Human iPSC-derived alveolar and airway epithelial cells can be cultured at air-liquid interface and express SARS-CoV-2 host factors

Abstract

Development of an anti-SARS-CoV-2 therapeutic is hindered by the lack of physiologically relevant model systems that can recapitulate host-viral interactions in human cell types, specifically the epithelium of the lung. Here, we compare induced pluripotent stem cell (iPSC)-derived alveolar and airway epithelial cells to primary lung epithelial cell controls, focusing on expression levels of genes relevant for COVID-19 disease modeling. iPSC-derived alveolar epithelial type II-like cells (iAT2s) and iPSC-derived airway epithelial lineages express key transcripts associated with lung identity in the majority of cells produced in culture. They express ACE2 and TMPRSS2, transcripts encoding essential host factors required for SARS-CoV-2 infection, in a minor subset of each cell sub-lineage, similar to frequencies observed in primary cells. In order to prepare human culture systems that are amenable to modeling viral infection of both the proximal and distal lung epithelium, we adapt iPSC-derived alveolar and airway epithelial cells to two-dimensional air-liquid interface cultures. These engineered human lung cell systems represent sharable, physiologically relevant platforms for SARS-CoV-2 infection modeling and may therefore expedite the development of an effective pharmacologic intervention for COVID-19.

SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

“…Therefore, we adapted our SFTPC tdTomato+ iAT2s (SPC2-ST-B2 iPSC line) to 2D ALI cultures ( To quantify protein-level expression frequency of the viral ACE2 receptor in iAT2s at ALI, we employed flow cytometry, observing that 13.2 ± 6.5% of live iAT2s demonstrated cell surface expression of ACE2 ( Fig. 1E) and indicating more frequent expression at the protein level than had been predicted from analysis of published primary AT2 or ALI cultured iAT2 scRNA-Seq profiles 6 . We validated the sensitivity and specificity of the ACE2 antibody by staining controls consisting of human 293T cells, which lack ACE2, and 293T cells lentivirally transduced to overexpress ACE2 18 (Fig.…”

“…We and others 6,17 have recently profiled the frequency of ACE2 mRNA expressing primary adult and iPSC-derived AT2-like cells by single cell RNA sequencing (scRNA-Seq), finding that mRNA expression occurs in a minority of cells (1-3%) at any given time with similar frequencies observed in primary AT2s compared to iAT2s. In contrast, the gene encoding the protease utilized for viral entry, TMPRSS2, is expressed more robustly in both AT2s and iAT2s 6 and is less developmentally variable, being stably expressed by week 16 of distal fetal lung development (Fig. 1C).…”

“…Thus, SARS-CoV-2 infection modeling has to this point been predominantly performed using either human airway (non-alveolar) cells, non-human cell lines that naturally express the ACE2 viral receptor and propagate the virus, such as the African Green Monkey Vero E6 cell line 5 , or transformed human cell lines with or without forced over-expression of ACE2. Although some of these transformed cell lines, such as A549 and Calu-3 cells, were originally generated from transformed cancerous lung epithelial cells, they no longer express NKX2-1, the master transcriptional regulator required for differentiated lung epithelial gene expression 6 , and thus are limited in their capacity to simulate an accurate lung cellular response to most perturbations, including viral infections.…”

“…To provide alternative sources of self-renewing human lung epithelial lineages, our group and others have recently developed human lung epithelial organoids and 2D air-liquid interface (ALI) lung cultures through the directed differentiation of induced pluripotent stem cells (iPSCs) in vitro [6][7][8][9][10][11][12][13][14][15][16] . Here we report the successful infection of a pure population of human iPSC-derived AT2-like cells (iAT2s) with SARS-CoV-2, providing a reductionist model that reveals the cell-intrinsic distal lung epithelial global transcriptomic responses to infection.…”

Rapid endothelial infection, endothelialitis and vascular damage characterise SARS-CoV-2 infection in a human lung-on-chip model

“…There is therefore an urgent need for a better understanding of the pathogenesis of SARS-CoV-2 in alveolar epithelial cells and in a more realistic model of the alveolar space that is vascularized. The lung-onchip model is well-suited to this purpose because it includes a vascular compartment maintained under flow, and infection can occur at the air-liquid interface, two key physiological features that are lacking in organoid models [17][18][19] . We therefore establish a human lung-on-chip model for SARS-CoV-2 infections, and probe the viral growth kinetics, cellular localization and responses to a low dose infection using qRT-PCR, ELISA, RNAscope, immunofluorescence and confocal imaging (Fig.…”