Human embryonic stem cell-derived cardiomyocyte platform screens inhibitors of SARS-CoV-2 infection

Abstract

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated.

Fig. 1. Detection of host cell proteins and genes associated with SARS-CoV-2 viral infection.

a–f Representative fluorescent confocal images (n = 3 independent experiments performed in duplicate) of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (upper) and representative fluorescent images (n = 6 from 6 different donors) of human left ventricle (human LV) tissue sections (lower). Both cells and tissue were fixed with 4% formaldehyde and immunolabelled with primary antibodies raised against ACE2 a, TMPRSS2 b, B⁰AT1 c, cathepsin B d, cathepsin L e, and furin f, before visualisation with secondary antibody conjugated to Alexa Fluor 555 (yellow) and Hoechst 33342 nuclear marker (blue). g shows control cells (upper) and tissue (lower) treated with secondary antibody only and Hoechst 33342 nuclear marker. Scale bars show 50 μm. h Graphical data showing the percentage of the observed hESC-CM population positively immunolabelled (above background) after visualisation with a secondary antibody targeting primary antibodies raised against the outlined protein targets. i Graphical data showing the reads per million (RPM) ± SEM for expression of viral entry and processing genes in hESC-CMs (n = 3 replicates across three distinct differentiations) and human left ventricle (n = 5 individuals). SLC6A19, CTSB, and CTSL are the genes that encode B⁰AT1, cathepsin B, and cathepsin L, respectively. All graphical data are mean±SEM, with individual data points indicated.

Fig. 2. SARS-CoV-2 spike-pseudotyped viral infection, and pharmacological inhibition, in hESC-CMs.

a Schematic showing the experimental workflow in brief for generating human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and taking them into the pseudotyped lentiviral infection drug screen before conducting quantitative imaging (see Methods for further details). The schematic was generated using templates from Servier Medical Art (https://smart.servier.com/) b Representative fluorescent confocal images (n = 2 independent experiments performed in triplicate) of hESC-CMs pretreated with small molecule inhibitors (camostat, benztropine, and E64d), peptide antagonist (DX600), or antibody (ACE2 Ab) targeting protein components involved in SARS-CoV-2 infection. Control cells were treated with DMSO (0.6%) or media. Cells were treated with drugs for 1 h before incubation with SARS-CoV-2 spike-pseudotyped GFP-expressing (green) lentivirus for 4 h. After removal of viral particles, cells were washed and maintained in the presence of drugs for 5 days before fixation with 4% formaldehyde and staining with Hoechst 33342 nuclear marker (blue). Scale bar shows 200 μm. c Representative fluorescent confocal images (n = 2 independent experiments performed in triplicate) of control human embryonic stem cell-derived cardiomyocytes (hESC-CMs) treated with VSV-G pseudotyped GFP-expressing (green) lentivirus, in the absence (upper) or presence (middle) of antibody (ACE2 Ab). Uninfected controls were not treated with viral particles (bottom). Again, cells were stained with Hoechst 33342 nuclear marker. d Graphical data showing the percentage of observed hESC-CMs infected with either SARS-CoV-2 spike or VSV-G (control) pseudotyped lentivirus in the presence of drugs or DMSO (0.6%) as indicated. Uninfected cells were not treated with viral particles. **p < 0.005; ***p < 0.0005; ****p < 0.00005; and ns = no significant difference (as determined by one-way ANOVA) for each condition versus the DMSO treated control cells. # = no significant difference for condition versus the VSV-G control. e Graphical data showing the overall count of observed hESC-CMs for each condition, as indicated. No condition showed a count significantly different (as determined by one-way ANOVA) from the DMSO treated control cells. All graphical data are mean ± SEM, with individual data points indicated.