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Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

View ORCID ProfileAmeen A. Salahudeen, View ORCID ProfileShannon S. Choi, View ORCID ProfileArjun Rustagi, Junjie Zhu, View ORCID ProfileSean M. de la O, View ORCID ProfileRyan A. Flynn, View ORCID ProfileMar Margalef-Català, António J. M. Santos, Jihang Ju, Arpit Batish, Vincent van Unen, View ORCID ProfileTatsuya Usui, View ORCID ProfileGrace X.Y. Zheng, View ORCID ProfileCaitlin E. Edwards, View ORCID ProfileLisa E. Wagar, View ORCID ProfileVincent Luca, View ORCID ProfileBenedict Anchang, Monica Nagendran, Khanh Nguyen, Daniel J. Hart, Jessica M. Terry, Phillip Belgrader, Solongo B. Ziraldo, Tarjei S. Mikkelsen, View ORCID ProfilePehr B. Harbury, Jeffrey S. Glenn, K. Christopher Garcia, Mark M. Davis, Ralph S. Baric, Chiara Sabatti, Manuel R. Amieva, View ORCID ProfileCatherine A. Blish, View ORCID ProfileTushar J. Desai, View ORCID ProfileCalvin J. Kuo
doi: https://doi.org/10.1101/2020.07.27.212076
Ameen A. Salahudeen
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Shannon S. Choi
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Arjun Rustagi
2Infectious Disease and Geographic Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
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Junjie Zhu
3Stanford University School of Engineering, Department of Electrical Engineering, Stanford, CA 94305, USA
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Sean M. de la O
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Ryan A. Flynn
4Stanford ChEM-H and Department of Chemistry, Stanford University, Stanford, CA 94305, USA
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Mar Margalef-Català
5Departments of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA
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António J. M. Santos
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Jihang Ju
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Arpit Batish
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Vincent van Unen
6Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Tatsuya Usui
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Grace X.Y. Zheng
710x Genomics Inc., Pleasanton, California, 94566, USA
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  • ORCID record for Grace X.Y. Zheng
Caitlin E. Edwards
8Departments of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA
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Lisa E. Wagar
6Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
9Stanford Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305, USA
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Vincent Luca
10Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA
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  • ORCID record for Vincent Luca
Benedict Anchang
11Biomedical Data Science, Stanford University School of Medicine, Stanford, CA 94305, USA
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  • ORCID record for Benedict Anchang
Monica Nagendran
12Pulmonary, Allergy and Critical Care, Stanford University School of Medicine, Stanford, CA 94305, USA
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Khanh Nguyen
13Gastroenterology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Daniel J. Hart
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Jessica M. Terry
710x Genomics Inc., Pleasanton, California, 94566, USA
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Phillip Belgrader
710x Genomics Inc., Pleasanton, California, 94566, USA
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Solongo B. Ziraldo
710x Genomics Inc., Pleasanton, California, 94566, USA
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Tarjei S. Mikkelsen
710x Genomics Inc., Pleasanton, California, 94566, USA
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Pehr B. Harbury
14Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA
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Jeffrey S. Glenn
13Gastroenterology, Stanford University School of Medicine, Stanford, CA 94305, USA
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K. Christopher Garcia
10Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA
15Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305
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Mark M. Davis
6Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
9Stanford Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305, USA
15Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305
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Ralph S. Baric
8Departments of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA
16Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, USA
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Chiara Sabatti
11Biomedical Data Science, Stanford University School of Medicine, Stanford, CA 94305, USA
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Manuel R. Amieva
5Departments of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA
6Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
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Catherine A. Blish
2Infectious Disease and Geographic Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
17Chan Zuckerberg Biohub, San Francisco, CA 94158
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  • For correspondence: cjkuo@stanford.edu tdesai@stanford.edu cblish@stanford.edu
Tushar J. Desai
12Pulmonary, Allergy and Critical Care, Stanford University School of Medicine, Stanford, CA 94305, USA
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  • For correspondence: cjkuo@stanford.edu tdesai@stanford.edu cblish@stanford.edu
Calvin J. Kuo
1Department of Medicine, Divisions of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA
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  • For correspondence: cjkuo@stanford.edu tdesai@stanford.edu cblish@stanford.edu
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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 TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi 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.

Competing Interest Statement

The authors are pursuing patents on methods described in this manuscript.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures
Ameen A. Salahudeen, Shannon S. Choi, Arjun Rustagi, Junjie Zhu, Sean M. de la O, Ryan A. Flynn, Mar Margalef-Català, António J. M. Santos, Jihang Ju, Arpit Batish, Vincent van Unen, Tatsuya Usui, Grace X.Y. Zheng, Caitlin E. Edwards, Lisa E. Wagar, Vincent Luca, Benedict Anchang, Monica Nagendran, Khanh Nguyen, Daniel J. Hart, Jessica M. Terry, Phillip Belgrader, Solongo B. Ziraldo, Tarjei S. Mikkelsen, Pehr B. Harbury, Jeffrey S. Glenn, K. Christopher Garcia, Mark M. Davis, Ralph S. Baric, Chiara Sabatti, Manuel R. Amieva, Catherine A. Blish, Tushar J. Desai, Calvin J. Kuo
bioRxiv 2020.07.27.212076; doi: https://doi.org/10.1101/2020.07.27.212076
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Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures
Ameen A. Salahudeen, Shannon S. Choi, Arjun Rustagi, Junjie Zhu, Sean M. de la O, Ryan A. Flynn, Mar Margalef-Català, António J. M. Santos, Jihang Ju, Arpit Batish, Vincent van Unen, Tatsuya Usui, Grace X.Y. Zheng, Caitlin E. Edwards, Lisa E. Wagar, Vincent Luca, Benedict Anchang, Monica Nagendran, Khanh Nguyen, Daniel J. Hart, Jessica M. Terry, Phillip Belgrader, Solongo B. Ziraldo, Tarjei S. Mikkelsen, Pehr B. Harbury, Jeffrey S. Glenn, K. Christopher Garcia, Mark M. Davis, Ralph S. Baric, Chiara Sabatti, Manuel R. Amieva, Catherine A. Blish, Tushar J. Desai, Calvin J. Kuo
bioRxiv 2020.07.27.212076; doi: https://doi.org/10.1101/2020.07.27.212076

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