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

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 mu M) and an opener, diazoxide (100 mM), 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.