Long‐term expanding human airway organoids for disease modeling

Resource14 January 2019Open Access Transparent process Long-term expanding human airway organoids for disease modeling Norman Sachs Norman Sachs Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Angelos Papaspyropoulos Angelos Papaspyropoulos Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Domenique D Zomer-van Ommen Domenique D Zomer-van Ommen Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Inha Heo Inha Heo Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Lena Böttinger Lena Böttinger Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Dymph Klay Dymph Klay St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands Search for more papers by this author Fleur Weeber Fleur Weeber The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Guizela Huelsz-Prince Guizela Huelsz-Prince FOM Institute AMOLF, Amsterdam, The Netherlands Search for more papers by this author Nino Iakobachvili Nino Iakobachvili Maastricht University, Maastricht, The Netherlands Search for more papers by this author Gimano D Amatngalim Gimano D Amatngalim Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Joep de Ligt Joep de Ligt orcid.org/0000-0002-0348-419X UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Arne van Hoeck Arne van Hoeck orcid.org/0000-0002-6570-1452 UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Natalie Proost Natalie Proost Mouse Clinic for Cancer and Aging (MCCA) Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Marco C Viveen Marco C Viveen UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Anna Lyubimova Anna Lyubimova Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Luc Teeven Luc Teeven Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Sepideh Derakhshan Sepideh Derakhshan Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Jeroen Korving Jeroen Korving Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Harry Begthel Harry Begthel Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Johanna F Dekkers Johanna F Dekkers Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Kuldeep Kumawat Kuldeep Kumawat Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Emilio Ramos Emilio Ramos Hubrecht Organoid Technology, Utrecht, The Netherlands Search for more papers by this author Matthijs FM van Oosterhout Matthijs FM van Oosterhout St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands Search for more papers by this author G Johan Offerhaus G Johan Offerhaus UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Dominique J Wiener Dominique J Wiener Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Eduardo P Olimpio Eduardo P Olimpio FOM Institute AMOLF, Amsterdam, The Netherlands Search for more papers by this author Krijn K Dijkstra Krijn K Dijkstra The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Egbert F Smit Egbert F Smit The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Maarten van der Linden Maarten van der Linden Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Sridevi Jaksani Sridevi Jaksani Hubrecht Organoid Technology, Utrecht, The Netherlands Search for more papers by this author Marieke van de Ven Marieke van de Ven Mouse Clinic for Cancer and Aging (MCCA) Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Jos Jonkers Jos Jonkers orcid.org/0000-0002-9264-9792 Mouse Clinic for Cancer and Aging (MCCA) Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Anne C Rios Anne C Rios Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Search for more papers by this author Emile E Voest Emile E Voest The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Coline HM van Moorsel Coline HM van Moorsel St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands Search for more papers by this author Cornelis K van der Ent Cornelis K van der Ent Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Edwin Cuppen Edwin Cuppen orcid.org/0000-0002-0400-9542 UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Alexander van Oudenaarden Alexander van Oudenaarden Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Frank E Coenjaerts Frank E Coenjaerts UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Linde Meyaard Linde Meyaard Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Louis J Bont Louis J Bont Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Peter J Peters Peter J Peters Maastricht University, Maastricht, The Netherlands Search for more papers by this author Sander J Tans Sander J Tans FOM Institute AMOLF, Amsterdam, The Netherlands Search for more papers by this author Jeroen S van Zon Jeroen S van Zon FOM Institute AMOLF, Amsterdam, The Netherlands Search for more papers by this author Sylvia F Boj Sylvia F Boj Hubrecht Organoid Technology, Utrecht, The Netherlands Search for more papers by this author Robert G Vries Robert G Vries Hubrecht Organoid Technology, Utrecht, The Netherlands Search for more papers by this author Jeffrey M Beekman Jeffrey M Beekman Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Hans Clevers Corresponding Author Hans Clevers [email protected] orcid.org/0000-0002-3077-5582 Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Search for more papers by this author Norman Sachs Norman Sachs Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Angelos Papaspyropoulos Angelos Papaspyropoulos Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Domenique D Zomer-van Ommen Domenique D Zomer-van Ommen Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Inha Heo Inha Heo Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Lena Böttinger Lena Böttinger Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Dymph Klay Dymph Klay St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands Search for more papers by this author Fleur Weeber Fleur Weeber The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Guizela Huelsz-Prince Guizela Huelsz-Prince FOM Institute AMOLF, Amsterdam, The Netherlands Search for more papers by this author Nino Iakobachvili Nino Iakobachvili Maastricht University, Maastricht, The Netherlands Search for more papers by this author Gimano D Amatngalim Gimano D Amatngalim Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Joep de Ligt Joep de Ligt orcid.org/0000-0002-0348-419X UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Arne van Hoeck Arne van Hoeck orcid.org/0000-0002-6570-1452 UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Natalie Proost Natalie Proost Mouse Clinic for Cancer and Aging (MCCA) Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Marco C Viveen Marco C Viveen UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Anna Lyubimova Anna Lyubimova Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Luc Teeven Luc Teeven Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Sepideh Derakhshan Sepideh Derakhshan Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Jeroen Korving Jeroen Korving Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Harry Begthel Harry Begthel Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Johanna F Dekkers Johanna F Dekkers Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Kuldeep Kumawat Kuldeep Kumawat Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Emilio Ramos Emilio Ramos Hubrecht Organoid Technology, Utrecht, The Netherlands Search for more papers by this author Matthijs FM van Oosterhout Matthijs FM van Oosterhout St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands Search for more papers by this author G Johan Offerhaus G Johan Offerhaus UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Dominique J Wiener Dominique J Wiener Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Eduardo P Olimpio Eduardo P Olimpio FOM Institute AMOLF, Amsterdam, The Netherlands Search for more papers by this author Krijn K Dijkstra Krijn K Dijkstra The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Egbert F Smit Egbert F Smit The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Maarten van der Linden Maarten van der Linden Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Sridevi Jaksani Sridevi Jaksani Hubrecht Organoid Technology, Utrecht, The Netherlands Search for more papers by this author Marieke van de Ven Marieke van de Ven Mouse Clinic for Cancer and Aging (MCCA) Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Jos Jonkers Jos Jonkers orcid.org/0000-0002-9264-9792 Mouse Clinic for Cancer and Aging (MCCA) Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Anne C Rios Anne C Rios Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Search for more papers by this author Emile E Voest Emile E Voest The Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Coline HM van Moorsel Coline HM van Moorsel St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands Search for more papers by this author Cornelis K van der Ent Cornelis K van der Ent Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Edwin Cuppen Edwin Cuppen orcid.org/0000-0002-0400-9542 UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Alexander van Oudenaarden Alexander van Oudenaarden Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Frank E Coenjaerts Frank E Coenjaerts UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Linde Meyaard Linde Meyaard Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Louis J Bont Louis J Bont Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Peter J Peters Peter J Peters Maastricht University, Maastricht, The Netherlands Search for more papers by this author Sander J Tans Sander J Tans FOM Institute AMOLF, Amsterdam, The Netherlands Search for more papers by this author Jeroen S van Zon Jeroen S van Zon FOM Institute AMOLF, Amsterdam, The Netherlands Search for more papers by this author Sylvia F Boj Sylvia F Boj Hubrecht Organoid Technology, Utrecht, The Netherlands Search for more papers by this author Robert G Vries Robert G Vries Hubrecht Organoid Technology, Utrecht, The Netherlands Search for more papers by this author Jeffrey M Beekman Jeffrey M Beekman Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands Search for more papers by this author Hans Clevers Corresponding Author Hans Clevers [email protected] orcid.org/0000-0002-3077-5582 Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Search for more papers by this author Author Information Norman Sachs1, Angelos Papaspyropoulos1, Domenique D Zomer-van Ommen2, Inha Heo1, Lena Böttinger1, Dymph Klay3, Fleur Weeber4, Guizela Huelsz-Prince5, Nino Iakobachvili6, Gimano D Amatngalim2, Joep Ligt7, Arne Hoeck7, Natalie Proost8, Marco C Viveen7, Anna Lyubimova1, Luc Teeven1, Sepideh Derakhshan2, Jeroen Korving1, Harry Begthel1, Johanna F Dekkers1, Kuldeep Kumawat2, Emilio Ramos9, Matthijs FM Oosterhout3, G Johan Offerhaus7, Dominique J Wiener1, Eduardo P Olimpio5, Krijn K Dijkstra4, Egbert F Smit4, Maarten Linden2, Sridevi Jaksani9, Marieke Ven8, Jos Jonkers8, Anne C Rios10, Emile E Voest4, Coline HM Moorsel3, Cornelis K Ent2, Edwin Cuppen7, Alexander Oudenaarden1, Frank E Coenjaerts7, Linde Meyaard2, Louis J Bont2, Peter J Peters6, Sander J Tans5, Jeroen S Zon5, Sylvia F Boj9, Robert G Vries9, Jeffrey M Beekman2 and Hans Clevers *,1,10 1Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands 2Wilhelmina Children's Hospital and UMC Utrecht, Utrecht, The Netherlands 3St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands 4The Netherlands Cancer Institute, Amsterdam, The Netherlands 5FOM Institute AMOLF, Amsterdam, The Netherlands 6Maastricht University, Maastricht, The Netherlands 7UMC Utrecht, Utrecht, The Netherlands 8Mouse Clinic for Cancer and Aging (MCCA) Preclinical Intervention Unit, The Netherlands Cancer Institute, Amsterdam, The Netherlands 9Hubrecht Organoid Technology, Utrecht, The Netherlands 10Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands *Corresponding author. Tel: +31 30 2121800; E-mail: [email protected] The EMBO Journal (2019)38:e100300https://doi.org/10.15252/embj.2018100300 See also: M Paschini & CF Kim (February 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease. Synopsis To date, persistent in vitro culture of adult human lung epithelium remains elusive. In this methods resource article, culture conditions to maintain three-dimensional pulmonary tissue long-term are reported and applied to recapitulate related diseases. Culture conditions for long-term expansion of healthy, hereditary disease and malignant human airway epithelial organoids. Airway organoids are amenable for medium-throughput drug screening. Airway organoids readily allow modeling of viral infection. Introduction To date, several approaches have been explored to generate mammalian airway organoids (Barkauskas et al, 2017). In 1993, Puchelle and colleagues described the first self-organizing 3D structures of adult human airway epithelium in collagen (Benali et al, 1993). A first description of the generation of lung organoids from human iPS (induced pluripotent stem) cells was given by Rossant and colleagues and included the use of CFTR-mutant iPS cells as a proof of concept for modeling CF (Wong et al, 2012). Snoeck and colleagues designed an improved four-stage protocol (Huang et al, 2014) and later generated lung bud organoids from human pluripotent stem cells that recapitulate fetal lung development (Chen et al, 2017). Spence and colleagues (Dye et al, 2015) followed a modified trajectory to generate mature lung organoids, containing basal, ciliated, and club cells. These cultures were stable for up to several months and resembled proximal airways. Konishi et al (2016) improved on the iPS cell-derived generation of multi-ciliated airway cells in 3D, and McCauley et al (2017) generated CF patient iPS cell-derived airway organoids for disease modeling. Hogan and colleagues reported the first adult stem cell-based murine bronchiolar lung organoid culture protocol, involving Matrigel supplemented with EGF (Rock et al, 2009). Single basal cells isolated from the trachea grew into tracheospheres consisting of a pseudostratified epithelium with basal and ciliated luminal cells. These organoids could be passaged at least twice. No mature club, neuroendocrine, or mucus-producing cells were observed (Rock et al, 2009). In a later study, this clonal 3D organoid assay was used to demonstrate that IL-6 treatment resulted in the formation of ciliated cells at the expense of secretory and basal cells (Tadokoro et al, 2014). Tschumperlin and colleagues combined human adult primary bronchial epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in 3D to generate airway organoids (Tan et al, 2017). Under these conditions, randomly seeded mixed cell populations underwent rapid condensation to self-organize into discrete epithelial and endothelial structures that were stable up to 4 weeks of culture (Tan et al, 2017). Hild and Jaffe have described a protocol for the culture of bronchospheres from primary human airway basal cells. Mature bronchospheres are composed of functional multi-ciliated cells, mucin-producing secretory cells, and airway basal cells (Hild & Jaffe, 2016). Mou et al (2016) expanded basal cells of mouse and human airway epithelium in 2D that allowed subsequent differentiation under air–liquid interphase conditions. And finally, Nikolic et al (2017) designed conditions to expand human fetal lung epithelium as self-renewing organoids. Since none of these approaches allows long-term expansion of pseudostratified airway epithelium from adult human individuals in vitro, we set out to establish such culture conditions and model a variety of pulmonary diseases. Results Generation and characterization of human airway organoids We collected macroscopically inconspicuous lung tissue from non-small-cell lung cancer (NSCLC) patients undergoing medically indicated surgery and isolated epithelial cells through mechanical and enzymatic tissue disruption (see Materials and Methods). Following our experience with generating organoids from other adult human tissues (Sato et al, 2011; Karthaus et al, 2014; Boj et al, 2015; Huch et al, 2015; van de Wetering et al, 2015) and recent developments in the field (Mou et al, 2016; Tadokoro et al, 2016; Balasooriya et al, 2017), we embedded isolated cells in basement membrane extract (BME) and activated/blocked signaling pathways important for airway epithelium (Table EV1). Under optimized conditions, 3D organoids formed within several days (94% success rate, n = 18). The organoids were composed of a polarized, pseudostratified airway epithelium containing basal, secretory, and multi-ciliated cells (Fig 1A and B, Appendix Fig S1A, Movie EV1) and were termed airway organoids (AOs). Cells that stained for basal cell marker keratin-5 (KRT5), club cell marker secretoglobin family 1A member 1 (SCGB1A1), cilia marker acetylated α-tubulin, or secretory cell marker mucin 5AC (MUC5AC) localized to their corresponding in vivo positions (Fig 1C, Appendix Fig S1B). Secretory cells as well as cilia were functional as evidenced by time-lapse microscopy showing beating cilia and whirling mucus (Movies EV2 and EV3). Figure 1. Characterization of airway organoids Transmission electron micrograph of an AO cross section showing the polarized, pseudostratified epithelium containing basal, secretory, brush, and multi-ciliated cells. Details display apical microvilli and cilia with their characteristic microtubule structure. Scale bars equal 10 μm, 2 μm, and 500 nm. See also Appendix Fig S1A and Movies EV1–EV3. Scanning electron micrograph of a partially opened AO visualizing its 3D architecture, as well as basal and apical ultrastructure. Details display apical surfaces of secretory and multi-ciliated cells. Scale bars equal 50 μm (overview) and 2 μm (details). Immunofluorescent sections of AOs showing markers for basal cells (KRT5), cilia (acetylated α-tubulin), secretory cells (MUC5AC), and club cells (SCGB1A1). KRT5 is present exclusively in basally localized cells, while cilia, MUC5AC, and SCGB1A1 localize luminally. Counterstained is the actin cytoskeleton (red). Scale bar equals 10 μm. See Appendix Fig S1B for IHC images. Luminescent cell viability assay comparing proliferative capacity of two independently generated AO lines at early, mid-, and late passage numbers. Per group, 3,000 cells were seeded and their expansion was measured at the indicated time points. Error bars represent standard deviations of technical triplicates. Quantification of cell types in AO lines at early and late passage (P5 vs. P19) as determined by immunofluorescence using the indicated markers. The number of basal cells, club cells, ciliated cells, and secretory cells does not differ significantly between early and late passage AOs. Data shown are representatives of at least three independent experiments. Error bars indicate s.e.m. Download figure Download PowerPoint Airway organoids were passaged by mechanical disruption at 1:2 to 1:4 ratios every other week for > 1 year, proliferating at comparable rates regardless of passage number (Fig 1D) while retaining similar frequencies of basal, club, multi-ciliated, and secretory cells (Fig 1E, Appendix Fig S1C). Comparative RNA sequencing of early and late passage AOs confirmed these findings with dozens of airway cell type-specific genes retaining their respective expression patterns (Appendix Fig S1D and E, Table EV2). The airway epithelial composition of 10 independently established AO lines was validated by quantitative PCR (qPCR): While expressing the general lung marker NKX2-1 and several airway-specific markers, AOs expressed virtually no HOXA5 [a bona fide lung mesenchyme gene (Hrycaj et al, 2015)] or alveolar transcripts (Appendix Fig S2A). While AO transcriptomes were strongly enriched for bulk lung and small airway epithelial signature (Appendix Fig S2B) as shown by gene set enrichment analysis (GSEA), cell type-specific signatures were limited to basal, club, and ciliated cells (Appendix Fig S2C, Table EV3). Accordingly, hallmark lung genes encoding for keratins, secretoglobins, dyneins, and others were consistently among the highest AO enriched genes (Appendix Fig S2D). Elevated levels of WNT3A transcripts explained why AOs—in contrast to intestinal organoids (Sato et al, 2011)—did not require the addition of exogenous WNT3A to the culture media. Manipulation of WNT signaling resulted in dramatic changes in the expression of WNT target genes (Appendix Fig S2E). Withdrawal of the Wnt amplifier R-spondin terminated AO expansion after 3–4 passages (Appendix Fig S2E), similar to withdrawal of fibroblast growth factors (Appendix Fig S2F). Taken together, our culture conditions allow long-term expansion of AOs while retaining major characteristics of the in vivo epithelium. Airway organoids from patients with cystic fibrosis recapitulate central disease features and swell upon modulation of CFTR as well as activation of TMEM16A Rectal organoids are being successfully used as functional model for cystic fibrosis (CF; Noordhoek et al, 2016), a multi-organ disease with extensive phenotypic variability caused by mutations in the CF transmembrane conductance regulator gene (CFTR; Ratjen et al, 2015). Following opening of the CFTR channel by cAMP-inducing agents (e.g., forskolin), anions and fluid are transported to the organoid lumen resulting in rapid organoid swelling (Dekkers et al, 2013), allowing personalized in vitro drug screenings (Dekkers et al, 2016). The current gold standard for modeling the primarily affected CF lung epithelium is air–liquid interface (ALI) culture of human bronchial epithelial cells, a system with limited cell expansion and lengthy differentiation protocols (Fulcher et al, 2005). While iPS cell-derived AOs have been used for functional assessment of CFTR, their generation is also considerably long (McCauley et al, 2017). To assess adult stem cell AOs for CF disease modeling, we applied forskolin and observed a dose-dependent swelling response that was largely, but not entirely, abrogated upon chemical inhibition of CFTR (Fig 2A, Appendix Fig S3A), indicating the presence of additional ion channels. Indeed, AOs—but not rectal organoids—swell upon addition of Eact (Fig 2B, Appendix Fig S3A), an activator of the chloride channel TMEM16A (Namkung et al, 2011; Sondo et al, 2014). Figure 2. Airway organoids to study cystic fibrosis Box-and-whisker plot showing concentration-dependent forskolin-induced swelling of AOs in the absence and presence of CFTR inhibitors CFTRinh-172 and GlyH101. Upon CFTR inhibition, swelling is noticeably decreased but not absent. Shown are pooled data from three different AO lines used in each of three independent experiments. Whiskers indicate smallest and largest values, boxes indicate 25th to 75th percentile, and horizontal solid line indicates median. AUC, area under the curve. Box-and-whisker plot showing concentration-dependent Eact-induced swelling of AOs, but not rectal organoids (black outlines). Forskolin causes swelling in both organoid types (gray outlines). Shown are pooled data from three different AO and two different rectal organoid lines used in three to four independent experiments. Whiskers indicate smallest and largest values, boxes indicate 25th to 75th percentile, and horizontal solid line indicates median. Swelling was linear for 2 h for AOs, but only 1 h for rectal organoids. See Appendix Fig S3A for respective time course plots. Representative histological sections of periodic acid–Schiff (PAS)-stained organoids from a CF patient with CFTRF508del/F508del mutation. Note the thick layer of PAS-positive polysaccharides apically lining the airway epithelium. Rectal organoids were generated from rectal biopsies; AOs were generated from broncho-alveolar lavages (BALs). Scale bars equal 50 μm. See Appendix Fig S3B for PAS-stained wild-type and CFTRR334W/R334W organoid sections. Box-and-whisker plot showing swelling assays of several CF patient AO lines carrying the indicated CFTR mutations (G542X is a premature stop associated with severe disease and no functional CFTR protein; F508del is the most common CFTR mutation in subjects with CF and severely reduces apical trafficking and function, leading to severe disease (high sweat chloride, high pancreas insufficiency, high pseudomonas infection rate); R334W is a milder CFTR mutation associated as indicated by lower pseudomonas infection rates and pancreas sufficiency with reduced ion channel conductivity, normal apical expression, and some residual function). Forskolin-induced swelling rarely exceeds vehicle control