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

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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 beta-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.
Author(s)
김수미이훈정보영차흥원황창모
Issued Date
2021
Type
Article
Keyword
mesenchymal stem cell-derived conditioned mediumrabbit corneal endothelial cellsROCK inhibitorsenescenceY-27632
DOI
10.3390/cells10061463
URI
https://oak.ulsan.ac.kr/handle/2021.oak/7668
https://ulsan-primo.hosted.exlibrisgroup.com/primo-explore/fulldisplay?docid=TN_cdi_doaj_primary_oai_doaj_org_article_a28d773d0d2a4bb6b74b182096c56850&context=PC&vid=ULSAN&lang=ko_KR&search_scope=default_scope&adaptor=primo_central_multiple_fe&tab=default_tab&query=any,contains,Effect%20of%20Rho-Associated%20Kinase%20Inhibitor%20and%20Mesenchymal%20Stem%20Cell-Derived%20Conditioned%20Medium%20on%20Corneal%20Endothelial%20Cell%20Senescence%20and%20Proliferation&offset=0&pcAvailability=true
Publisher
CELLS
Location
미국
Language
영어
ISSN
2073-4409
Citation Volume
10
Citation Number
6
Citation Start Page
0
Citation End Page
0
Appears in Collections:
Medicine > Medicine
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