SARS-CoV-2 Infection of Ocular Cells from Human Adult Donor Eyes and hESC-Derived Eye Organoids

Abstract

The outbreak of COVID-19 caused by the SARS-CoV-2 virus has created an unparalleled disruption of global behavior and a significant loss of human lives. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible viral entry routes is essential. As aerosol transmission is thought to be the primary route of spread, we sought to investigate whether the eyes are potential entry portals for SARS-CoV-2. While virus has been detected in the eye, in order for this mucosal membrane to be a bone fide entry source SARS-CoV-2 would need the capacity to productively infect ocular surface cells.  As such, we conducted RNA sequencing in ocular cells isolated from adult human cadaver donor eyes as well as from a pluripotent stem cell-derived whole eye organoid model to evaluate the expression of ACE2 and TMPRSS2, essential proteins that mediate SARS-CoV-2 viral entry. We also infected eye organoids and adult human ocular cells with SARS-CoV-2 and evaluated virus replication and the host response to infection. We found the limbus was most susceptible to infection, whereas the central cornea exhibited only low levels of replication. Transcriptional profiling of the limbus upon SARS-CoV-2 infection, found that while type I or III interferons were not detected in the lung epithelium, a significant inflammatory response was mounted. Together these data suggest that the human eye can be directly infected by SARS-CoV-2 and thus is a route warranting protection. Funding: The National Eye Institute (NEI), Bethesda, MD, USA, extramural grant 1R21EY030215-01 and the Icahn School of Medicine at Mount Sinai supported this study.

Figure 1.. Zone 3 in the SEAM eye organoids composed of cells expressing ocular surface ectoderm gene profile.

(A) hESC-derived SEAM organoids were differentiated for 55 days, then processed for single cell RNA-seq and immunohistochemical analysis (n=1 biological replicate). B) Unbiased clustering was conducted using the Seurat Package in R. C) SEAM eye organoids were evaluated for the presence of ocular surface ectoderm markers in Zone 3. D) Cells possessing ocular surface ectoderm annotation were further clustered, presented as UMAP and PCA. E) UMAP presentation of relative expression of known corneal markers across the ocular surface ectoderm cell clusters. F) Heatmap of genes distinguishing each cluster by relative expression. G) Violin plots of known markers of corneal, limbal and conjunctival cells.

Figure 2.. Presumptive corneal cell clusters from SEAM eye organoids express ACE2 and TMPRSS2.

A) Relative expression of ACE2 in corneal clusters from SEAM eye organoids presented as UMAP and violin plot. B) ACE2 positive cells evaluated by Jensen TISSUES, Mouse Gene Atlas and Gene Ontology analyses. C) Relative expression of TMPRSS2 in corneal clusters from SEAM eye organoids presented as UMAP and violin plot. D) TMPRSS2 positive cells evaluated by Jensen TISSUES, Mouse Gene Atlas and Gene Ontology analyses. E) Table showing total cell number and percentage of the corneal cells from SEAM eye organoids expressing potential SARS-CoV-2 targets. F) Violin plots of cell clusters and their respective relative expression of genes central to corneal function as well as inflammatory responses to viral entry.

Figure 3.. SARS-CoV-2 induces an inflammatory response in infected human adult corneal tissues and hESC-derived SEAM corneal cells.

A) Adult human corneal cells isolated from two genetically different adult human donor eyes were infected with SARS-CoV-2, then sequenced for the presence of viral genome transcripts and mapped (n=2 biological replicates). B) RNA-sequencing analysis comparison between non-infected controls and infected cells uncovers the upregulation of an inflammatory network. The five most up regulated genes are labeled by name. Vertical lines indicate a log2 fold change of +/− 0.5 and the horizontal line indicates an adjusted p-value = 0.05. C) Gene network analysis of upregulated genes from (B) identifies an inflammatory complex and a complex involved in mitosis. D) hESC-derived SEAM eye organoids were infected with SARS-CoV-2, then sequenced for presence of viral genome transcripts and mapped (n = 1 biological replicate). E) Table of reads per million (RPM) of genes associated with SARS-CoV-2 infection.

Figure 4.. Ocular cell types isolated from adult human eyes infected with SARS-CoV-2.

A selection of ocular tissues (cornea (n=4), limbus (n=3), sclera (n=4), iris (n=3), RPE (n=2), choroid (=3) biological replicates) were isolated from adult human cadaver donor eyes and cultured. Cells were exposed to 0.1units/cell of active SARS-CoV-2 virus for 48 hrs. A) Immunofluorescence imaging of ocular cells upon staining for active SARS-CoV-2 virus and ACE-2 receptor expression. B) RNA was isolated and qPCR analysis was conducted to evaluate expression of SARS-CoV-2, C) ACE-2 and D) TMPRSS2 relative to internal controls. Y-axis scaled in log¹⁰ format. Scale bars = 100μm. Error bar indicates SEM.