An organoid‐derived bronchioalveolar model for SARS‐CoV‐2 infection of human alveolar type II‐like cells
2020; Springer Nature; Volume: 40; Issue: 5 Linguagem: Inglês
10.15252/embj.2020105912
ISSN1460-2075
AutoresMart M. Lamers, Jelte van der Vaart, Kèvin Knoops, Samra Riesebosch, Tim I. Breugem, Anna Z. Mykytyn, Joep Beumer, Debby Schipper, Karel Bezstarosti, Charlotte D. Koopman, Nathalie Gröen, Raimond B. G. Ravelli, Hans Q. Duimel, Jeroen Demmers, Georges M. G. M. Verjans, Marion Koopmans, Mauro J. Muraro, Peter J. Peters, Hans Clevers, Bart L. Haagmans,
Tópico(s)Polyomavirus and related diseases
ResumoArticle11 January 2021Open Access Transparent process An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells Mart M Lamers Mart M Lamers orcid.org/0000-0002-1431-4022 Viroscience Department, Erasmus University Medical Center, Rotterdam, The NetherlandsThese authors contributed equally to this work Search for more papers by this author Jelte van der Vaart Jelte van der Vaart orcid.org/0000-0002-4126-8420 Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The NetherlandsThese authors contributed equally to this work Search for more papers by this author Kèvin Knoops Kèvin Knoops The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Samra Riesebosch Samra Riesebosch Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Tim I Breugem Tim I Breugem Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Anna Z Mykytyn Anna Z Mykytyn Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Joep Beumer Joep Beumer orcid.org/0000-0002-2055-4444 Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The Netherlands Search for more papers by this author Debby Schipper Debby Schipper Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Karel Bezstarosti Karel Bezstarosti Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Charlotte D Koopman Charlotte D Koopman Single Cell Discoveries, Utrecht, The Netherlands Search for more papers by this author Nathalie Groen Nathalie Groen Single Cell Discoveries, Utrecht, The Netherlands Search for more papers by this author Raimond B G Ravelli Raimond B G Ravelli The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Hans Q Duimel Hans Q Duimel The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Jeroen A A Demmers Jeroen A A Demmers Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Georges M G M Verjans Georges M G M Verjans orcid.org/0000-0002-2465-2674 Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Marion P G Koopmans Marion P G Koopmans Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Mauro J Muraro Mauro J Muraro Single Cell Discoveries, Utrecht, The Netherlands Search for more papers by this author Peter J Peters Peter J Peters The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, 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, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The NetherlandsThese authors contributed equally to this work Search for more papers by this author Bart L Haagmans Corresponding Author Bart L Haagmans [email protected] orcid.org/0000-0001-6221-2015 Viroscience Department, Erasmus University Medical Center, Rotterdam, The NetherlandsThese authors contributed equally to this work Search for more papers by this author Mart M Lamers Mart M Lamers orcid.org/0000-0002-1431-4022 Viroscience Department, Erasmus University Medical Center, Rotterdam, The NetherlandsThese authors contributed equally to this work Search for more papers by this author Jelte van der Vaart Jelte van der Vaart orcid.org/0000-0002-4126-8420 Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The NetherlandsThese authors contributed equally to this work Search for more papers by this author Kèvin Knoops Kèvin Knoops The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Samra Riesebosch Samra Riesebosch Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Tim I Breugem Tim I Breugem Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Anna Z Mykytyn Anna Z Mykytyn Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Joep Beumer Joep Beumer orcid.org/0000-0002-2055-4444 Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The Netherlands Search for more papers by this author Debby Schipper Debby Schipper Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Karel Bezstarosti Karel Bezstarosti Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Charlotte D Koopman Charlotte D Koopman Single Cell Discoveries, Utrecht, The Netherlands Search for more papers by this author Nathalie Groen Nathalie Groen Single Cell Discoveries, Utrecht, The Netherlands Search for more papers by this author Raimond B G Ravelli Raimond B G Ravelli The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Hans Q Duimel Hans Q Duimel The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, The Netherlands Search for more papers by this author Jeroen A A Demmers Jeroen A A Demmers Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Georges M G M Verjans Georges M G M Verjans orcid.org/0000-0002-2465-2674 Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Marion P G Koopmans Marion P G Koopmans Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Mauro J Muraro Mauro J Muraro Single Cell Discoveries, Utrecht, The Netherlands Search for more papers by this author Peter J Peters Peter J Peters The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, 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, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The NetherlandsThese authors contributed equally to this work Search for more papers by this author Bart L Haagmans Corresponding Author Bart L Haagmans [email protected] orcid.org/0000-0001-6221-2015 Viroscience Department, Erasmus University Medical Center, Rotterdam, The NetherlandsThese authors contributed equally to this work Search for more papers by this author Author Information Mart M Lamers1, Jelte Vaart2, Kèvin Knoops3, Samra Riesebosch1, Tim I Breugem1, Anna Z Mykytyn1, Joep Beumer2, Debby Schipper1, Karel Bezstarosti4, Charlotte D Koopman5, Nathalie Groen5, Raimond B G Ravelli3, Hans Q Duimel3, Jeroen A A Demmers4, Georges M G M Verjans1, Marion P G Koopmans1, Mauro J Muraro5, Peter J Peters3, Hans Clevers *,2 and Bart L Haagmans *,1 1Viroscience Department, Erasmus University Medical Center, Rotterdam, The Netherlands 2Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, The Netherlands 3The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, Maastricht, The Netherlands 4Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands 5Single Cell Discoveries, Utrecht, The Netherlands *Corresponding author. Tel: +31 30 2121800; E-mail: [email protected] *Corresponding author. Tel: +31 10 7044004; E-mail: [email protected] The EMBO Journal (2021)40:e105912https://doi.org/10.15252/embj.2020105912 See also: SL Leibel & X Sun (March 2021) 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 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air–liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens. Synopsis In vitro investigation of human lung alveolar physiology has remained difficult. Here, establishment and profiling of an organoid-derived bronchioalveolar tissue culture allows analysis of SARS-CoV-2 cellular tropism and testing of treatment strategies for respiratory syndromes. Bronchioalveolar-like cells derived from self-renewing human fetal lung organoids give rise to a 2D human bronchioalveolar-like model with air-exposed apical side. In these cultures, alveolar type-2-like cells are particularly permissive to SARS-CoV-2 infection. SARS-CoV-2 infection induces a type-I/III interferon host response in vitro. SARS-CoV-2 viral titers are sensitive to low dose interferon lamda 1 treatment. Introduction The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally within several months after an initial outbreak in Wuhan, China, in December 2019 (Zhu et al, 2020). The World Health Organization (WHO) declared SARS-CoV-2 a pandemic on March 11, 2020. As of November 1, 2020, > 46,000,000 patients were confirmed including > 1,200,000 deaths (WHO, 2020). SARS-CoV-2 belongs to the Sarbecovirus subgenus (genus Betacoronavirus, family Coronaviridae), together with SARS-CoV (Andersen et al, 2020; Coronaviridae Study Group of the International Committee on Taxonomy of V, 2020; Lu et al, 2020). SARS-CoV-2 causes coronavirus disease 2019 (COVID-19), an influenza-like illness associated with a broad spectrum of clinical respiratory syndromes, ranging from mild upper airway symptoms to a life-threatening viral pneumonia (Chen et al, 2020; Wang et al, 2020; Wolfel et al, 2020; Xu et al, 2020). This pneumonia fulfills the radiographic and histological criteria for acute respiratory distress syndrome (ARDS). Classically, ARDS is caused by damage to the alveoli, more specifically to the alveolar type I cells that are critical in the oxygen transport from the alveoli to the blood, triggered through an inflammatory response to pathogens or toxins (Ware & Matthay, 2000; Thompson et al, 2017; Ackermann et al, 2020; Barton et al, 2020; Menter et al, 2020; Raptis et al, 2020). The early events that lead to respiratory failure as a result of coronavirus infection are initiated following virus replication (tenOever, 2016). After virus entry, which is determined by receptor availability, viral replication generates pathogen-associated molecular patterns, specifically RNA structures, that can be detected by pattern recognition receptors (PRRs). PRR signaling subsequently leads to the transcription of antiviral genes; the response triggered varies between pulmonary cell types (preprint: Ravindra et al, 2020). Therefore, disease outcome can be greatly influenced by the viral target cell, which is determined largely by the presence of CoV entry receptor. In the lungs, angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor (Hoffmann et al, 2020; Walls et al, 2020; Wrapp et al, 2020; Zhou et al, 2020), is expressed mainly in ciliated cells and alveolar type II cells (Jia et al, 2005; Hikmet et al, 2020; Qi et al, 2020). Ciliated cells, alveolar type II cells, but also alveolar type I cells, have been identified as SARS-CoV-2 target cells in animal models and in COVID-19 patients (Hou et al, 2020; Rockx et al, 2020; Xu et al, 2020). In previous work, we confirmed ciliated cells as a viral target cell using organoid-derived bronchial airway cultures (Lamers et al, 2020). In contrast to ciliated cells, alveolar cells are notoriously difficult to culture, thus limiting our understanding of COVID-19, but also of other respiratory virus infections. Currently, standardized methods use immortal cell lines or primary alveolar cells to study disease (van den Bogaard et al, 2009). Immortal cell lines, however, do not fully recapitulate the complexity of the alveolar space and epithelium. Primary alveolar cells partially capture this complexity but remain incapable of undergoing passaging and quickly lose their in vivo phenotype which restricts the model by the availability of donor material (Logan & Desai, 2015; Zacharias et al, 2018). Importantly, primary adult alveolar cultures were recently shown to be poorly permissive to SARS-CoV-2 infection, emphasizing the urgent need to develop a susceptible SARS-CoV-2 alveolar infection model (Hou et al, 2020; Hui et al, 2020). Some induced pluripotent stem cell-derived models have been able to show alveolar differentiation (Gotoh et al, 2014; Dye et al, 2015; Jacob et al, 2017; Yamamoto et al, 2017; de Carvalho et al, 2019; van Riet et al, 2020), yet differentiation from primary lung cells has remained challenging. Here, we describe a bronchioalveolar-like 2D air–liquid interface cell system by confocal and electron microscopy and mRNA expression analysis. In this model, alveolar cells, but also basal cells, and rare neuroendocrine cells, are grown from 3D self-renewing lung bud tip progenitor organoids. In this system, SARS-CoV-2 replication competence, tropism, and induced host responses were determined and compared to 2D differentiated small airway epithelium. Results Replication competence and tropism of SARS-CoV-2 in 2D differentiated small airway epithelium Human bronchial airway epithelial (HAE) cell cultures are an established primary epithelial lung cell model to study respiratory virus infections. In these cultures, primary basal cells can be differentiated at air–liquid interface into mature airway cell types. It has already been shown that 3D self-renewing airway organoids can also be used as a source of basal cells in this culture system (Zhou et al, 2018; Sachs et al, 2019; Lamers et al, 2020). We first established this system using small airway basal cells (Appendix Fig S1A–C) and demonstrated that SARS-CoV-2 readily infects human small airway organoid-derived epithelium cultured in 2D at air–liquid interface (ALI) (Fig 1). SARS-CoV-2 grew to relatively high titers on these cells, as shown by titration of VeroE6 cells (Fig 1A and B) and viral RNA quantification (Appendix Fig S1D). Shedding of virus occurred predominantly apically (Appendix Fig S1E and F). As we have previously shown for large airway (bronchial) cultures (Lamers et al, 2020), ciliated cells were extensively targeted by SARS-CoV-2 (Fig 1C). In addition, we noted rare infection of CC10+ club cells (Fig 1D), but no infection of MUC5AC+ goblet cells (Fig 1E). Figure 1. SARS-CoV-2 infects human organoid-derived 2D small airway cultures A, B. Infectious virus titers can be observed by virus titrations on VeroE6 cells of apical washes at 2, 24, 48, and 72 h after infection at MOI 0.01 (A) or 0.1 (B) with SARS-CoV-2 (red). The dotted line indicates the lower limit of detection. Error bars represent SEM. N = 4. H p.i. = hours post-infection. C–E. Immunofluorescent staining of SARS-CoV-2 infected differentiated small airway cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with the ciliated cell marker AcTUB (green; (C, D, E)) and club cell marker CC10 (blue; (D)). Phalloidin was included to stain actin (blue; (C)). Goblet cells are identified by MUC5AC (blue; (E)). Scale bars indicate 50 μm. Data information: Nuclei are stained with Hoechst or TOPRO and shown in white (C-E). Scale bars indicate 50 μm. Download figure Download PowerPoint Establishment of a 2D differentiated bronchioalveolar-like model Another 3D lung organoid system was developed by Nikolic et al (2017), but this system has not yet been applied in virology. In this system, culture conditions were established to support long-term self-renewal of multipotent SOX2+SOX9+ lung bud tip progenitor cells which in vivo differentiate into both airway and alveolar cells. We grew these lung bud tip organoids (LBT) from canalicular stage human fetal lungs 16–17 pcw (post-conception weeks). In expansion medium, which activates EGF, FGF, and WNT signaling, and inhibits BMP and TGFβ, the vast majority of cells were SOX2+SOX9+ (Fig 2A), but rare ATII-L were also detected in a subpopulation of organoids using the HTII-280 antibody which exclusively stains ATII cells in the human lung (Gonzalez et al, 2010) (Fig 2B). Organoid lines were maintained for > 14 passages without apparent change in morphology, or SOX2 and SOX9 expression. Next, we established a model that contains alveolar-like cells, which could be accessed from the apical side for infection experiments. We plated SOX2+SOX9+ progenitor cells in Transwell inserts in differentiation medium. Based on recent literature, we reasoned that addition of human canalicular stage mesenchyme would provide cues for the survival of ATII-L cells and differentiation toward an alveolar fate (Barkauskas et al, 2013; Nikolic et al, 2017; Nikolic & Rawlins, 2017; Leeman et al, 2019). After reaching confluency, cells were differentiated for at least 14 days at ALI (Fig 2C). The addition of canalicular stage mesenchymal cells in the bottom compartment of the Transwell increased the frequency of HTII-280+ cells (Appendix Fig S2A and B). After 14 days of differentiation at ALI, cultures consisted of both multilayered and single-layered squamous epithelium. The areas with a single layer of mostly thin epithelium contained cells expressing the ATI markers HOPX (Fig 2D) and HTI-56 (Fig 2E), as well as the mature ATII marker HTII-280 (Fig 2F) and LPCAT1 (Fig 2G) (Dobbs et al, 1999; Gonzalez et al, 2010; Gonzales et al, 2015). SFTPC+ cells were present in the single-layered epithelium and the top layer of the multilayered areas (Fig 2H). The multilayered areas contained TP63+ basal cells as a bottom layer (Fig 2I). Canalicular stage mesenchyme from different donors resulted in similar expression of alveolar markers (Fig EV1-EV5), whereas some variation was observed in basal cell proliferation (Fig 1EVI and J). Using mass spectrometry on apical washes, we also detected secreted surfactant proteins SFTPA1, SFTPA2, SFTPB, and SFTPD (Appendix Fig S3). mRNA expression analysis of 3D LBT and confluent 2D cultures before (2D non-ALI) and after (2D ALI) exposure to air indicated that lung bud tip progenitor markers SOX9 and SOX2 were decreased during differentiation (Fig EV2-EV5A and B), while alveolar cell markers SFTPA2, SFTPB, and HOPX gradually increased during differentiation (Fig EV2-EV5). A general increase in ATI- and ATII-related genes from 3D LBT to 2D ALI cells was observed with intermediate levels at 2D non-ALI (Fig EV2F, Tables EV1 and EV2). The composition of cells in 2D ALI cultures was different from small airway ALI cultures, as indicated in differential expression analysis (Fig 3A, Table EV3). While small airway ALI cultures were mainly expressing ciliated cell markers like DNAH10 (Fig 3A), FOXJ1, and SNTN (Fig 3B), alveolar-like ALI cultures were enriched in ATI&II makers like HOPX and SFTPA1 (Fig 3). Moreover, 2D ALI cultures were enriched in markers for pulmonary neuroendocrine, tuft, and basal cells, while small airway cultures were enriched in goblet and ciliated cell markers (Fig 3B). All together, these data indicate that the 2D ALI cultures contain both alveolar-like and bronchiolar-like cells. These cultures could therefore be characterized as bronchioalveolar-like by confocal microscopy and mRNA expression analysis. The presence of pulmonary neuroendocrine cells (PNEC) was confirmed by confocal imaging (Appendix Fig S6B). The club and ATII cell marker SCGB3A2 was also detected in both single- and multilayered areas (Appendix Fig S6C). Figure 2. Self-renewing human fetal lung bud tip progenitor organoids differentiate at air–liquid interface to alveolar type I- and type II-like cells A. Immunofluorescent staining of fetal lung bud tip progenitor organoids grown in expansion medium co-expressing stem cell markers Sox2 (green) and Sox9 (red). Phalloidin (white) was used to stain actin. B. Immunofluorescent staining of rare HTII-280 + type II pneumocytes (green) in a subpopulation of fetal lung bud tip progenitor organoids grown in expansion medium. C. Schematic of the 2D air–liquid interface bronchioalveolar-like model. D–I. Differentiated lung bud tip organoids at air–liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air–liquid interface, cells express alveolar type I (HOPX, green, (D); HTI-56, green, (E)), type II cells (HTII-280, green, (F); LPCAT1, green, (G); SFTPC (SPC), green, (H)), and basal cell (TP63 (P63), green, (I)) markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single-layered epithelium. Data information: Nuclei are stained with Hoechst (blue in (A, B) or white in (D–I)). Scale bars indicate 50 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. A comparison of different mesenchymal donors on 2D bronchioalveolar-like differentiation A–J. Immunofluorescent staining of HTII-280 (A, B), LPCAT1 (C, D), SPC (E, F), HOPX (G, H), and TP63 (I, J) after 2 weeks of differentiation at air–liquid interface in the presence of mesenchyme in the basal compartment of the Transwell. Nuclei are stained with Hoechst (blue). Scale bars represent 50 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Transcriptomic analysis of differentiation toward bronchioalveolar-like cells from lung bud tip organoids A–E. Line graphs indicating normalized read count of 3D lung bud tip organoids (3D LBT), airway organoids on transwell system with apical medium (2D non-ALI), and 2D bronchioalveolar-like air–liquid cultures (2D ALI). n = 2 and error bars represent stdev. (A, B) Graphs depicting fetal bud tip progenitor markers SOX9 and SOX2. (C, D) Graphs depicting ATII markers SFTPA2 and SFTPB. (E) Graphs depicting ATI marker HOPX. F. Heatmaps depicting top 50 enriched genes in 2D bronchioalveolar-like air–liquid cultures (2D ALI) compared to 3D lung bud tip organoids (3D LBT). N = 2 of 3D LBT, airway organoids on transwell system with apical medium (2D non-ALI), and 2D ALI. Colored bars represent Z-scores of log2-transformed values. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Comparison of bronchioalveolar culture to published datasets of AT2 cells Heatmaps depicting marker genes in 2D small airway air–liquid cultures (small airway ALI), bronchioalveolar-like air–liquid cultures (alveolar ALI), and published dataset of purified AT2 cells (purified AT2). Colored bars represent Z-scores of log2-transformed values. Barplot indicating percentage of annotated cells within a cluster. Color represents annotated cell identity. Combined current dataset and of GSM2855485 are presented. Indicated annotation is based on separate clustering. Published dataset GSM2855485 is annotated as "Purified AT2". Violin plots visualizing expression levels of marker genes for AT2 cells. AT2-like cells comprise cells annotated as AT2-like cells in current dataset (clusters 0 and 2). Purified AT2 cells are from published dataset GSM2855485. Data information: Error bars represent SEM. N = 4 for donor 1 and N = 3 for donor 2. H p.i. = hours post-infection. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Apical viral RNA and basal shedding in differentiated 2D bronchioalveolar-like cultures A, B. Viral SARS-CoV-2 RNA titers of apical washes at 2, 24, 48, and 72 h after infection at MOI 0.1 for donor 1 (A) and donor 2 (B). C, D. Infectious virus titers as observed by virus titrations on VeroE6 cells (C) and qRT–PCR (D) in basal compartments. The dotted line indicates the lower limit of detection. Data information: Error bars represent SEM. N = 4 for donor 1 and N = 3 for donor 2. H p.i. = hours post-infection. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Transmission electron microscopy analysis of 2D bronchioalveolar-like (A–F) and 2D small airway (G–K) cultures after 72 h of SARS-CoV-2 infection A–F. Pulmonary alveolar type II-like (ATII-L) cells were found in the 2D bronchioalveolar-like culture (A, B). The early infection of the ATII-L cell (C) is recognizable by the double-membranes vesicles (Dmv) and virus particles (circles). (D) Next to the lamellar bodies (Lb), the ATII-L also contained a lysosomal compartment with internal virus particles (circle). Extracellular virus particles were found in the intercellular space between the ATI-L and ATII-L cells (E), albeit at low concentration. Dashed circles indicate viral particles outside of the cell. The remnants of a dead detached cell (F) mainly showed apical-shed virus particles (in areas marked by dotted lines), whereas the basal side was relatively free of virus particles. G–K. Ciliated cells (CC; (H)) and goblet cells (GC; (K)) are visible in the cross section through the 2D small airway culture after 8 weeks of differentiation. SARS-CoV-2 replication (I) and virus shedding (J) were found to be associated particularly with ciliated cells, whereas no infections were found in goblet cells (K). Dashed circles indicate viral particles inside and outside the cell. Data information: Scale bars represent 5 μm (A, G), 2.5 μm (B, H, K), 1 μm (F), 500 nm (D, I, J), and 250 nm (C, E). Download figure Download PowerPoint Figure 3. Transcriptomic analysis of bronchioalveolar-like and small airway air–liquid interface cultures Heatmaps depicting top 30 up- and downregulated genes in 2D bronchioalveolar-like air–liquid cultures (bronchioalveolar) compared to small airway air–liquid interface cultures (small airway). Colored bars represent Z-scores of log2-transformed values. Heatmaps depicting pulmonary cell type marker genes in 2D bronchioalveolar-like air–liquid cultures (bronchioalveolar) and small airway air–liquid interface cultures (small airway). Color-coded bars indicate which cell type is marked by the presented gene. Colored bars represent Z-scores of log2-transformed values. Download figure Download PowerPoint To further investigate the diversity and frequency of the cell types in the bronchioalveolar system, we performed single-cell mRNA sequencing (Fig 4). This revealed a large alveolar-like cluster (94.3% of cells), a smaller basal cell cluster (4.9% of cells), and a rare population of PNECs (0.9% of cells) (Fig 4A). The alveolar cluster contained ATII-like cells (46.0% of cells, clusters 0 and 2) (Fig 4B–D; SFTPC, SFTPA1, LPCAT1; Appendix Fig S4A), proliferating ATII-like cells (6.4% of alveolar cells, cluster 4) (Fig 4E), and alveolar cells that showed an increased expression of several ATI markers (6.5% of alveolar-like cells, cluster 3) (Fig 4F–H; AGER, CAV1, HOPX; Appendix Fig S4B). The basal cell cluster (cluster 5) was characterized by KRT5 and TP63 expression (Fig 4I and J; Appendix Fig S4C). A rare PNEC population (cluster 7) expressed CHGA (Fig 4K; Appendix Fig S4D). To investigate whether the cultured cells were representative of adult alveolar cells, we compared our bulk RNA sequencing and single-cell RNA sequencing dataset to previously published bulk (Fig EV3A) and single-cell sequencing datasets (Figs 4L and M, and EV3B and C) of freshly isolated adult HTII-280+ ATII cells. The freshly isolated adult ATII cells did not form a separate cluster in the t-SNE map (Fig 4L and M), and overlapped with cultured alveolar, proliferating alveolar and ATII-like cells (Figs 4M and EV3B). The expression of a set of ATII marker g
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