SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells.

Ivo Djidrovski,Maria Georgiou,Marina Moya-Molina,Edward I Patterson,Martine J Smit,Jelle Bor,Grant L Hughes,Majlinda Lako,Giancarlo A Biagini,Shaun H Pennington,Lyle Armstrong,Git Chung,Aitor Casas-Sanchez

doi:10.1002/stem.3422

Abstract

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs.

Highlights

The outbreak of the novel coronavirus disease, COVID-19, caused by coronavirus SARS-CoV-2 has been designated as a pandemic by the World Health Organization and is currently a significant threat to human health
We established an efficient method of isolation of iPSCsderived basal-like cells from a mixed population of lung progenitors
With primary cells, when grown in BEGM and 3T3-J2 feeders, the Induced pluripotent stem cells (iPSCs)-derived basal cells can self-renew and form colonies. They express genes associated with a basal cell phenotype (KRT14, deltaNp63, NGFR, Integrin alpha 6) while maintaining their multipotent capacity

Summary

| INTRODUCTION

The outbreak of the novel coronavirus disease, COVID-19, caused by coronavirus SARS-CoV-2 has been designated as a pandemic by the World Health Organization and is currently a significant threat to human health. The pathways involved in differentiation into proximal and distal airway lineages have already been established by previous groups, who successfully differentiated functional ciliated cells, mucus-producing cells, and alveolar cells.[22,23,24,25,26] The current methods of lung differentiation use 3D self-forming spheroids or 2D cultures of mixed population of cells, which are more challenging when performing experiments similar to the primary airway ALI models.[27,28,29] In this study, we isolated a population of basal-like cells from differentiating iPSCs and used these to generate airway epithelial equivalents by ALI culture We show that these comprise the cell types found in the human upper airway epithelium including functional ciliated cells, are capable of secreting mucus, and are readily infected by SARS-CoV-2 as demonstrated by the replication within the cells of the airway construct, release of virions into the supernatant growth media, and the presence of SARSCoV-2 spike protein in specific cells. Infected constructs secrete cytokines at levels corresponding to the behavior of the airway epithelial in vivo following SARS-CoV-2 infection

| MATERIALS AND METHODS

| RESULTS

Findings

| DISCUSSION