| Gene pathway analysis of the mechanism by which the Rho-associated kinase inhibitor Y-27632 inhibits apoptosis in isolated thawed human embryonic stem cells. | |
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MedLine Citation:
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PMID: 22133891 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Cryopreservation is an essential technique in basic research and clinical applications of human embryonic stem (hES) cells. Cryopreserved hES cells are fragile and undergo post-thaw apoptosis. We performed gene pathway analysis on cryopreserved and thawed hES cells to examine the effect of Y-27632, a Rho-associated kinase (ROCK) inhibitor, on apoptosis and associated molecular events. Y-27632 was added to the cryopreservation solution and/or the post-thaw medium of two hES cell lines (KhES-1, KhES-3). Post-thaw apoptosis was recorded as a function of time using Giemsa staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis plateaued 12h after the untreated hES cells were thawed. Gene pathway analysis showed the activation of IL-1β, TGF-β, and their respective receptors (IL-1R, ACVR1C) in the mitogen-activated protein kinase (MAPK) pathway, which resulted in the upregulation of caspase-8 and -10. Quantitative RT-PCR confirmed the upregulation of IL-1β, TGF-β, their respective receptors, and caspase-10 and -3. As these molecules were suppressed by Y-27632, gene pathways involving these molecules probably depend on ROCK activation. The TGF-β receptor antagonist, SB-431542, and an inhibitor of p38MAPK, SB-203580, did not affect apoptosis. Combining Y-27632 with SB-203580, however, resulted in an increase in the survival rate compared with the control. This suggests that the initiation of apoptosis depends on cytokine interactions and multiple ways exist to reduce post-thaw apoptosis in hES cells. Y-27632 can suppress cytokine interactions and the MAPK pathway, thereby reducing the occurrence of apoptosis, and is an effective cryoprotectant for hES cells. |
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Authors:
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Hinako Ichikawa; Naohiro Nakata; Youichi Abo; Sakiko Shirasawa; Tadayuki Yokoyama; Susumu Yoshie; Fengming Yue; Daihachiro Tomotsune; Katsunori Sasaki |
Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-11-23 |
Journal Detail:
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Title: Cryobiology Volume: - ISSN: 1090-2392 ISO Abbreviation: - Publication Date: 2011 Nov |
Date Detail:
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Created Date: 2011-12-2 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0006252 Medline TA: Cryobiology Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Copyright Information:
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Copyright © 2011. Published by Elsevier Inc. |
Affiliation:
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Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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