A single-cell RNA-seq analysis unravels the heterogeneity of primary cultured human corneal endothelial cells

The cornea is a transparent and avascular tissue located in front of the eye. Its inner surface is lined by a monolayer of corneal endothelial cells (CECs), which maintain the cornea transparency. CECs remain arrested in a non-proliferative state and damage to these cells can compromise their function leading to corneal opacity. The primary culture of donor-derived CECs is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single-cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo-temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies.

Automated summary

The primary culture of corneal endothelial cells (CECs) has limitations in terms of limited expansion and assessing the quality of the culture. Through single-cell RNA sequencing, this study identified the transcriptomic changes and suggested markers to evaluate the quality of primary CEC cultures. This research provides a better understanding of cellular heterogeneity and lays the foundation for improving culture protocols and therapies.