Abstract:

BACKGROUND: Cell marker technology has been widely applied in many studies concerning cell transplantation. Chlormethylbenzamido-1,1-dioctadecyl-3,3,3’3’-tetramethylin-docarbocyamine (CM-DIL) and 4’,6-diamidino-2-phenylindole (DAPI) are commonly used for labeling cells. To our knowledge, there are few reports on comparing the two fluorescent dyes.
OBJECTIVE: To compare the effects of CM-DIL and DAPI on labeling bone marrow mesenchymal stem cells in vitro and in vivo.
METHODS: Isolation and expansion of bone marrow-derived mesenchymal stem cells were performed according to attachment culture. The cells were labeled by CM-DIL and DAPI, respectively. Cell viability was assessed via trypan blue exclusion assay. Growth curves of bone marrow-derived mesenchymal stem cells were depicted using MTS assay. The reduction of fluorescent intensity was observed under an inverted fluorescent contrast phase microscope from passage 1 to passage 3 after labeling. Myocardial infarction was induced by left anterior artery ligation in Sprague-Dawley rats. One week later, bone marrow-derived mesenchymal stem cells labeled by CM-DIL or DAPI were injected randomly into the border area of infarct myocardium. After 3 days, transplanted cell distribution was examined under the fluorescent microscope through paraffin sections and frozen sections respectively.
RESULTS AND CONCLUSION: In vitro, bone marrow-derived mesenchymal stem cells labeled by both CM-DIL and DAPI showed decreased cell proliferation during the early period; the percentage of fluorescent-positive cells was approximately 100% in the two groups; however, the fluorescent intensity was significantly reduced from passage 1 to passage 3 in bone marrow-derived mesenchymal stem cells labeled by DAPI. In vivo, the transplanted cells were detected in a concentrated way both on the paraffin sections and frozen ones; the background color of frozen sections was lower in the CM-DIL group than in the DAPI group; false positive results of fluorescent expression could be eliminated in the CM-DIL group by using fluorescent mounting medium with the fluorescence of DNA staining. These data indicates that CM-DIL is more appropriate to in vivo tracing cells than DAPI.

Key words: stem cells, staining and labeling, mesenchymal stem cells, cell transplantation, cell differentiation, cell proliferation