Abstract
Purpose :
Protocols for the primary culture of human corneal endothelial cells (hCECs) have several limitations: cultures are heterogeneous, have a limited capacity to grow without negatively affecting the cells, are only successful if derived from donors younger than 40, and there is a lack of markers to identify the high quality from inferior cells. We hypothesized that by gaining transcriptomic information from individual cells, markers to enrich for high quality clinical-grade CECs can be identified and protocols improved using the molecular pathway–level information gained.
Methods :
Four human donor corneas, with ages ranging from 24 to 34 years were used for this study. hCECs were isolated and cultured as described by Peh et al. 2015. hCECs from all donors at culture time points day 2 (passage 0), day 5 (passage 0), day 14 (passage 0), day 30 (passage 1) and day 46 (passage 2) were sequenced with 10x Genomics 3’ V3.1 protocol. The data from all samples were loaded in R (v.3.6.2), processed with Seurat package (v.3.2.0), and cells clustered using graph-based clustering.
Results :
Approximately 85,000 hCECs were loaded to 10x genomics. Data of 62,000 sequenced cells revealed the main clusters observed during primary expansion of hCECs. The sequenced cells expressed typical endothelial markers such as ALCAM, PRDX6, TJP1, and ATP1A1. Pseudotime analysis revealed the dynamics and variations in hCEC during primary expansion. Differential expression analysis will be used to identify the major differences between cell clusters at early and late time points. This will allow the identification of markers to differentiate high quality to lower quality hCECs. Furthermore, differential analysis will allow the identification of altered pathways during primary expansion of hCECs.
Conclusions :
This work provides the first single cell analysis of primary cultured hCECs depicting their heterogeneity. This research allows deep understanding on the process of primary expansion of hCECs and sets the basis for further improvement of protocols and therapies based on primary hCECs.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.