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• W3204755594 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Background: SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. Methods: We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Results: Infected ALO monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (AT2→AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Conclusions: Findings validate a human lung model of COVID-19, which can be immediately utilized to investigate COVID-19 pathogenesis and vet new therapies and vaccines. Funding: This work was supported by the National Institutes for Health (NIH) grants 1R01DK107585-01A1, 3R01DK107585-05S1 (to SD); R01-AI141630, CA100768 and CA160911 (to PG) and R01-AI 155696 (to PG, DS and SD); R00-CA151673 and R01-GM138385 (to DS), R01- HL32225 (to PT), UCOP-R00RG2642 (to SD and PG), UCOP-R01RG3780 (to P.G. and D.S) and a pilot award from the Sanford Stem Cell Clinical Center at UC San Diego Health (P.G, S.D, D.S). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists. L.C.A's salary was supported in part by the VA San Diego Healthcare System. This manuscript includes data generated at the UC San Diego Institute of Genomic Medicine (IGC) using an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929). Introduction SARS-CoV-2, the virus responsible for COVID-19, causes widespread inflammation and injury in the lungs, giving rise to diffuse alveolar damage (DAD) (Andrea Valeria Arrossi and Farver, 2020; Damiani et al., 2021; Borczuk et al., 2020; Li et al., 2021; Roden, 2020), featuring marked infection and viral burden leading to apoptosis of alveolar pneumocytes (Hussman, 2020), along with pulmonary edema (Bratic and Larsson, 2013; Carsana et al., 2020). DAD leads to poor gas exchange and, ultimately, respiratory failure; the latter appears to be the final common mechanism of death in most patients with severe COVID-19 infection. How the virus causes so much damage remains unclear. A particular challenge is to understand the out-of-control immune reaction to the SARS-CoV-2 infection known as a cytokine storm, which has been implicated in many of the deaths from COVID-19. Although rapidly developed preclinical animal models have recapitulated some of the pathognomonic aspects of infection, for example, induction of disease, and transmission, and even viral shedding in the upper and lower respiratory tract, many failed to develop severe clinical symptoms (Lakdawala and Menachery, 2020). Thus, the need for preclinical models remains both urgent and unmet. To address this need, several groups have attempted to develop human preclinical COVID-19 lung models, all within the last few months (Duan et al., 2020; Mulay et al., 2020; Salahudeen et al., 2020). While a head-to-head comparison of the key characteristics of each model can be found in Table 1, what is particularly noteworthy is that most of the models do not recapitulate the heterogeneous epithelial cellularity of both proximal and distal airways, that is, airway epithelia, basal cells, secretory club cells, and alveolar pneumocytes. Although induced pluripotent stem cells (iPSC)-derived AT2 cells be differentiated into proximal and distal cell types, including AT1, ciliated, and club cells (Kawakita et al., 2020; Dye et al., 2015; Huang et al., 2020), these iPSC-derived models lack propagability and cannot be reproducibly generated for biobanking; nor can they be scaled up in cost-effective ways for use in drug screens. More specifically, adult lung organoid models that can be grown in a sustainable mode and are complete with proximo-distal epithelia are yet to emerge. Besides the approaches described so far, there are a few more approaches used for modeling COVID-19: (i) 3D organoids from bronchospheres and tracheospheres have been established before (Hild and Jaffe, 2016; Rock et al., 2009; Tadokoro et al., 2016) and are now used in apical-out cultures for infection with SARS-COV-2 (Suzuki, 2020); (ii) the most common model used for drug screening is the air-liquid interphase (ALI model) in which pseudo-stratified primary bronchial or small airway epithelial cells are used to recreate the multilayered mucociliary epithelium (Mou et al., 2016; Randell et al., 2011); (iii) several groups have also generated 3D airway models from iPSCs or tissue-resident stem cells (Dye et al., 2015; Wong et al., 2012; Ghaedi et al., 2013; Konishi et al., 2016; McCauley et al., 2017; Miller et al., 2019); (iv) others have generated AT2 cells from iPSCs using closely overlapping protocols of sequential differentiation starting with definitive endoderm, anterior foregut endoderm, and distal alveolar expression (Gotoh et al., 2014; Jacob et al., 2017; Jacob et al., 2019; Yamamoto et al., 2017; Chen et al., 2017; Huang et al., 2014); and (v) finally, long-term in vitro culture conditions for pseudo-stratified airway epithelium organoids, derived from healthy and diseased adult humans suitable to assess virus infectivity (Sachs et al., 2019; van der Vaart and Clevers, 2021; Zhou et al., 2018), have been pioneered; unfortunately, these airway organoids expressed virtually no lung mesenchyme or alveolar signature. What remains unclear is if any of these models accurately recapitulate the immunopathological phenotype that is seen in the lungs in COVID-19. Table 1 A comparison of current versus existing lung organoid models available for modeling COVID-19. AuthorSource of stem cellsPropagabilityCell typesSARS-COV-2 infectionDemonstrated reproducibility using more than one patientCost-effective (use of conditioned media)NotesAT1AT2ClubBasalCiliatedGobletZhou et alPMID: 29891677Small pieces of normal lung tissue adjacent to the diseased tissue from patients undergoing surgical resection for clinical conditions.Long term culture > 1 yInfection with H1N1 pandemic Influenza virusProximal differentiation (PD) of human Adult Stem Cell-derived airway organoid (AO) culture. Differentiation conditions (PneumaCult-ALI medium) increase ciliated cells. Serine proteases known to be important for productive viral infection, were elevated after PD.Sachs et alPMID: 30643021Generation of normal and tumor organoids from resected surplus lung tissue of patients with lung cancers.long term culture for over 1 yearNot clearly mentionedairway organoid (AO) expressed no mesenchyme or alveolar transcripts. Strongly enriched for bulk lung and small airway epithelial signature limited to basal, club, and ciliated cells Withdrawal of R-spondin terminated AO expansion after 3–4 passages similar to the withdrawal of FGFsDuan et alPMID: 32839764hPSC derived lung cells and macrophagesLowSARS-CoV-2 infection mediated damage onset by macrophages.Co-culture of lung cells and macrophages. Protocol followed enables alveolar differentiation process, although described presence of almost all lung cell types.Salahudeen et alPMID: 33238290Cells sorted from human peripheral lung tissues.Distal Lung organoid with possibility of long-term cultureFrom differentiation of AT2After diff of basal cellsInfection and presence of dsRNA and nucleocapsidNo RNA seq of infected samples to compare with COVID Differentiation to different cell types SARS CoV2 infection in apical-out organoids (not polarized monolayers). The combination of EGF and the Noggin was optimal, without any additional growth-promoting effects of either WNT3A or R-spondinHan et alPMID: 33116299hPSC-derived lung organoidsOrganoids were generated by 50 days of differentiationSARS-CoV-2 and SARS-CoV-2-Pseudo-Entry Viruses.AT1, AT2, stromal cells, low number of pulmonary neuroendocrine cells, proliferating cells, and airway epithelial cells were reported. Mostly AT2 based ACE2 receptor was used for virus infection. High throughput screen using hPSC-derived lung organoids identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry.Youk et alPMID: 33142113Adult alveolar stem cells isolated from distal lung parenchymal tissues by collagenase, dispase and sortingMultiple passages upto 10 monthsFrom AT2; Lost in higher passagesIn the organoid formSingle cell transcriptomic profiling identified two clusters and type I interferon signal pathway are highly elevated at three dpiMulay et alPMID: 32637946doi.org/10.1101/2020.06.29.174623Alv organoids with distal lung epithelial cells with lung fibroblast cellsIn the organoid formInfection of AT2 cells trigger apoptosis that may contribute to alveolar injury. Alteration of innate immune response genes from AT2 cellsProximal airway ALI with heterogenous cellsInfection of ciliated and goblet cells Two separate models for SARS-CoV2 infectionHuang JPMID:32979316iPSC derived AT2 cell ALI modelBulk RNA seq after day 1 and day four infection. The infection induces rapid inflammatory responses.Abo et alPMID: 32577635doi: 10.1101/2020.06.03.132639iPSC derived basal cells as oranoids or 2D ALIiPSCs transcripts match human lung better than cancer cell lines. iPSC AT2 cells express host genes mportant for SARS-CoV-2 infection.iPSC AT2 cells as organoids or 2D ALIRock et alPMID: 19625615Bronchospheres were isolated from human lung tissue.Bronchospheres derived from human lung can act as stem cells and can differentiate into other cell types.Lamers et alPMID: 33283287Lung organoids derived from fetal Lung epithelial bud tips and differentiated ALI model.14 passagesDetected SCGB3A2(ATII/club marker)2 subjects were mentionedOrganoid model derived from fetal lung bud tip tissue consists primarily of SOX2+ SOX9+ progenitor cells. Differentiation under ALI conditions is necessary to achieve mature alveolar epithelium. ALI model was found to contain mostly ATII and ATI cells, with small basal and rare neuroendocrine populations. SFTPC + Alveolar type II like cells were most readily infected by SARS-CoV-2. The infectious virus titer is much higher (five log) compared to other established model.Suzuki et aldoi: https://doi.org/10.1101/2020.05.25.115600Commercially available adult HBEpC cells were used to generate human bronchial organoids.In the organoid formOrganoids derived from HBEpC cells undergo differentiation process to achieve mature phenotype. Organoids are lacking distal epithelial cell types SARS-CoV-2 infection was performed on organoids and only the basolateral region came in to contact with the virus. Treatment with a TMPRSS2 inhibitor prior to infection demonstrated a reduction in infectivity.Tiwari et alPMID: 33631122Differentiated human iPSCs into lung organoids.80 daysIn the organoid formOrganoids originated from iPSC cells and have proximal and distal epithelial cells. Infected organoids with SARS-CoV-2 and pseudovirus. SARS-CoV-2 pseudovirus entry was blocked by viral entry inhibitors.Tindle et al [Current study]Deep lung tissue sections surgically obtained from patients undergoing lobe resections for lung cancers.RNA Seq and cross-validation of COVID-19 model. Single model with all the cells types and infection of SARS-CoV2 in the 2D form with Apical accessibility that close to physiologic state. ACE2: angiotensin-converting enzyme II; ALI: air-liquid interphase; TMPRSS2: transmembrane serine protease 2. Blue color cells denote the presence of the features. Red color cells denote the absence of the features. Grey color cells denote information not found. We present a rigorous transdisciplinary approach that systematically assesses an adult lung organoid model that is propagable, personalized, and complete with both proximal airway and distal alveolar cell types against existing models that are incomplete, and we cross-validate them all against COVID-19 patient-derived respiratory samples. Findings surprisingly show that cellular crosstalk between both proximal and distal components is necessary to emulate how SARS-CoV-2 causes diffuse alveolar pneumocyte damage; the proximal airway mounts a sustained viral infection, but it is the distal alveolar pneumocytes that mount the overzealous host response that has been implicated in a fatal disease. Results A rationalized approach for creating and validating acute lung injury in COVID-19 To determine which cell types in the lungs might be most readily infected, we began by analyzing a human lung single-cell sequencing dataset (GSE132914) for the levels of expression of angiotensin-converting enzyme II (ACE2) and transmembrane serine protease 2 (TMPRSS2), the two receptors that have been shown to be the primary sites of entry for the SARS-CoV-2 (Hoffmann et al., 2020). The dataset was queried with widely accepted markers of all the major cell types (see Table 2). Alveolar epithelial type 2 (AT2), ciliated and club cells emerged as the cells with the highest expression of both receptors (Figure 1A, Figure 1—figure supplement 1A). These observations are consistent with published studies demonstrating that ACE2 is indeed expressed highest in AT2 and ciliated cells (Mulay et al., 2020; Zhao et al., 2020; Jia et al., 2005). In a cohort of deceased COVID-19 patients, we observed by H&E (Figure 1—figure supplement 1B) that gas-exchanging flattened AT1 pneumocytes are virtually replaced by cuboidal cells that were subsequently confirmed to be AT2-like cells via immunofluorescent staining with the AT2-specific marker, surfactant protein C (SFTPC; Figure 1B, upper panel, Figure 1—figure supplement 1C, top). We also confirmed that club cells express ACE2 (Figure 1—figure supplement 1C, bottom), underscoring the importance of preserving these cells in any ideal lung model of COVID-19. When we analyzed the lungs of deceased COVID-19 patients, the presence of SARS-COV-2 in alveolar pneumocytes was also confirmed, as determined by the colocalization of viral nucleocapsid protein with SFTPC (Figure 1B, lower panel, Figure 1—figure supplement 1D). Immunohistochemistry studies further showed the presence of SARS-COV-2 virus in alveolar pneumocytes and in alveolar immune cells (Figure 1—figure supplement 1E). These findings are consistent with the gathering consensus that alveolar pneumocytes support the interaction between the epithelial cells and inflammatory cells recruited to the lung; via mechanisms that remain unclear, they are generally believed to contribute to the development of acute lung injury and acute respiratory distress syndrome (ARDS), the severe hypoxemic respiratory failure during COVID-19 (Hou et al., 2020; Spagnolo et al., 2020). Because prior work has demonstrated that SARS-CoV-2 infectivity in patient-derived airway cells is highest in the proximal airway epithelium compared to the distal alveolar pneumocytes (AT1 and AT2) (Hou et al., 2020), and yet, it is the AT2 pneumocytes that harbor the virus, and the AT1 pneumocytes that are ultimately destroyed during DAD, we hypothesized that both proximal airway and distal (alveolar pneumocyte) components might play distinct roles in the respiratory system to mount the so-called viral infectivity and host immune response phases of the clinical symptoms observed in COVID-19 (Chen and Li, 2020). Table 2 Markers used to identify various cell types in the lung. Cell typeMarkersAT1AQP5*$, PDPN*$$, Carboxypeptidase M, CAV-1, CAV-2, HTI56, HOPX, P2R × 4*$$, Na+/K + ATPase$, TIMP3*++, SEMA3F PDPN* AQP5* P2R × 4* TIMP3* SERPINE*AT2ABCA3*$$, CC10 (SCGB1A1*)+, CD44v6, Cx32, gp600++, ICAM-1++, KL-6, LAMP3*$$, MUC1, SFTPA1*$$, SFTPB*$, SFTPC*+, SFTPD*, SERPINE1ClubCC10 (SCGB1A1*)+, CYP2F2*, ITAG6*$$, SCGB3A2*$$, SFTPA1*$$, SFTPB*$, SFTPD*GobletCDX-2*, MUC5AC*, MUC5B*, TFF3*, UEA1+CiliatedACT (ACTG2*)$, BTub4 (TUBB4A*), FOXA3*++, FOXJ1*, SNTN*BasalCD44v6 (CD44*), ITGA6*$$, KRT5*$, KRT13*, KRT14*, p63 (CKAP4*), p75 (NGFR*)$$Generic Lung LineageCx43 (GJA1*), TTF-1 (TTF1*; Greatest in AT2 & Club), EpCAM (EPCAM*) *Markers used for single-cell gating (Figure 1A). $ denotes markers used in this work for Immunofluorescence (IF). $$ denotes markers used in this work for qPCR. + denotes markers used in both IF and qPCR. ++ denotes obscure markers (Not a lot of research relative to lung). Figure 1 with 1 supplement see all Download asset Open asset A rationalized approach to building and validating human preclinical models of COVID-19. A) Whisker plots display relative levels of angiotensin-converting enzyme II (ACE2) expression in various cell types in the normal human lung. The cell types were annotated within a publicly available single-cell sequencing dataset (GSE132914) using genes listed in Table 1. p-values were analyzed by one-way ANOVA and Tukey’s post hoc test. (B) Formalin-fixed paraffin-embedded sections of the human lung from normal and deceased COVID-19 patients were stained for SFTPC, alone or in combination with nucleocapsid protein and analyzed by confocal immunofluorescence. Representative images are shown. Scale bar = 20 µm. (C) Schematic showing key steps generating an adult stem cell-derived, propagable, lung organoid model, complete with proximal and distal airway components for modeling COVID-19-in-a-dish. See Materials and methods for details regarding culture conditions. (D) A transcriptome-based approach is used for cross-validation of in vitro lung models of SARS-CoV-2 infection (left) versus the human disease, COVID-19 (right), looking for a match in gene expression signatures. Because no existing lung model provides such proximodistal cellular representation (Table 1), and hence, may not recapitulate with accuracy the clinical phases of COVID-19, we first sought to develop a lung model that is complete with both proximal and distal airway epithelia using adult stem cells that were isolated from deep lung biopsies (i.e., sufficient to reach the bronchial tree). Lung organoids were generated using the work flow outlined in Figure 1C and a detailed protocol that had key modifications from previously published (Sachs et al., 2019; Zhou et al., 2018) methodologies (see Materials and methods). Organoids grown in 3D cultures were subsequently dissociated into single cells to create 2D monolayers (either maintained submerged in media or used in ALI model) for SARS-CoV-2 infection, followed by RNA seq analysis. Primary airway epithelial cells and hiPSC-derived alveolar type II (AT2) pneumocytes were used as additional models (Figure 1D, left panel). Each of these transcriptomic datasets was subsequently used to cross-validate our ex vivo lung models of SARS-CoV-2 infection with the human COVID-19 autopsy lung specimens (Figure 1D, right panel) to objectively vet each model for their ability to accurately recapitulate the gene expression signatures in the patient-derived lungs. Creation of a lung organoid model, complete with both proximal and distal airway epithelia Three lung organoid lines were developed from deep lung biopsies obtained from the normal regions of lung lobes surgically resected for lung cancer; both genders, smokers and non-smokers, were represented (Figure 2—figure supplement 1A; Table 3). Three different types of media were compared (Figure 2—figure supplement 1B); the composition of these media was inspired either by their ability to support adult-stem cell-derived mixed epithelial cellularity in other organs (like the gastrointestinal [GI] tract [Miyoshi and Stappenbeck, 2013; Sato et al., 2009; Sayed et al., 2020c]) or rationalized based on published growth conditions for proximal and distal airway components (Gotoh et al., 2014; Sachs et al., 2019; van der Vaart and Clevers, 2021). A growth condition that included conditioned media from L-WRN cells that express Wnt3, R-spondin, and Noggin, supplemented with recombinant growth factors, which we named as ‘lung organoid expansion media,’ emerged as superior compared to alveolosphere media-I and II (Jacob et al., 2019; Yamamoto et al., 2017) (details in Materials and methods), based on its ability to consistently and reproducibly support the best morphology and growth characteristics across multiple attempts to isolate organoids from lung tissue samples. Three adult lung organoid lines (ALO1-3) were developed using the expansion media, monitored for their growth characteristics by brightfield microscopy and cultured with similar phenotypes until P10 and beyond (Figure 2—figure supplement 1C and D). The 3D morphology of the lung organoid was also assessed by H&E staining of slices cut from formalin-fixed paraffin-embedded (FFPE) cell blocks of HistoGel-emb`edded ALO1-3 (Figure 2—figure supplement 1E). Table 3 Characteristics of patients enrolled into this study for obtaining lung tissues to serve as source of stem cells to generate lung organoids. NameDate of surgeryAgeSexSmoking historyReason for surgeryHistologyALO14/17/202064MaleCurrent, chronic smokerPacks/day: 0.50Years: 53Pack years: 26.5Lung carcinomaInvasive squamous cell carcinoma, non-keratinizingALO24/17/202059MaleNon-smokerLung carcinomaInvasive adenocarcinomaALO37/7/202046FemaleNon-smokerLeft lower lobe noduleInvasive adenocarcinoma To determine if all the six major lung epithelial cells (illustrated in Figure 2A) are present in the organoids, we analyzed various cell-type markers by qRT-PCR (Figure 2B–H and Figure 2—figure supplement 2A-H). All three ALO lines had a comparable level of AT2 cell surfactant markers (compared against hiPSC-derived AT2 cells as positive control) and a significant amount of AT1, as determined using the marker AQP5. ALOs also contained basal cells (as determined by the marker ITGA6, p75/NGFR, TP63), ciliated cells (as determined by the marker FOXJ1), and club cells (as determined by the marker SCGB1A1). As expected, the primary normal human bronchial epithelial cells (NHBE) had significantly higher expression of basal cell markers than the ALO lines (hence, served as a positive control), but they lacked stemness and club cells (hence, served as a negative control). Figure 2 with 5 supplements see all Download asset Open asset Adult stem cell-derived lung organoids are propagatable models with both proximal and distal airway components. (A) Schematic lists the various markers used here for qPCR and immunofluorescence to confirm the presence of all cell types in the 3D lung organoids here and in 2D monolayers later (in Figure 3). (B–H) Bar graphs display the relative abundance of various cell-type markers (normalized to 18S) in adult lung organoids (ALO), compared to the airway ( normal human bronchial epithelial cell [NHBE]) and/or alveolar (AT2) control cells, as appropriate. p-values were analyzed by one-way ANOVA. Error bars denote SEM; n = 3–6 datasets from three independent ALOs and representing early and late passages. See also Figure 2—figure supplement 2 for individual ALOs. (I, J). H&E-stained cell blocks were prepared using HistoGel (I). Slides were stained for the indicated markers and visualized by confocal immunofluorescence microscopy. Representative images are shown in (J). Scale bar = 50 µm. (K) 3D organoids grown in 8-well chamber slides were fixed, immunostained, and visualized by confocal microscopy as in (J). Scale bar = 50 µm. See also Figure 2—figure supplement 2. Top row (ACE2/KRT5-stained organoids) displays the single and merged panels as max projections of z-stacks (top) and a single optical section (bottom) of a selected area. For the remaining rows, the single (red/green) channel images are max projections of z-stacks; however, merged panels are optical sections to visualize the centers of the organoids. All immunofluorescence images showcased in this figure were obtained from ALO lines within passage #3–6. See also Figure 2—figure supplements 3–5 for additional evidence of mixed cellularity of ALO models, their similarity to lung tissue of origin, and stability of cellular composition during early (#1–8) and late (#8–15) passages, as determined by qPCR and flow cytometry. The presence of all cell types was also confirmed by assessing protein expression of various cell types within organoids grown in 3D cultures. Two different approaches were used—(i) slices cut from FFPE cell blocks of HistoGel-embedded ALO lines (Figure 2I and J) or (ii) ALO lines grown in 8-well chamber slides were fixed in Matrigel (Figure 2K), stained, and assessed by confocal microscopy. Such staining not only confirmed the presence of more than one cell type (i.e., mixed cellularity) of proximal (basal-KRT5) and distal (AT1/AT2 markers) within the same ALO line, but also, in some instances, demonstrated the presence of mixed cellularity within the same 3D structure. For example, AT2 and basal cells, marked by SFTPB and KRT5, respectively, were found in the same 3D structure (Figure 2J, interrupted curved lines). Similarly, ciliated cells and goblet cells stained by Ac-Tub and Muc5AC, respectively, were found to coexist within the same structure (Figure 2J, interrupted box; Figure 2K, arrow). Intriguingly, we also detected 3D structures that co-stained for CC10 and SFTPC (Figure 2J, bottom panel) indicative of mixed populations of club and AT2 cells. Besides the organoids with heterogeneous makeup, each ALO line also showed homotypic organoid structures that were relatively enriched in one cell type (Figure 2J, arrowheads pointing to two adjacent structures that are either KRT5- or SFTPB-positive). Regardless of their homotypic or heterotypic cellular organization into 3D structures, the presence of mixed cellularity was documented in all three ALO lines (see multiple additional examples in Figure 2—figure supplement 2I). It is noteworthy that the coexistence of proximal and distal epithelial cells in lung organoids has been achieved in one another instance prior; Lamers et al. showed such mixed cellular composition in fetal lung bud tip-derived organoids Lamers et al., 2021. However, their model lacked ciliated and goblet cells (Lamers et al., 2021), something that we could readily detect in our 3D organoids. Finally, using qRT-PCR of various cell-type markers as a measure, we confirmed that the ALO models overall recapitulated the cell-type composition in the adult lung tissues from which they were derived (Figure 2—figure supplement 3) and retained such composition in later passages without significant notable changes in any particular cell type (Figure 2—figure supplement 4). The mixed proximal and distal cellular composition of the ALO models and their degree of stability during in vitro culture was also confirmed by flow cytometry (Figure 2—figure supplement 5). Organoid cellularity resembles tissue sources in 3D cultures but differentiates in 2D cultures To model respiratory infections such as COVID-19, it is necessary for pathogens to be able to access the apical surface. It is possible to microinject into the lumens of 3D organoids, as done previously with pathogens in the case of gut organoids (Engevik et al., 2015; Forbester et al., 2015; Leslie et al., 2015; Williamson et al., 2018), or FITC-dextran in the case of lung organoids (Porotto et al., 2019), or carry out infection in apical-out 3D lung organoids with basal cells (Salahudeen et al., 2020). However, the majority of the researchers have gained apical access by dissociating 3D organoids into single cells and plating them as 2D-monolayers (Duan et al., 2020; Mulay et al., 2020; Huang et al., 2020; Sachs et al., 2019; Zhou et al., 2018; Han et al., 2020a; Han et al., 2020b. As in any epithelium, the differentiation of airway epithelial cells relies upon dimensionality (apicobasal polarity); because the loss of dimensionality can have a major impact on cellular proportions and impact disease-modeling in unpredictable ways, we assessed the impact of the 3D-to-2D conversion on cellularity by RNA seq analyses. Two commonly encountered methods of growth in 2D monolayers were tested: (i) monolayers polarized on trans-well inserts but submerged in growth media (Figure 3A and Figure 3—figure supplement 1A-D) and (ii) monolayers were grown at the air-liquid interface (popularly known as the ‘ALI model’; Prytherch et al., 2011; Dvorak et al., 2011) for 21 days to differentiate into the mucociliary epithelium (Figure 3A and Figure 3—figure supplement 1E-G). The submerged 2D monolayers had several regions of organized vacuolated-appearing spots (Figure 3—figure supplement 1D, arrow), presumably due to morphogenesis and cellular organization even in 2D. Consistent with this morphological appearance, the epithelial barrier formed in the submerged condition was leakier, as determined by relatively lower transepithelial electrical resistance (TEER; Figure 3—figure supplement 1B) and the flux of FITC-dextran from apical to basolateral chambers (Figure 3—figure supplement 1C), and corroborated by morphological assessment by confocal immunofluorescence of localization of occludin, a bona fide TJ marker. We chose occludin because it is a shared and constant marker throughout the" @default.
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• W3204755594 title "Author response: Adult stem cell-derived complete lung organoid models emulate lung disease in COVID-19" @default.
• W3204755594 doi "https://doi.org/10.7554/elife.66417.sa2" @default.
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