title: Gene pathway analysis of the mechanism by which the Rho-associated kinase inhibitor Y-27632 inhibits apoptosis in isolated thawed human embryonic stem cells.
abstract: 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.
authors: Department of Histology and Embryology, Shinshu University School of Medicine, Matsumoto, Nagano, Japan. h.ichikawa@thu.ac.jp
authors: Hinako
authors: H
authors: Ichikawa
authors: Naohiro
authors: N
authors: Nakata
authors: Youichi
authors: Y
authors: Abo
authors: Sakiko
authors: S
authors: Shirasawa
authors: Tadayuki
authors: T
authors: Yokoyama
authors: Susumu
authors: S
authors: Yoshie
authors: Fengming
authors: F
authors: Yue
authors: Daihachiro
authors: D
authors: Tomotsune
authors: Katsunori
authors: K
authors: Sasaki
mesh_terms: D000577:Amides / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001549:Benzamides / Q000494:pharmacology; D053455:Caspase 10 / Q000096:biosynthesis; D053148:Caspase 3 / Q000096:biosynthesis; D053181:Caspase 8 / Q000096:biosynthesis; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000378:metabolism; D004149:Dioxoles / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D053583:Interleukin-1beta / Q000096:biosynthesis; D011725:Pyridines / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D000577:Amides; D001549:Benzamides; D003451:Cryoprotective Agents; D004149:Dioxoles; D007093:Imidazoles; D053583:Interleukin-1beta; D011725:Pyridines; D016212:Transforming Growth Factor beta; C108830:Y 27632; D054460:rho-Associated Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D053455:Caspase 10; D053148:Caspase 3; D053181:Caspase 8; C093642:SB 203580