Unambiguous identification of implanted cells after cellular cardiomyoplasty: a critical issue.

After Cellular Cardiomyoplasty: A Critical Issue To the Editor: In their article describing cell implantation in the myocardium of rats, Davani et al1 stress the attention on integration of the transplanted syngenic mesenchymal progenitor cells within myocardial architecture and on their differentiation into smooth muscle and endothelial cells. Their conclusions are based on experimental work using 4 ,6-diamidino-2-phenylindole (DAPI) as a cell marker. They raise the legitimate concern of falsenegative cells resulting from the dilution of the marker on cell proliferation. We think, however, that the occurrence of falsepositive is also a concern with this type of cell labeling. Our group has conducted research on an autologous muscle cell grafting procedure in canine urethra as well as in ovine myocardium. To validate the best tools for non-genetic cell labeling, we performed in vitro and in vivo assessment of chemical fluorescent markers, one of which was DAPI (Sigma). Cells were marked with 50 g/mL of DAPI during 30 minutes and then washed off 3 times to remove unbound DAPI. Those cells were suspended in Dulbecco’s Modified Eagle Medium and destroyed through 3 rounds of temperature variations (from 160°C to 80°C). One hundred percent cellular mortality was obtained. The dead cells (30 million cells) were then poured onto one Petri dish containing adherent muscle cells (1 million cells per dish) that had never been exposed to DAPI. Cultures were observed after 2, 12, and 24 hours under fluorescent microscopy. Nuclei of adherent muscle cells were all marked with DAPI starting as early as 2 hours after the experiment. In addition to these in vitro assays, we carried out skeletal muscle cell implantation in the urethra of a dog and harvested the grafted area 24 hours post-surgery. We found a pocket of DAPI-labeled cells (presumably the grafted muscle cells) within the urethral wall. Many of these cells looked necrotic. The outer muscle layer was intensely marked with DAPI. Because implanted cells could not have possibly had time to colonize the whole urethra and change their phenotype to smooth muscle cells, this seriously raises the issue of resident cell labeling with DAPI released by necrotic donor cells. This issue has also been raised when grafting 1,1 -dioctadecyl-3,3,3 ,3 tetramethylindocarbocyanine perchlorate (DiI) labeled cells in the central nervous system.2 This is not unlikely when considering the massive cell death reported after cell transplantation.3 We also performed in vivo implantation of skeletal muscle derived cells as follows: We implanted either live DAPI-labeled cells, destroyed DAPI-labeled cells, or free DAPI in sheep myocardium. The grafted areas were harvested 1 hour after implantation. We did observe the live grafted cells and intensely DAPI-labeled neighboring cardiomyocytes. We also found numerous marked resident cardiomyocytes in the destroyed DAPIlabeled cell or free DAPI assays. Small DAPI-labeled capillaries were observed as well. We conclude that re-uptake of DAPI disqualifies the use of this reagent for accurate tracking of implanted cells. We think that only genetic labeling will permit unambiguous identification of the implanted cells and analysis of their fate.