Differentiation of immortalized human MLPC to alveolar type 2 (AT2)-like cells: ACE-2 expression and binding of SARS-CoV-2 spike and spike 1 proteins

Collins, Daniel.P.; Osborn, Mark.J.; Steer, Clifford.J.

Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the ACE-2 receptor to facilitate entry into respiratory epithelium. Alveolar type 2 (AT2) cells are committed respiratory progenitor cells responsible for the integrity and regeneration of the respiratory epithelium and production of respiratory surfactant proteins. They express high levels of surface ACE-2 and thus are a leading target for primary infection by the SARS-CoV-2. This study describes a method to directly differentiate TERT-immortalized human cord blood-derived multilineage progenitor cells (MLPC) to AT2-like cells for the purpose of generating an in vitro cellular platform for viral studies. Differentiation was confirmed with the acquisition of AT2 and absence of alveolar type 1 (AT1) specific markers by confocal microscopy. Expression of the ACE-2 receptor was confirmed by immunofluorescence antibody staining, qRT-PCR, and binding of biotinylated SARS-CoV-2 spike (S and S1) proteins. The binding of biotinylated spike proteins was specifically blocked by unlabeled spike proteins and neutralizing antibodies. Additionally, it was demonstrated that the spike protein was internalized after binding to the surface membrane of the cells. We defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. Our method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies.