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 angiotensin-converting enzyme 2 (ACE-2) receptor to facilitate entry into the 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. AT2 cells express high levels of surface ACE-2 and thus are a leading target for primary infection by SARS-CoV-2. This study describes a method for directly differentiating telomerase reverse transcriptase-immortalized human cord blood-derived multi-lineage progenitor cells (MLPCs) 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, quantitative reverse transcription polymerase chain reaction and binding of biotinylated SARS-CoV-2 spike and spike 1 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. The authors defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. The authors’ method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies.
|Original language||English (US)|
|Number of pages||10|
|Early online date||Aug 17 2021|
|State||Published - Dec 2021|
Bibliographical noteFunding Information:
CytoMedical Design Group, LLC, provided funding for this study in the form of salary for DPC as well as facility resources. Additional funding was provided by BioE, LLC, which had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Conception and design of the study: DPC, MJO and CJS. Acquisition of data: DPC and MJO. Analysis and interpretation of data: DPC, MJO and CJS. Drafting or revising the manuscript: DPC, MJO and CJS. All authors have approved the final article. The authors thank Peter W. Collins and Joel H. Hapke for their contributions to tissue culture and analytical methodologies.
© 2021 International Society for Cell & Gene Therapy
- spike binding