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Nature
12, 2020;588 (7839): 670-675.

Progenitor identification and SARS-CoV-2 infection in human distal lung organoids.

Ameen A Salahudeen, Shannon S Choi, Arjun Rustagi, Junjie Zhu, Vincent van Unen, Sean M de la O, Ryan A Flynn, Mar Margalef-Català, António J M Santos, Jihang Ju, Arpit Batish, 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
Author Information
  1. Ameen A Salahudeen: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. ORCID
  2. Shannon S Choi: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  3. Arjun Rustagi: Division of Infectious Disease and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. ORCID
  4. Junjie Zhu: Stanford University School of Engineering, Department of Electrical Engineering, Stanford, CA, USA. ORCID
  5. Vincent van Unen: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. ORCID
  6. Sean M de la O: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  7. Ryan A Flynn: Stanford ChEM-H, Stanford University, Stanford, CA, USA. ORCID
  8. Mar Margalef-Català: Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA. ORCID
  9. António J M Santos: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  10. Jihang Ju: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  11. Arpit Batish: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  12. Tatsuya Usui: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  13. Grace X Y Zheng: 10x Genomics, Pleasanton, CA, USA.
  14. Caitlin E Edwards: Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. ORCID
  15. Lisa E Wagar: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
  16. Vincent Luca: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA.
  17. Benedict Anchang: Division of Biomedical Data Science, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  18. Monica Nagendran: Division of Pulmonary, Allergy and Critical Care, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  19. Khanh Nguyen: Division of Gastroenterology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  20. Daniel J Hart: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  21. Jessica M Terry: 10x Genomics, Pleasanton, CA, USA.
  22. Phillip Belgrader: 10x Genomics, Pleasanton, CA, USA.
  23. Solongo B Ziraldo: 10x Genomics, Pleasanton, CA, USA.
  24. Tarjei S Mikkelsen: 10x Genomics, Pleasanton, CA, USA.
  25. Pehr B Harbury: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
  26. Jeffrey S Glenn: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
  27. K Christopher Garcia: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. ORCID
  28. Mark M Davis: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. ORCID
  29. Ralph S Baric: Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. ORCID
  30. Chiara Sabatti: Division of Biomedical Data Science, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  31. Manuel R Amieva: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
  32. Catherine A Blish: Division of Infectious Disease and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. cblish@stanford.edu. ORCID
  33. Tushar J Desai: Division of Pulmonary, Allergy and Critical Care, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. tdesai@stanford.edu. ORCID
  34. Calvin J Kuo: Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. cjkuo@stanford.edu. ORCID
PMID: 33238290 DOI: 10.1038/s41586-020-3014-1

Abstract

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5 basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5 cells in basal organoids revealed a distinct population of ITGA6ITGB4 mitotic cells, whose offspring further segregated into a TNFRSF12A subfraction that comprised about ten per cent of KRT5 basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.

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Grants

  1. T32 GM007365/NIGMS NIH HHS
  2. R01 AI157155/NIAID NIH HHS
  3. DK11572802/NIH HHS
  4. U24 DK085532/NIDDK NIH HHS
  5. U01 DE025188/NIDCR NIH HHS
  6. UH3 CA255135/NCI NIH HHS
  7. U01 DK085532/NIDDK NIH HHS
  8. UG3 HL145623/NHLBI NIH HHS
  9. T32 AI007502-23/NIH HHS
  10. /Wellcome Trust
  11. T32 AI007502/NIAID NIH HHS
  12. R01 HL142549/NHLBI NIH HHS
  13. T32 GM007365-44/NIH HHS
  14. U01 CA176299/NCI NIH HHS
  15. R56 AI111460/NIAID NIH HHS
  16. 5R01HL14254902/NIH HHS
  17. /Howard Hughes Medical Institute
  18. U19 AI057229/NIAID NIH HHS
  19. K08 DE027730/NIDCR NIH HHS
  20. U01 DK085527/NIDDK NIH HHS
  21. U01 CA217851/NCI NIH HHS
  22. U19 AI116484/NIAID NIH HHS

MeSH Term

Alveolar Epithelial Cells
COVID-19
Cell Differentiation
Cell Division
Clone Cells
Humans
In Vitro Techniques
Influenza A Virus, H1N1 Subtype
Integrin alpha6
Integrin beta4
Keratin-5
Lung
Models, Biological
Organoids
Pneumonia, Viral
SARS-CoV-2
Single-Cell Analysis
TWEAK Receptor
Tissue Culture Techniques

Chemicals

ITGA6 protein, human
ITGB4 protein, human
Integrin alpha6
Integrin beta4
KRT5 protein, human
Keratin-5
TNFRSF12A protein, human
TWEAK Receptor
Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

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