ISL1 Protein Transduction Promotes Cardiomyocyte Differentiation from Human Embryonic Stem Cells
Figure 3
Optimization of the effect of rISL1 protein on hESCs.
(A) To evaluate the effect of discontinuous (2 h/day) or continuous rISL1 protein addition on hESCs (Royan H5) differentiation, cells were treated continuously or discontinuously from days 1–8 post initiation of differentiation. Isl1 qRT-PCR analysis of differentiated cells at day 8 showed higher significant endogenous Isl1 expression in hESCs in the continuous protocol.Thus continuous treatment was applied in the next steps. )* : P<0.05((B) To determine the best concentration of rISL1 protein for cardiac differentiation, cells were treated with four different concentrations of recombinant protein: 10, 20, 30, and 40 µg/ml in continuous treatment of hESCs during days 1–8 after initiation of differentiation. During differentiation, cells that were treated by 10 and 20 µg/ml rISL1 protein were morphologically similar to hematopoietic and endothelial progenitors, while the 30 and 40 µg/ml concentrations showed cardiomyocyte and muscular appearances. It seems that 30 and 40 µg/ml rISL1 protein are better concentrations for cardiac differentiation. )* : P<0.05((C) qRT-PCR analysis of differentiated cells at day 8 by different concentrations of rISL1 also showed that 40 µg/ml of the rISL1 protein induced more endogenous Isl1, but less Mef2c and Nkx2.5 expressions. )* : P<0.05((D) Schematic diagram of the differentiation protocol by the addition of rISL1 protein (40 µg/ml), which was added after induction with Activin A (days 1–8). qRT-PCR analysis of endogenous Isl1 expression in hESCs demonstrated that treated cells expressed higher significant endogenous Isl1 than the untreated control. )* : P<0.05((E) The percentage of beating clusters in continuous treatment of hESCs by 40 µg/ml rISL1 protein during days 1–8 after differentiation initiation in comparison with the control (vehicle-treated) group. The percentage of beating clusters in the rISL1-treated group was significantly higher than the untreated group at day 14 after plating (75±10% vs. 20±2.5%). )* : P<0.05((F) rISL1 treatment resulted in a 3.2±0.5 fold increase in the number of beating areas in comparison with untreated control group. rISL1 also caused a 2.2±0.4 fold increase in the other hESC line, Royan H6, which shows the reproducibility of this protocol for another hESC line. )* : P<0.05((G) In order to assess the expression of cardiac-specific genes, we collected samples at 3 stages: day 3 after plating (the day of rISL1 removal); day 14 after plating (day of maximum beating); and day 20 after plating (day that beating decreased and cells were mature) by qRT-PCR in two hESC lines. Target genes were normalized by the reference gene Gapdh. The relative expression was calculated by dividing the normalized target gene expression of treated hESCs with rISL1 protein and elution buffer (as control) with that of the undifferentiated state (day 0). All data are statistically significant in comparison with undifferentiated state (day 0) otherwise marked with “ns” (ns: P>0.05). a: P<0.05 in comparison with control group (elution buffer treated group). All data were represented as log2-linear plots.