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Isoflurane decreases death of human embryonic stem cell-derived, transcriptional marker Nkx2.5+ cardiac progenitor cells

Authors

  • J. H. KIM,

    1. Department of Anesthesiology and Pain Medicine, Seoul National University Medical College, Seoul, Korea
    2. Department of Anesthesiology and Pain Medicine, Seoul National University Bundang Hospital, Seonggnam-si, Korea
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  • A. Y. OH,

    1. Department of Anesthesiology and Pain Medicine, Seoul National University Medical College, Seoul, Korea
    2. Department of Anesthesiology and Pain Medicine, Seoul National University Bundang Hospital, Seonggnam-si, Korea
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  • Y. M. CHOI,

    Corresponding author
    1. Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea
    • Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea
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  • S. Y. KU,

    1. Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea
    2. Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea
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  • Y. Y. KIM,

    1. Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea
    2. Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea
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  • N. J. LEE,

    1. Department of Anesthesiology and Pain Medicine, Seoul National University Bundang Hospital, Seonggnam-si, Korea
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  • A. SEPAC,

    1. Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI
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  • Z. J. BOSNJAK

    1. Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI
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  • Supported by grant (04-2008-007) from the SNUBH Research Fund and grant (SC1150) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Korea.

Address:

Young-Min Choi

Department of Obstetrics and Gynecology

Seoul National University College of Medicine

28 Yungun-dong

Chongno-gu

Seoul 110-744

Korea

e-mail: ymchoi@snu.ac.kr

Abstract

Background

Cardiac progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) can multiply and generate cardiomyocytes, offering their tremendous potential for cardiac regenerative therapy. However, poor survival under stressful conditions is a major hurdle in the regeneration. We investigated whether isoflurane-induced preconditioning can increase hESC-derived CPC survival under oxidative stress.

Methods

Undifferentiated hESCs were cultured in suspension with 20% FBS (fetal bovine serum) and 20 ng/ml of BMP-4 (bone morphogenetic protein-4) to form embryoid bodies and grown onto Matrigel-coated plates for 2–3 weeks. To characterise the differentiated CPCs, immunostaining for Nkx2.5 (nonspecific transcriptional marker) and Isl-1 was performed. hESC-derived CPCs were exposed to oxidative stress induced by H2O2 and FeSO4. For anaesthetic preconditioning, CPCs were exposed to isoflurane (0.25, 0.5, 1.0 mM). CPC survival was determined by trypan blue exclusion. A mitoKATP channels inhibitor, 5-hydroxydecanoic acid (200 μM) and an opener, diazoxide (100 μM), were used to investigate the involvement of mitoKATP channels.

Results

hESC-derived CPCs stained with Nkx2.5 were 95 ± 3% of total cell number. Isoflurane (0.5 and 1.0 mM)-preconditioned CPCs showed a significantly lower death rate compared with control (0.5 mM: 30.6 ± 10.7% and 1.0 mM: 28.5 ± 6.2% vs. control: 43.2 ± 9.9%). Inhibition of mitoKATP channels with 5-HD completely abolished the protective effects of isoflurane. Diazoxide significantly decreased CPC death (29.5 ± 12.4%). However, when diazoxide was applied to CPC preconditioned with isoflurane, CPC death did not decrease further (28.7 ± 10.9%).

Conclusion

Isoflurane increased hESC-derived Nkx2.5+CPC survival under oxidative stress, and mitoKATP channels may be involved in the protective effect.

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