Figure 2
Phenotypic identification of c-KitPOS/NKX2.5POS bone marrow mesenchymal stem cells. (A) Confluent, c-KitPOS/NKX2.5POS bone marrow mesenchymal stem cells (BMSCs) were passaged and third-passage cells were subjected to immunofluorescence staining for c-Kit, NKX2.5, and α-sarcomeric actin (α-SA). Unsorted total BMSCs served as control. (B) Assessment of c-Kit expression in total BMSCs before and after magnetic activated cell sorting by flow cytometric analysis. (C) Semi-quantitative RT-PCR analysis of markers for stem cells (c-Kit), cardiomyocytes (NKX2.5, GATA-4, and cardiac troponin T (cTnT)), smooth muscle cells ( SM22α) and endothelial cells (von Willebrand factor (vWF)). A sample from a neonatal Sprague–Dawley rat heart was used as positive control. (D) Flow cytometry was performed to detect the presence of Notch1 to Notch4 receptors. For immunofluorescence staining, target proteins were detected with fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated IgG. Images were captured by fluorescence microscopy. Scale bar: 50 μm. For semi-quantitative RT-PCR analysis, β-actin mRNA was used as an internal control. For flow cytometric analysis, isotype control IgG was used to set the threshold.