Files in this Data Supplement:

• Supplementary Methods - (Word) (68.5 KB)
• Figure I - (LSM) (20.7 MB) The LSM-files contain original data created by confocal microscopy. They can be viewed using the “Import” option of the Zeiss LSM Image browser, which can be downloaded free of charge under: www.zeiss.de/lsm. Manual 3D navigation within the images is possible using the “ortho” mode, 3D animation is possible using the xy / slice or the split xy / slice function.A representative image of a coculture of DiI-labeled huEPCs with NRCMs is shown. In such cases, even 3D confocal microscopy was not able to discriminate between 1) transdifferentiation of DiIpos huEPCs into cardiomyocytes, 2) fusion of DiIpos huEPC / EPC-fragments with NRCMs, 3) adhesion of DiIpos EPC fragments to NRCMs, and 4) cardiac actininpos NRCMs growing above or around DiIpos EPC fragments.
• Figure II - (LSM) (14.3 MB) A representative image of a coculture of DiI-labeled MLE-12 cells with NRCMs is shown. MLE-12 is a non cardiogenic terminally differentiated type II pneumocyte like cell line. Obtained images were very similar to those obtained from cocultures of DiI-labeled huEPCs with NRCMs, suggesting that also other mechanisms than transdifferentiation can account for the detected DiI-stained cardiomyocytes.
• Figure III - (TIF) (21.2 MB) A representative image of a TUNEL assay performed with an NRCM-EPC coculture is shown. The TUNEL assay used as positive control NRCMs incubated with 100 μM H2O2 for 18 h for induction of apoptosis (left panel). Cocultures on day 2 (middle) and day 6 (right panel) were examined for incorporation of FITC-labeled dUTP (green), cardiomyocytes were stained with anti sarcomeric α-actinin (Cy^TM3, red) and DAPI (blue). On day 6 in coculture, a number of dying cardiomyocytes show condensed nuclei (see arrows), only a minority of these cells is positive in the TUNEL assay (see arrowheads).
• Figure IV - (TIF) (4.87 MB) No expression of human cardiac transcription factors GATA-4 and Nkx2.5 or human cardiac troponin I in cocultures of human endothelial progenitor cells (huEPCs) and neonatal rat cardiomyocytes (NRCMs). Prior to cocultivation, EPCs were grown in endothelial cell growth medium for 3 or 7 days (indicated as d3 or d7, respectively). Expression of human cardiac transcripts in cocultures of huEPCs and NRCM was analyzed by species-specific PCR after 6 or 10 days of coculture (indicated as +6 or +10, respectively). To avoid contaminations with genomic DNA, RNA was treated with DNAse I prior to reverse transcription. Species-specificity is demonstrated using RNA of adult human heart tissue and neonatal rat cardiomyocytes. Reactions without reverse transcriptase were performed in parallel to control for remaining contaminations of genomic DNA. Integrity of NRCM RNA was confirmed by species specific rat β-actin and rat GATA-4 RT-PCR. Some analyses showed unspecific amplification with human GATA-4 primers (product > 300 bp; specific product 188 bp). Some analyses showed hardly detectable expression of human FOG-2. As FOG-2 is also expressed in lymphocytes, these results most likely represent low level contaminations of EPC preparations with lymphocytic cells. For β-actin and cardiac troponin I, primer pairs were designed to enclose intron sequences, thereby resulting in different product sizes in from genomic and cDNA templates. Product sizes: human β-actin, genomic: 348 bp; human β-actin, cDNA: 254 bp, rat β-actin, genomic: 400 bp; rat β-actin, cDNA: 305 bp; human FOG-2 cDNA: 203 bp; human GATA-4 cDNA: 188 bp; human Nkx2.5 cDNA: 168 bp; rat GATA-4 cDNA 284 bp; human cTnI genomic: 908 bp; human cTnI cDNA: 250 bp.