title: No evidence of transdifferentiation of human endothelial progenitor cells into cardiomyocytes after coculture with neonatal rat cardiomyocytes.
abstract: BACKGROUND
  Recent studies have suggested the differentiation of human endothelial progenitor cells (huEPCs) isolated from peripheral blood into cardiomyocytes. This study investigates whether, when cocultured, neonatal rat cardiomyocytes (NRCMs) can induce transdifferentiation of huEPCs into cardiomyocytes.
  
  
  RESULTS
  Coculture experiments with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI)-labeled huEPCs and NRCMs have been performed. Cocultures have been analyzed by means of flow cytometry, 3D confocal laser microscopy, species-specific reverse transcriptase-polymerase chain reaction for the expression of human cardiac marker genes, and electron microscopy. Although fluorescence-activated cell sorting (FACS) analysis and conventional wide-field fluorescence microscopy suggested the existence of DiIpos cardiomyocytes in cocultures, no convincing evidence of cardiac differentiation of huEPCs has been obtained. Apparently, DiIpos cardiomyocytes were identified as necrotic NRCMs or NRCM-derived vesicles with high levels of autofluorescence or, alternatively, as NRCMs lying on top of or below labeled huEPCs or huEPC fragments. Accordingly, no expression of human Nkx2.5, GATA-4, or cardiac troponin I was detected.
  
  
  CONCLUSIONS
  No convincing evidence of transdifferentiation of huEPCs into cardiomyocytes was obtained. Although we cannot exclude that recent contrary data may be due to slightly different culture protocols, our study has revealed that recently applied standard analysis tools including FACS and wide-field fluorescence microscopy are not sufficient to demonstrate transdifferentiation in coculture settings and can lead to misinterpretation of the data obtained solely with these methods.
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School, Hannover, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Gruh
authors.forename: Janina
authors.initials: J
authors.lastname: Beilner
authors.forename: Ulrike
authors.initials: U
authors.lastname: Blomer
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmiedl
authors.forename: Ingrid
authors.initials: I
authors.lastname: Schmidt-Richter
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Kruse
authors.forename: Axel
authors.initials: A
authors.lastname: Haverich
authors.forename: Ulrich
authors.initials: U
authors.lastname: Martin
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000166:cytology*; D051381:Rats; D013234:Stem Cells / Q000166:cytology*