Paired t-test, *p 0.05; ns, non-significant. Spectral flow cytometry analyses with single tube staining of fourteen markers (termed spectral flow here), including a panel of recently developed airway TA-02 epithelial markers31 revealed diversified compositions of cell subsets in fifteen Rabbit Polyclonal to GRAK lung organoids. not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with contamination rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 contamination. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19. Introduction Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has caused a global pandemic of coronavirus disease (COVID-19) with over 170M cases worldwide (https://coronavirus.jhu.edu/map.html) as of May 2021. Clinically, SARS-CoV-2 can lead to lethal acute respiratory distress syndrome (ARDS)1. Many SARS-CoV-2 studies have focus on the immune system, currently, we do not understand how the lung epithelium itself may play a role in variation in disease spectrum of COVID-19. The lung airway epithelium defends against pollutants, allergens, and pathogens and is composed of a variety of cell types including basal cells, TA-02 ciliated cells, mucus-producing goblet cells, secretory cells, and neuroendocrine cells. The distal lung epithelium also includes alveolar type 1 (AT1) cells, which mediate gas exchange, and AT2 cells, which secrete surfactant2, 3. In the case of COVID-19, studies thus far suggest that SARS-CoV-2 infects mostly ciliated cells, goblet cells, AT2 cells but also basal stem cells4, 5, 6, 7, 8. Comprehensive SARS-CoV-2 infection studies focusing on lung epithelial cell subsets in different individuals have not been reported TA-02 to date. Coronavirus (CoV) particles are spherical with three viral proteins anchored in the envelope: the triple-spanning membrane (M) protein, the envelope (E) protein, and the spike (S) protein, which form the characteristic trimeric spikes9, 10, 11. The surface spike glycoprotein of SARS-CoV-2 binds to human ACE2 (Angiotensin-converting enzyme 2)12. Binding of the S protein to ACE2 mediates membrane fusion and viral entry. The S protein is usually cleaved by host cell type II trans-membrane serine proteases resulting in spike protein activation and viral entry13, 14, 15, 16. As such, ACE2 and Transmembrane protease, serine 2 (TMPRSS2) are critical for SARS-CoV-2 entry into the cell17. Viruses typically co-opt various host proteins to maximize infectious potential18. The wide variation in SARS-CoV-2 contamination rates and COVID-19 severity suggests that there must be facilitators other than ACE2 and TMPRSS-2 that have yet to be discovered. Epithelial organoids have gained traction as a physiological platform for personal medicine because these organoids retain patient-specific traits19. A handful of studies have used alveolospheres and organoids to study SARS-CoV-2 epithelial response20, 21, 22, 23, 24, 25 and tropism7, 26, 27, 28, 29, 30. A comprehensive analysis of SARS-CoV-2 contamination in a large panel of lung organoids has not been reported to date. Here, we performed a comprehensive analysis with the goal of obtaining insights for different subjects as well as discovery of new molecules that play a role in infection. Results A comprehensive lung organoid biobank We generated organoids from biopsies from healthy donors or adjacent normal tissue from lung cancer patients undergoing medical procedures (Table 1). 3D lung organoids from twenty subjects were expanded through passaging and cryopreserved, generating a biobank (Figs. 1A and ?and1B,1B, and Suppl. Fig S1A and S1B). Open in a separate window Physique 1: Donor-derived lung organoids are stable and distinctive.A) Workflow of lung organoid generation. B) Brightfield images of lung organoids derived from different donors. C) Spectral Flow Cytometry gating strategy to define seven cell populations: (1)TUBA+ CD271? as ciliated cells. (2) MUC5AC+ TUBA? as goblet-like cells. (3) TSPAN8+ MUC5AC? TUBA? as pre-goblet cells. (4) CD49f+ CD271+, (5) CD49f? CD271+, (6) CD49f+ CD271+ as basal cells and (7) CD49f? CD271? as undefined cells. D) Scheme of cell types found in the 3D lung organoids. E) Pie charts representing spectral flow cytometry analyses of lung organoids from indicated donors. FCG) Percentage of F) TA-02 ACE2+ and G) TMPRSS2+ cells in lung organoids based on spectral flow cytometry analyses. Bars represent mean, error bars are SEM, n=3. Paired t-test, *p 0.05; ns, non-significant. Spectral flow.