Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation
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 endot...
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MDPI AG
2021-06-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/10/6/1463 |
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| author | Boyoung Jung Hun Lee Sumi Kim Hungwon Tchah Changmo Hwang |
| author_facet | Boyoung Jung Hun Lee Sumi Kim Hungwon Tchah Changmo Hwang |
| author_sort | Boyoung Jung |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T10:30:29Z |
| format | Article |
| id | doaj.art-a28d773d0d2a4bb6b74b182096c56850 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T10:30:29Z |
| publishDate | 2021-06-01 |
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| record_format | Article |
| series | Cells |
| spelling | doaj.art-a28d773d0d2a4bb6b74b182096c568502023-11-21T23:40:58ZengMDPI AGCells2073-44092021-06-01106146310.3390/cells10061463Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and ProliferationBoyoung Jung0Hun Lee1Sumi Kim2Hungwon Tchah3Changmo Hwang4Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, KoreaDepartment of Ophthalmology, University of Ulsan College of Medicine, Seoul 05505, KoreaBiomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, KoreaDepartment of Ophthalmology, University of Ulsan College of Medicine, Seoul 05505, KoreaDepartment of Convergence Medicine, University of Ulsan College of Medicine, Seoul 05505, KoreaThis 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.https://www.mdpi.com/2073-4409/10/6/1463rabbit corneal endothelial cellsROCK inhibitorY-27632mesenchymal stem cell-derived conditioned mediumsenescence |
| spellingShingle | Boyoung Jung Hun Lee Sumi Kim Hungwon Tchah Changmo Hwang Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation Cells rabbit corneal endothelial cells ROCK inhibitor Y-27632 mesenchymal stem cell-derived conditioned medium senescence |
| title | Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation |
| title_full | Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation |
| title_fullStr | Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation |
| title_full_unstemmed | Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation |
| title_short | Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation |
| title_sort | effect of rho associated kinase inhibitor and mesenchymal stem cell derived conditioned medium on corneal endothelial cell senescence and proliferation |
| topic | rabbit corneal endothelial cells ROCK inhibitor Y-27632 mesenchymal stem cell-derived conditioned medium senescence |
| url | https://www.mdpi.com/2073-4409/10/6/1463 |
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