 60 mL syringe (BD, 309653) Sterile 20 mL syringe (BD, 309661) Membrane Filter 0.22μm (Millipore, GSWP04700) DPBS (Thermofisher, 14190144) 50 mL Falcon Tubes (Corning, 352070) 15 mL Falcon Tubes (Corning, 352096) 10cm Tissue Culture Dish (Corning, 353003) 100μm cell strainer (Corning, 352360) 40μm cell strainer (Corning, 352340) 5ml round bottom tubes with cell strainer (Corning, 352235) Sterile Low-bind 1.5 mL tubes (Eppendorf, 022431081) Dissociation Buffer •Collagenase I 100U/mL (Thermofisher, 17100017) •Collagenase II 100U/mL (Thermofisher, 17101015) •Dnase 100U/mL (Roche, 04716728001) •Trypsin 50U/mL (Thermofisher, 27250018) •Phenol red-free Leibovitz L-15 (Thermofisher, 21083027) Collection Buffer •Sterile 20% FBS (Hyclone, SH30071) •Phenol red-free Leibovitz L-15 (Thermofisher, 21083027) Suspension Buffer •Sterile 2% FBS (Hyclone, SH30071) •HEPES 25mM (Thermofisher, 15630080) •EDTA 2mM (Thermofisher, 15575020) Nature Methods: doi:10.1038/nmeth.4498 4 •Phenol red-free Leibovitz L-15 (Thermofisher, 21083027) •DAPI 0.1μg/mL (Sigma, D9542) (before FACS sorting only) FACS Buffer •BSA 0.2% (Sigma, A9418) •Sterile 2% FBS (Hyclone, SH30071) •Phenol red-free Leibovitz L-15 (Thermofisher, 21083027) Post-Sorting Collection Buffer •Suspension Buffer •Sterile 20% FBS (Hyclone, SH30071) •RNasin Ribonuclease Inhibitor 40U/μL (Promega, N2115) •G418 50μg/mL (Thermofisher, 11811031) Antibodies •anti-Cdh1 e660 (eBioscience, 50-3249-82) •anti-Pecam1/CD31 e450 (eBioscience, 480311) •anti-Ptprc/CD45 e450 (eBioscience, 480451) •anti-Ter119 e450 (eBioscience, 485921) Primary Sox9 lung progenitor cell culture, passaging and cryopreservation 15 mL Falcon Tubes (Corning, 352096) 24 well Plates (Corning, 353047) Advanced DMEM/F12 (Thermofisher, 12634010) Growth Factor Reduced, Phenol Red-Free Matrigel 10mg/mL (Corning, 356231) LPM3D Medium (store at 4°C, use before 10days) •Advanced DMEM/F12 (Thermofisher, 12634010) •Fgf10 50ng/mL (R&D, 6224-FG-025) •Fgf9 50ng/mL (R&D, 7399-F9-025) •Egf 50ng/mL (R&D, 2028-EG-200) •CHIR99021 3μM (Tocris, 4423) •BIRB796 1μM (Tocris, 5989) •Y27632 10μM (Tocris, 1254) •A8301 1μM (Tocris, 2939) Nature Methods: doi:10.1038/nmeth.4498 5 •Heparin 5μg/mL (Sigma, H3149) •Insulin 10μg/mL (Roche, 11376497001) •Transferrin 15μg/mL (Roche, 10652202001) •PenStrep 1x (Thermofisher, 15140122) •Glutamine 1x (Thermofisher, 25030081) •Anti-Anti (optional) 1x (Thermofisher, 15240062) •G418 (optional) 50μg/mL (Thermofisher, 11811031) Dissociation Buffer for Passaging •Dispase II (Thermofisher, 17105041) 0.5mg/mL •Collagenase IV (Thermofisher, 17104019) 0.5mg/mL •Papain (Worthington, LS003118) 0.5mg/mL •Advanced DMEM/F12 (Thermofisher, 12634010) Freezing Medium •90% FBS (Hyclone, SH30071) •10% DMSO (Sigma, C6295) •Advanced DMEM/F12 (Thermofisher, 12634010) CryoTube vials 1.8 mL (Thermo Scientific Nunc, 377267) Equipment Sterile Binocular Stereo Zoom Microscope (Leica) Sterile dissection forceps (Dumont, 11251-10) Sterile dissecting scissors (Thermofisher) Disposable Transfer Pipettes (VWR) Refrigerated Microcentrifuge (Eppendorf) BD FACS Aria II cytometer or higher Hemocytometer Water bath (shaking) set at 37°C Refrigerated centrifuge 5804R (Eppendorf) Incubator at 37°C (20% O2 and 5% CO2) Laminar flow hood Mr. FrostyTM freezing container (Thermo Fisher Scientific, 5100-0001) Nature Methods: doi:10.1038/nmeth.4498

3D culture of multipotent Sox9+ mouse embryonic lung progenitors: Isolation, Expansion and Cryopreservation