title: Susceptibility of murine induced pluripotent stem cell-derived cardiomyocytes to hypoxia and nutrient deprivation.
abstract: BACKGROUND
  Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) may be suitable for myocardial repair. While their functional and structural properties have been extensively investigated, their response to ischemia-like conditions has not yet been clearly defined.
  
  
  METHODS
  iPS-CMs were differentiated and enriched from murine induced pluripotent stem cells expressing enhanced green fluorescent protein (eGFP) and puromycin resistance genes under the control of an α-myosin heavy chain (α-MHC) promoter. iPS-CMs maturity and function were characterized by microscopy, real-time PCR, calcium transient recordings, electrophysiology, and mitochondrial function assays, and compared to those from neonatal murine cardiomyocytes. iPS-CMs as well as neonatal murine cardiomyocytes were exposed for 3 hours to hypoxia (1% O2) and glucose/serum deprivation, and viability, apoptosis markers, reactive oxygen species, mitochondrial membrane potential and intracellular stress signaling cascades were investigated. Then, the iPS-CMs response to mesenchymal stromal cell-conditioned medium was determined.
  
  
  RESULTS
  iPS-CMs displayed key morphological and functional properties that were comparable to those of neonatal cardiomyocytes, but several parameters indicated an earlier iPS-CMs maturation stage. During hypoxia and glucose/serum deprivation, iPS-CMs exhibited a significantly higher proportion of poly-caspase-active, 7-aminoactinomycin D-positive and TUNEL-positive cells than neonatal cardiomyocytes. The average mitochondrial membrane potential was reduced in "ischemic" iPS-CMs but remained unchanged in neonatal cardiomyocytes; reactive oxygen species production was only increased in "ischemic" iPS-CMs, and oxidoreductase activity in iPS-CMs dropped more rapidly than in neonatal cardiomyocytes. In iPS-CMs, hypoxia and glucose/serum deprivation led to upregulation of Hsp70 transcripts and decreased STAT3 phosphorylation and total PKCε protein expression. Treatment with mesenchymal stromal cell-conditioned medium preserved oxidoreductase activity and restored pSTAT3 and PKCε levels.
  
  
  CONCLUSIONS
  iPS-CMs appear to be particularly sensitive to hypoxia and nutrient deprivation. Counteracting the ischemic susceptibility of iPS-CMs with mesenchymal stromal cell-conditioned medium may help enhance their survival and efficacy in cell-based approaches for myocardial repair.
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies, Föhrer Str.15, Berlin, 13353, Germany. andreja.brodarac@charite.de.
authors.forename: Andreja
authors.initials: A
authors.lastname: Brodarac
authors.affiliation: Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany. tomo.saric@uni-koeln.de.
authors.forename: Tomo
authors.initials: T
authors.lastname: Šarić
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies, Föhrer Str.15, Berlin, 13353, Germany. barbara.oberwallner@alumni.charite.de.
authors.forename: Barbara
authors.initials: B
authors.lastname: Oberwallner
authors.affiliation: Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany. Shokoufeh.Mahmoodzadeh@mdc-berlin.de.
authors.forename: Shokoufeh
authors.initials: S
authors.lastname: Mahmoodzadeh
authors.affiliation: Department of Cardiothoracic Surgery, Heart Center, University Hospital Cologne, Cologne, Germany. klaus.neef@uk-koeln.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Neef
authors.affiliation: Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany. julie.albrecht@uk-koeln.de.
authors.forename: Julie
authors.initials: J
authors.lastname: Albrecht
authors.affiliation: Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany. karsten.burkert@uni-koeln.de.
authors.forename: Karsten
authors.initials: K
authors.lastname: Burkert
authors.affiliation: Max-Planck-Institute for Metabolism Research, Cologne, Germany. Matteo.Oliverio@sf.mpg.de.
authors.forename: Matteo
authors.initials: M
authors.lastname: Oliverio
authors.affiliation: Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany. filo.nguemo@uni-koeln.de.
authors.forename: Filomain
authors.initials: F
authors.lastname: Nguemo
authors.affiliation: Department of Cardiothoracic Surgery, Heart Center, University Hospital Cologne, Cologne, Germany. yh.choi@uk-koeln.de.
authors.forename: Yeong-Hoon
authors.initials: YH
authors.lastname: Choi
authors.affiliation: Department of Anatomy I, Medical Faculty, University of Cologne, Cologne, Germany. wolfnag.neiss@uni-koeln.de.
authors.forename: Wolfram F
authors.initials: WF
authors.lastname: Neiss
authors.affiliation: Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany. imorano@mdc-berlin.de.
authors.forename: Ingo
authors.initials: I
authors.lastname: Morano
authors.affiliation: Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany. j.hescheler@uni-koeln.de.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hescheler
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies, Föhrer Str.15, Berlin, 13353, Germany. stamm@dhzb.de.|Deutsches Herzzentrum Berlin, Berlin, Germany. stamm@dhzb.de.
authors.forename: Christof
authors.initials: C
authors.lastname: Stamm
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D015687:Cell Hypoxia; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D018840:HSP70 Heat-Shock Proteins; D017382:Reactive Oxygen Species; D050796:STAT3 Transcription Factor; D051744:Protein Kinase C-epsilon; D002118:Calcium