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. 2016 Mar 8;7:10774. doi: 10.1038/ncomms10774

Figure 3. In vitro differentiation of ISL1+ CVP clones into cardiomyocytes, as well as endothelial and smooth muscle cells.

Figure 3

Undifferentiated ISL1+ CVP clones were maintained in medium supplemented with EDN1 and WNT3A. Differentiation of the CVPs into various cardiac lineages was achieved using protocols described under material and method. (a) Immunocytochemistry demonstrating the presence of endothelial cell (EC; CD31+), smooth muscle cell (SM; SMMHC+) and cardiomyocyte (CM; CTNT+) on differentiation with various differentiation media. EC was stained with mouse anti-CD31 primary antibody while SM and CM were stained with mouse anti-SMMHC and mouse anti-CTNT, respectively, at 1:100 dilutions. Cell nuclei (blue) were stained with 4,6-diamindino-2-phenylindole (DAPI). Scale bar, 100 μm. (b) Graphical representation illustrating differentiation efficiencies of ISL1+ CVP into various cardiac lineages. Flow cytometric analyses for EC (CD31+), SM (SMMHC+) and CM (CTNT+) differentiation were performed on two progenitor clones (clones 1 and 2). Bars, s.d. of n=3 experiments. *P<0.05 and **P<0.001, evaluated by Student's t-test. (c–e) Quantitative PCR confirming the expression of CD31 and VWF (EC markers); SM22 and SMMHC (SM markers); and NKX2.5, GATA4, MEF2C, TBX5, CX43 and TNNT2 (CM markers) when ISL1+ CVP clones were differentiated into the various cardiac lineages. Gene expressions were normalized to the respective undifferentiated ISL1+ CVP clones. n=3 experiments. *P<0.05 and **P<0.01, evaluated by Student's t-test.