Multicolor-immunofluorescence - an Approach to Identify Human Mesenchymal Stem Cells

Introduction: Human mesenchymal stem cells (hMSCs) are most suitable for tissue engineering applications. However, because they lack distinctive phenotypic features, their identification is only possible retrospectively, i.e. by differentiation assays. Until now, no specific marker is known that allows the definitive characterization of undifferentiated stem cells. Materials and Methods: In order to characterize hMSCs without performing differentiation assays, we established a simultaneous four-color immunofluorescence of the putative stem cell markers CD 44, CD 105, CD 106, and collagen IV. Detection of antibody binding was performed by immunofluorescence using fluorochrome conjugated secondary antibodies (AMCA, TexasRed, FITC, and Alexa546, respectively). Because a clear distinction of more than three different fluorochromes is not possible with conventional image analysis systems, we performed spectral image acquisition that enables discrimination of four fluorescent spectra plus nuclear stain (DAPI). Results: We found that the surface proteins CD 44, CD 105, and CD 106 were positive on almost every cell within the hMSC population. In contrast, collagen IV revealed heterogeneous labeling results. Conclusions: We present a four-color immunofluorescence of human mesenchymal stem cells with a characteristic labeling profile. This technique appears a suitable approach in order to characterize mesenchymal stem cells on single cell level without performing differentiation assays.