Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures
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Abstract
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5 + basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5 + cells contained a distinct ITGA6 + ITGB4 + mitotic population whose proliferation segregated to a TNFRSF12A hi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12A hi subset of FACS-purified ITGA6 + ITGB4 + basal cells from human lung or derivative organoids. In vivo , TNFRSF12A + cells comprised ~10% of KRT5 + basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 “apical-out” organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6545855.
This study may provide useful information on COVID-19-associated pneumonia
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SciScore for 10.1101/2020.07.27.212076: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Human tissue procurement and processing: All material used in this work was approved by the Stanford School of Medicine’s Institutional Review Board and performed under protocol #28908.
Consent: Standard informed consent for research was obtained in writing prior to tissue procurement.Randomization not detected. Blinding Confocal images were acquired in a blinded manner and organoids were defined as a cluster of 3 or greater DAPI nuclei. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 107 cells were incubated with Fc Block (Biolegend 422301) and diluted 1:100 … SciScore for 10.1101/2020.07.27.212076: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Human tissue procurement and processing: All material used in this work was approved by the Stanford School of Medicine’s Institutional Review Board and performed under protocol #28908.
Consent: Standard informed consent for research was obtained in writing prior to tissue procurement.Randomization not detected. Blinding Confocal images were acquired in a blinded manner and organoids were defined as a cluster of 3 or greater DAPI nuclei. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 107 cells were incubated with Fc Block (Biolegend 422301) and diluted 1:100 in FACS buffer (2 mM EDTA and 0.2% fetal calf serum in 1X PBS pH 7.4), for ten minutes followed by APC conjugated anti-CD45 antibodies at 1 μg/ml in FACS buffer for 30 minutes, washed, and subjected to two rounds of depletion with magnetic beads according to manufacturer’s protocol (Miltenyi: anti-human fibroblast 130-050-601, anti-CD31 130-091-935, anti-APC 130-090-855, LS column 130-042-401). anti-CD45suggested: Noneanti-human fibroblast 130-050-601suggested: Noneanti-CD31suggested: Noneanti-APCsuggested: (Miltenyi Biotec Cat# 130-090-855, RRID:AB_244367)Unlabeled cells were then centrifuged at 300 x g and labeled with a cocktail of 1 μg/ml of PerCP-Cy5.5 anti-EPCAM antibody and Zombie Aqua viability stain (Biolegend 423101) diluted 1:400 from stock concentration in FACS buffer. anti-EPCAMsuggested: NoneFixed and permeabilized cells were then incubated with 1:400 dilution of Alexa Fluor 647 conjugated mouse anti-human cytokeratin 5 antibody (Abcam) for 24 hours at 4C in permeabilization buffer. anti-human cytokeratin 5suggested: NoneCells were then washed with FACS buffer and labeled with PE conjugated mouse anti-human TNFRSF12A antibody (clone ITEM-4, Biolegend) for 30 minutes on ice, followed by washing and analysis on a BD Aria Fusion instrument. anti-human TNFRSF12Asuggested: NoneExperimental Models: Cell Lines Sentences Resources Titers were determined by plaque assay on VeroE6 cells using Avicel (FMC Biopolymer) and crystal violet (Sigma), viral genome sequence was verified, and all infections were done with passage 3 virus. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Software and Algorithms Sentences Resources Screening exogenous growth factors in organoid culture: Distal airway cells were isolated and plated as above with the following exceptions: ADMEM/F12 was used instead of organoid medium during elastase digestion of lung tissue, cells were serially diluted and filtered through a 40 micron cell strainer and counted with a hemocytometer. ADMEM/F12suggested: NoneData preprocessing and Principle Component Analysis were carried out with CellRanger v1.2. CellRangersuggested: None3D rendering of confocal image stacks was performed using Volocity Image Analysis software (Quorum Technologies Inc., Guelph, Ontario). Volocity Imagesuggested: NoneFor Figure 5j, requiring 5 colors, cilia were distinguished by staining with two fluorescent secondary antibodies and merging the colocalized voxels into a pseudocolored channel using Volocity software. Volocitysuggested: (Volocity 3D Image Analysis Software, RRID:SCR_002668)Antiviral dose response curves were generated using four-parameter nonlinear regression curve fitting with GraphPad Prism 7 (GraphPad Software, San Diego, CA). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Z-stacks were collected and images were processed in ImageJ and maximum intensity Z projections were used to quantitate SCGB1A1 and SFTPC RNA in situ in Cell Profiler74 using the RNA Proximity Ligase Assay counting pipeline (https://github.com/tischi/cellprofiler-practical-NeuBIAS-Lisbon-2017). ImageJsuggested: (ImageJ, RRID:SCR_003070)Digital videos were collected by a Hamamatsu high-resolution ORCA-285 digital camera and rendered using OpenLab 5.5.2 software (Improvision). OpenLabsuggested: (Openlab, RRID:SCR_012158)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 32, 29 and 31. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.07.27.212076: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 29, 31, 31, 31, 31, 31, 31, 32, 32 and 32. At least one figure is not …
SciScore for 10.1101/2020.07.27.212076: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 29, 31, 31, 31, 31, 31, 31, 32, 32 and 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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SciScore for 10.1101/2020.07.27.212076: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 29, 31, 31, 31, 31, 31, 31, 32, 32 and 32. At least one figure is not …
SciScore for 10.1101/2020.07.27.212076: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 29, 31, 31, 31, 31, 31, 31, 32, 32 and 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
-
SciScore for 10.1101/2020.07.27.212076: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources To functionally validate this observation, we fractionated total distal lung organoids by anti-TNFRSF12A monoclonal antibody FACS into EPCAM+ITGA6+ITGB4+ cells and then into TNFRSF12Ahi and TNFRSF12Aneg subsets. anti-TNFRSF12Asuggested: NoneSoftware and Algorithms Sentences Resources B.A. and S.P. performed SPADE analysis. SPADEsuggested: (SPADE, SCR_001810)Data from …
SciScore for 10.1101/2020.07.27.212076: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources To functionally validate this observation, we fractionated total distal lung organoids by anti-TNFRSF12A monoclonal antibody FACS into EPCAM+ITGA6+ITGB4+ cells and then into TNFRSF12Ahi and TNFRSF12Aneg subsets. anti-TNFRSF12Asuggested: NoneSoftware and Algorithms Sentences Resources B.A. and S.P. performed SPADE analysis. SPADEsuggested: (SPADE, SCR_001810)Data from additional tools added to each annotation on a weekly basis.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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