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Updated: Oct 30, 2025
In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition
Published on: August 20, 2016
Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial
Boyoung Jung1,2, Hun Lee3,4, Sumi Kim2
1Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, Korea.
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.
Insights
Combining a ROCK inhibitor with stem cell-derived medium significantly boosts corneal endothelial cell proliferation and reduces senescence. This approach enhances cell numbers for regenerative applications, showing promise for tissue regeneration.
Area of Science:
- Ophthalmology
- Regenerative Medicine
- Cell Biology
Background:
- Corneal endothelial dysfunction is a leading cause of blindness.
- Current treatments often involve corneal transplantation, with limited donor availability.
- Regenerative approaches using cultured corneal endothelial cells are being explored.
Purpose of the Study:
- To evaluate the synergistic effects of a Rho-associated kinase (ROCK) inhibitor (Y-27632) and mesenchymal stem cell-derived conditioned medium (MSC-CM) on rabbit corneal endothelial cell (rCEC) proliferation and senescence.
- To determine the optimal culture conditions for generating sufficient rCECs for regenerative applications.
Main Methods:
- rCECs were cultured in four conditions: control medium, Y-27632 alone, MSC-CM alone, and a combination of Y-27632 and MSC-CM.
- Cells were analyzed for morphology, size, proliferation (Ki-67), senescence (β-galactosidase), and gene expression (MKI67, CIP2A, PCNA, LMNB1, MAP2K6, MMP2, CDC25C, CDCA2).
Main Results:
- The combination of Y-27632 and MSC-CM significantly increased rCEC passage number, proliferation, and cell numbers while reducing senescence.
- Combined treatment maintained high expression of cell proliferation markers and cell cycle checkpoint genes.
- Senescence markers and related genes were significantly reduced in combined and single-treatment groups compared to control.
Conclusions:
- The synergistic effect of Y-27632 and MSC-CM promotes robust corneal endothelial cell proliferation and reduces senescence, yielding a higher cell yield.
- This combined culture strategy holds significant potential for the development of human corneal endothelial tissue regeneration therapies.
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