Abstract
The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing 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.
Highlights
The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm
We identified new potential targets for suppressing cellular senescence, and confirmed that a relatively low mitogenic environment is better at maintaining the CEnC phenotype in vitro[22]
Corneal clarity depends on the corneal endothelium, and disease or injury to this tissue causes loss of visual acuity that may necessitate corneal transplantation
Summary
The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. Donor age and other factors (e.g., days in preservation medium, donor medical history, cell count) dictate the success of establishing in vitro cultures[11] This makes identifying an optimal in vitro culture protocol essential for ensuring consistent establishment and expansion of CEnC. We identified new potential targets for suppressing cellular senescence, and confirmed that a relatively low mitogenic environment is better at maintaining the CEnC phenotype in vitro[22]. These findings form the basis for continued development of in vitro culture and expansion of primary CEnC for their eventual use in cell replacement therapy for the management of corneal endothelial loss or dysfunction
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