SRA

The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided an available source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell state identity remain, and a comprehensive characterization of the cultured CEnC has not been performed. To this end, we compared two established CEnC culture methods by assessing cell function and characterizing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion. Overall design: Seven independent primary CEnC cultures were established from donor corneas. For each culture, half the cells were prepared and isolated using Tryp-LN-F99 protocol and the other half of cells were isolated and prepared using ColA-ColIV-M4M5. Five passages (P0-P4) were established and total RNA was collected from 70 samples.