Differentiation of immortalized human multi-lineage progenitor to alveolar type 2-like cells: angiotensin-converting enzyme 2 expression and binding of severe acute respiratory syndrome coronavirus 2 spike and spike 1 proteins.

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.

CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics.

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.