Results: 4

1.
Figure 4

Figure 4. Marker analysis of hESC-derived cardiomyocytes.. From: ISL1 Protein Transduction Promotes Cardiomyocyte Differentiation from Human Embryonic Stem Cells.

Cardiomyocytes were dissociated at differentiation day 14 post plating, cultured for an additional 2 days, and assessed by (A) immunofluorescence staining and (B) flow cytometry for cardiac-specific markers. Flow cytometric analysis showed significant increases in the levels of cTnT, CONNEXIN43, ACTININ, and GATA4 protein expression (P<0.05). ). The experiment was performed for at least 3 independent biological replicates. *: P<0.05.

Hananeh Fonoudi, et al. PLoS One. 2013;8(1):e55577.
2.
Figure 1

Figure 1. Daily qRT-PCR analysis in aggregate differentiation of hESCs.. From: ISL1 Protein Transduction Promotes Cardiomyocyte Differentiation from Human Embryonic Stem Cells.

Undifferentiated aggregates of hESCs were treated by Activin A for 1 day and then for 4 days by BMP4. At day 5, the aggregates were plated without cytokines. The data show the maximum expression of the mesoendodermal marker, Brachyury, one day after Activin A treatment (day 2 after differentiation initiation). By continuing differentiation with BMP4 for the next 4 days Isl1, a marker of precardiac mesoderm, and Actinin were reached to their highest expression level. Isl1 expression was remained at high level for the next 3 days and by decreasing its expression, Mef2c, a cardiac progenitor marker showed its maximum expression and after that, other cardiac progenitor genes, Gata4 Nkx2.5 and Tbx5 reached to their highest expression level respectively. Finally, the expression of MHC and cTnT, which are structural cardiomyocytes markers, got to maximum level (Fig. 1). These data shows that 3-dimentional structures of the cells are very important for cardiac differentiation and aggregated differentiation method enhances cardiac differentiation and functionality.Target genes were normalized by the reference gene Gapdh. The relative expression was calculated by dividing the normalized target gene expression of the treated sample with that of the undifferentiated state (day 0). All data represented as log2-linear plots. All data are statistically significant otherwise marked with “ns” (P>0.05).

Hananeh Fonoudi, et al. PLoS One. 2013;8(1):e55577.
3.
Figure 3

Figure 3. Optimization of the effect of rISL1 protein on hESCs.. From: ISL1 Protein Transduction Promotes Cardiomyocyte Differentiation from Human Embryonic Stem Cells.

(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.

Hananeh Fonoudi, et al. PLoS One. 2013;8(1):e55577.
4.
Figure 2

Figure 2. Generation, purification, and penetration of rISL1 protein.. From: ISL1 Protein Transduction Promotes Cardiomyocyte Differentiation from Human Embryonic Stem Cells.

(A) Construction of the ISL1 protein expression vector in fusion with the TAT transduction domain for penetration into the cells and N-terminus histidine tag for purification of recombinant protein. (B) Over-expressing rISL1fusion protein (42 kDa) was purified from the bacterial expression system as demonstrated by Coomassie blue staining by SDS-PAGE (M: Size marker; T: Total protein; E: Elution ). (C) Western blot analysis using anti-ISL1 antibody. (D) Gel shift assay confirmed that native rISL1 could bind to digested genomics DNA. The first lane is the ladder. The second lane shows DNA. Lanes 3 and 4 are rISL1 proteins at 2 different concentrations (5 and 10 µg). The other lines are DNA treated by different amounts of protein. The band of rISL1 is detectable in protein-loaded lines, which disappeared in the DNA-treated group although the other non-specific proteins were still visible. This indicated that only the ISL1 protein had the ability to interact with DNA. (E) The stability of the rISL1 protein was evaluated under cell culture conditions in the presence and absence of hESCs for 2 days by Western blot analysis. Temporal analysis showed that the rISL1 protein was detectable in medium up to 48 h in the presence or absence of cells; no significant decrease in the amount of protein was observed. Cellular uptake of the recombinant protein was also analyzed by Western blot analysis of cell lysates from vehicle (glycerol)- or rISL1-treated hESCs. (F) Quantification of Western blot analysis of experiments in Fig. 2E. The percentage volume of ISL1 protein was calculated by dividing the percentage volume of ISL1 protein at a defined time by the percentage volume of β-TUBULIN. Expression of the ISL1 protein was significantly higher when hESCs were treated with rISL1 in comparison with vehicle-treated cells. (G) Quantitative RT-PCR of ISL1 gene expression at different time points after addition of the rISL1 protein. Data is represented as log2-linear plot. These data show significant increase in the level of Isl1 expression in comparison to untreated group in all the time points after addition of the rISL1 protein (P<0.05). (H) ISL1-GFP reporter assay. Histograms show the percentages of GFP+ cells in rISL1 treated and control groups and indicate that after addition of rISL1 protein on undifferentiated cells, the percentages of ISL1 expressing cells are increased (P<0.05). This result suggests that rISL1 enhances its own gene expression. The experiment was performed for at least 3 independent biological replicates. (I) Cellular uptake of rISL1 protein into the adherent and sphere hESCs, visualized by immunostaining with both anti-TAT and ISL1 antibodies. *: P<0.05.

Hananeh Fonoudi, et al. PLoS One. 2013;8(1):e55577.

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