Unknown,Transcriptomics,Genomics,Proteomics

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Effect of Rho-associated kinase inhibitor on cryopreservation of hES cells

ABSTRACT: After thawing, the mechanism of apoptosis advancing in human ES cells was invesitigated mainly by Gene conpath analysis. As time passed after thawing, nuclear fragmented cells increased more and more in cR-/mR- and cR+/mR-, but in cR+/mR+ and cR-/mR+, thawed cells formed cluster markedly, which was seen among three cell lines. Flow cytometry using TUNEL assay supported final colony formation ratio, which defined that such cell death was due to apoptosis. Gene map analysis 12 hours later after thawing of cR-/mR- showed activation of IL-1? & its receptor IL-1R and TGF-? & its receptor ACVR1C required for activation of caspase-8, initiation of caspase-8 and 10 and marked activation of ARHGDIB promoting actin reorganization comparing to cR+/mR+. Recovered cell lines in any condition did not lose proliferation and pluripotency regardless of ROCK inhibitor treatment.?These results showed apoptotic events after thawing were more marked in cR- and advanced through autoenhanced cascaded mechanism followed by initiation of self-cytokain activity, and were suppressed by ROCK inhibitor Y-27632. To investigate the protective mechanism caused by a novel cryo-technique of human embryonic stem (hES) cells using ROCK inhibitor Y-27632 against the cell death due to cryopreservation and thawing with Gene map analysis in addition to estimations of survival rate, apoptosis, undifferentiated states and pluripotency, the dissociated hES cells were cryopreserved in a freezing container in the following four conditions: addition of 10µM of Y-27632 to 1: cryoprotection solution and post-thawing medium (cR+/mR+), 2: only addition to cryoprotection (cR+/mR-), 3: only addition to post-thawing medium (cR-/mR+), 4: no addition to any medium (cR-/mR-).

ORGANISM(S): Homo sapiens

SUBMITTER: Katsunori Sasaki

PROVIDER: E-GEOD-21796 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although the consequences of genotoxic injury include cell cycle arrest and apoptosis, cell survival responses after genotoxic injury can produce intrinsic death-resistance and contribute to the development of a transformed phenotype. Protein tyrosine phosphatases (PTPs) are integral components of key survival pathways, and are responsible for their inactivation, while PTP inhibition is are often associated with enhanced cell proliferation. Our aim was to elucidate signaling events that modulate cell survival after genotoxin exposure. Diploid human lung fibroblasts (HLF) were treated with Cr(VI) (as Na2CrO4), a well known human respiratory carcinogen that induces a wide spectrum of DNA damage, in the presence and absence of a broad-range PTP inhibitor, sodium orthovanadate. Notably, PTP inhibition abrogated Cr(VI)-induced clonogenic lethality. The enhanced survival of Cr(VI)-exposed cells after PTP inhibition was predominantly due to a bypass of cell cycle arrest and was not due to decreased Cr uptake as evidenced by unchanged Cr-DNA adduct burden. Additionally, the bypass of Cr-induced growth arrest by PTP inhibition, was accompanied by a decrease in Cr(VI)-induced expression of cell cycle inhibiting genes, and an increase in the Cr(VI)-induced expression of cell cycle promoting genes. Importantly, PTP inhibition resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of DNA damage may lead to genomic instability, via bypass of cell cycle checkpoints. Experiment Overall Design: Experimental factor was chemical treatment type (3 of them) Experiment Overall Design: (1) No: HLF 1-1, HLF 1-2, HLF 1-3, HLF 1-4 Experiment Overall Design: (2) 1uM Cr(VI): HLF 3-1, HLF 3-2, HLF 3-3, HLF 3-4 Experiment Overall Design: (3) 10uM SOV + 1uM Cr(VI): HLF 4-1, HLF 4-2, HLF 4-3, HLF 4-4 Experiment Overall Design: Biological replicates: 4 different RNA extractions from 4 different cell cultures=quadruplicate per chemical treatment type

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Effect of Rho-associated kinase inhibitor on cryopreservation of hES cells

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