title: Isoflurane decreases death of human embryonic stem cell-derived, transcriptional marker Nkx2.5(+)  cardiac progenitor cells.
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 H(2) O(2) and FeSO(4) . For anaesthetic preconditioning, CPCs were exposed to isoflurane (0.25, 0.5, 1.0 mM). CPC survival was determined by trypan blue exclusion. A mitoK(ATP) channels inhibitor, 5-hydroxydecanoic acid (200 μM) and an opener, diazoxide (100 μM), were used to investigate the involvement of mitoK(ATP) 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 mitoK(ATP) 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%).
  
  
  CONCLUSIONS
  Isoflurane increased hESC-derived Nkx2.5(+) CPC survival under oxidative stress, and mitoK(ATP) channels may be involved in the protective effect.
authors.affiliation: Department of Anesthesiology and Pain Medicine, Seoul National University Medical College, Seoul, Korea.
authors.forename: J H
authors.initials: JH
authors.lastname: Kim
authors.forename: A Y
authors.initials: AY
authors.lastname: Oh
authors.forename: Y M
authors.initials: YM
authors.lastname: Choi
authors.forename: S Y
authors.initials: SY
authors.lastname: Ku
authors.forename: Y Y
authors.initials: YY
authors.lastname: Kim
authors.forename: N J
authors.initials: NJ
authors.lastname: Lee
authors.forename: A
authors.initials: A
authors.lastname: Sepac
authors.forename: Z J
authors.initials: ZJ
authors.lastname: Bosnjak
mesh_terms: D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003652:Decanoic Acids / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology*; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000032:analysis*; D006801:Humans; D006880:Hydroxy Acids / Q000494:pharmacology; D007530:Isoflurane / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects*; D018384:Oxidative Stress; D015221:Potassium Channels / Q000187:drug effects; D013234:Stem Cells / Q000187:drug effects* / Q000502:physiology; D014157:Transcription Factors / Q000032:analysis*
chemical_list: D018685:Anesthetics, Inhalation; D003652:Decanoic Acids; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D006880:Hydroxy Acids; C095552:NKX2-5 protein, human; D015221:Potassium Channels; D014157:Transcription Factors; C117780:mitochondrial K(ATP) channel; C052853:5-hydroxydecanoic acid; D007530:Isoflurane; D003981:Diazoxide
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