High expression of SARS-CoV2 viral entry-related proteins in human limbal stem cells
Yuzuru Sasamoto1, Catherine A A Lee1, Masahito Yoshihara2
1Division of Genetics, Brigham and Women's Hospital, Boston, MA, United States; Transplant Research Program, Boston Children's Hospital, Boston, MA, United States.
Purpose:
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). While the ocular surface is considered one of the major SARS-CoV2 transmission routes, the specific cellular tropism of SARS-CoV2 is not fully understood. In the current study, we evaluated the expression and regulation of two SARS-CoV2 viral entry proteins, TMPRSS2 and ACE2, in human ocular epithelial cells and stem cells.
Methods:
TMPRSS2 and ACE2 expression in ABCB5-positive limbal stem cells (LSCs) were assessed by RNAseq, flow cytometry and immunohistochemistry. PAX6, TMPRSS2, and ACE2 mRNA expression values were obtained from the GSE135455 and DRA002960 RNA-seq datasets. siRNA-mediated PAX6 knockdown (KD) was performed in limbal and conjunctival epithelial cells. TMPRSS2 and ACE2 expression in the PAX6 KD cells was analyzed by qRT-PCR and Western blot.
Results:
We found that ABCB5-positive LSCs express high levels of TMPRSS2 and ACE2 compared to ABCB5-negative limbal epithelial cells. Mechanistically, gene knockout and overexpression models revealed that the eye transcription factor PAX6 negatively regulates TMPRSS2 expression. Therefore, low levels of PAX6 in ABCB5-positive LSCs promote TMPRSS2 expression, and high levels of TMPRSS2 and ACE2 expression by LSCs indicate enhanced susceptibility to SARS-CoV2 infection in this stem cell population.
Conclusions:
Our study points to a need for COVID-19 testing of LSCs derived from donor corneas before transplantation to patients with limbal stem cell deficiency. Furthermore, our findings suggest that expandable human ABCB5+ LSC cultures might represent a relevant novel model system for studying cellular SARS-CoV2 viral entry mechanisms and evaluating related targeting strategies.
Insights
Human limbal stem cells (LSCs) show high expression of SARS-CoV-2 entry proteins TMPRSS2 and ACE2, indicating susceptibility to COVID-19. Low PAX6 levels in LSCs drive this increased viral entry protein expression.
Area of Science:
- Ophthalmology
- Virology
- Cell Biology
Background:
- The ocular surface is a potential route for SARS-CoV-2 transmission.
- The specific cellular targets of SARS-CoV-2 on the ocular surface are not fully understood.
- Understanding viral entry mechanisms is crucial for preventing ocular infections.
Purpose of the Study:
- To investigate the expression and regulation of SARS-CoV-2 viral entry proteins (TMPRSS2 and ACE2) in human ocular epithelial cells and stem cells.
- To determine the role of PAX6 in regulating these viral entry proteins.
- To assess the susceptibility of limbal stem cells (LSCs) to SARS-CoV-2 infection.
Main Methods:
- Assessed TMPRSS2 and ACE2 expression in ABCB5-positive LSCs using RNAseq, flow cytometry, and immunohistochemistry.
- Analyzed RNA-seq datasets (GSE135455, DRA002960) for PAX6, TMPRSS2, and ACE2 mRNA expression.
- Utilized siRNA-mediated PAX6 knockdown in limbal and conjunctival cells, followed by qRT-PCR and Western blot analysis.
Main Results:
- ABCB5-positive LSCs express significantly higher levels of TMPRSS2 and ACE2 compared to ABCB5-negative cells.
- The transcription factor PAX6 negatively regulates TMPRSS2 expression.
- Low PAX6 levels in LSCs correlate with high TMPRSS2 and ACE2 expression, suggesting increased SARS-CoV-2 susceptibility.
Conclusions:
- COVID-19 testing of LSCs from donor corneas is recommended before transplantation.
- Expandable human ABCB5+ LSC cultures offer a valuable model for studying SARS-CoV-2 entry and evaluating therapeutic strategies.
