Abstract
Purpose :
Human corneal endothelial cells (HCECs) are known to have poor proliferative properties and to undergo cell state transition (CST; senescence, EMT, etc.) easily under in vitro culture conditions. Our group has been studying for many years to develop a culture method for high-quality human corneal endothelial cells that can be used in regenerative medicine, and we have established a highly efficient amplification culture method that suppresses phase transition. During this development process, it was found that the ease with which CST occurs depends not only on culture conditions but also on donor differences. In this study, we focused on the differences in stability under culture conditions depending on the donor, and aimed to search for factors that define the cell stability.
Methods :
HCECs were cultured from research grade human donor corneas and cryopreserved. These frozen cells were thawed and cultured under different culture conditions to select a group in which CST was easily induced and a group in which CST was not induced even under the same conditions. RNA was extracted from each group (n=3) and subjected to RNA-Seq using NovaSeq.
Results :
While there was no difference in the expression level of CD166 among all lots, the expression of CD44 was significantly higher in the lots which easily transitioned to CST cells, which was consistent with the results of cell surface marker analysis using a flow cytometer in our previous reports. In addition, DEG analysis was performed, and a group of genes mainly related to cell senescence, cell-cell/cell-ECM adhesion, and cytoskeleton formation were identified as the candidate factors.
Conclusions :
Frozen stocks of cultured HCECs having different characteristics were established and could be broadly classified into cells that maintain health (difficult phase transition lots) and cells that undergo phase transition (easy phase transition lots).
Comparative analysis of both groups identified several candidate factors that are thought to define the stability of HCECs in culture condition.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.