Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation

Abstract

This study aims to obtain sufficient corneal endothelial cells for regenerative application. We examined the combinatory effects of Rho-associated kinase (ROCK) inhibitor Y-27632 and mesenchymal stem cell-derived conditioned medium (MSC-CM) on the proliferation and senescence of rabbit corneal endothelial cells (rCECs). rCECs were cultured in a control medium, a control medium mixed with either Y-27632 or MSC-CM, and a combinatory medium containing Y-27632 and MSC-CM. Cells were analyzed for morphology, cell size, nuclei/cytoplasmic ratio, proliferation capacity and gene expression. rCECs cultured in a combinatory culture medium showed a higher passage number, cell proliferation, and low senescence. rCECs on collagen type I film showed high expression of tight junction. The cell proliferation marker Ki-67 was positively stained either in Y-27632 or MSC-CM-containing media. Genes related to cell proliferation resulted in negligible changes in MKI67, CIP2A, and PCNA in the combinatory medium, suggesting proliferative capacity was maintained. In contrast, all of these genes were significantly downregulated in the other groups. Senescence marker β-galactosidase-positive cells significantly decreased in either MSC-CM and/or Y-27632 mixed media. Senescence-related genes downregulated LMNB1 and MAP2K6, and upregulated MMP2. Cell cycle checkpoint genes such as CDC25C, CDCA2, and CIP2A did not vary in the combinatory medium but were significantly downregulated in either ROCK inhibitor or MSC-CM alone. These results imply the synergistic effect of combinatory culture medium on corneal endothelial cell proliferation and high cell number. This study supports high potential for translation to the development of human corneal endothelial tissue regeneration.

Cellular Senescence in Age-Related Diseases: Molecular Bases and Therapeutic Interventions

“…In contrast, cell cycle checkpoint genes such as CDC25C, CDCA2 and CIP2A did not vary in the combinatory medium but were significantly downregulated in either the ROCK inhibitor or MSC-CM alone. These results demonstrated that the synergistic effect of Y-27632 and MSC-CM on corneal endothelial cell proliferation and senescence could represent a significative potential intervention for the regeneration of human corneal endothelial tissue [3].…”

Recent advances in cell-based regenerative therapies for corneal disease

“…In all patients the procedure was combined with rho-associated protein kinase inhibitor (ROCKi) adjunctive therapy, which had previously been shown to promote CEC adhesion, prevent apoptosis, increase cell proliferation, restore cell morphology and function, and overall improve engraftment. [44–47]…”

“…In all patients the procedure was combined with rho-associated protein kinase inhibitor (ROCKi) adjunctive therapy, which had previously been shown to promote CEC adhesion, prevent apoptosis, increase cell proliferation, restore cell morphology and function, and overall improve engraftment. [44][45][46][47] Recently, 5-year follow-up for these patients was published [48 && ]. Ten of the 11 eyes demonstrated improvement in endothelial cell density (ECD), central corneal thickness (CCT) and BCVA.…”

Cell therapy in corneal endothelial disease

“…A dual-media approach with alternating stabilisation (low-mitogenic) and proliferation (high mitogenic) conditions has been shown to be effective [25,44,45]. More recently, human platelet lysate (HPL) [46][47][48][49], small molecules [50,51] and mesenchymal stem cell derived conditioned media [52,53] have been reported to be helpful.…”