Overexpression of GATA binding protein 4 and myocyte enhancer factor 2C induces differentiation of mesenchymal stem cells into cardiac-like cells

Figure 1 Isolation and morphology of human umbilical cord mesenchymal stem cells. A-D: A stepwise method of isolation and proliferation of human umbilical cord mesenchymal stem cells (hUC-MSCs), which show a spindle-shaped fibroblast-like cell morphology under the phase contrast microscope at P0 and P1. All images were captured under a phase contrast microscope (scale bar: 100 μm).

Figure 2 Human umbilical cord mesenchymal stem cell characterization by immunocytochemical analysis, flow cytometry, and lineage differentiation assays. A: Immunocytochemsitry of human umbilical cord mesenchymal stem cell (hUC-MSC) showing positive expression of CD44, CD90, CD105, and vimentin, and negative expression of CD45, a hematopoietic marker. Images were captured under a fluorescence microscope (scale bar: 50 μm); B: Flow cytometry of hUC-MSCs showing positive expression of CD44, CD73, and CD90. Data were analyzed using BD FACS Diva software; C: Adipogenic and osteogenic lineage differentiation of hUC-MSCs. Images were captured under a phase contrast microscope (scale bar: 100 μm).

Figure 3 Gene expression analysis of GATA binding protein 4 and myocyte enhancer factor 2C transfected human umbilical cord mesenchymal stem cells. Semiquantitative real-time polymerase chain reaction (RT-PCR) analysis was performed to show the gene expression levels of GATA binding protein 4 and myocyte enhancer factor 2C transfected mesenchymal stem cells, separately and in combination, in comparison to the control. Results are expressed as the mean ± SE (n = 3). Differences between groups are considered statistically significant where ^bP < 0.01 and ^cP < 0.001. GATA-4: GATA binding protein 4; MEF2C: Myocyte enhancer factor 2C.

Figure 4 Morphological changes and cardiac-specific gene expression in transfected human umbilical cord mesenchymal stem cells. A: Images showing human umbilical cord mesenchymal stem cells trasnfected with (b) GATA binding protein 4 (GATA-4), (c) myocyte enhancer factor 2C (MEF2C), and (d) GATA-4 + MEF2C, and (a) the corresponding untreated control. All images were captured at day 14 under a phase contrast microscope (scale bar: 100 μm); B: Bar diagrams showing fold change analysis of cardiac gene expression by semiquantitative real-time polymerase chain reaction (RT-PCR) in the transfected cells in comparison to the control cells after 14 d of culture. Results are expressed as the mean ± SE (n = 3). Differences between groups are considered statistically significant where ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. GATA-4: GATA binding protein 4; MEF2C: Myocyte enhancer factor 2C; MHC: Myosin heavy chain; NKX2.5: NK2 homeobox 5.

Figure 5 Cardiac-specific protein expression in transfected human umbilical cord mesenchymal stem cells. A: Fluorescence images showing human umbilical cord mesenchymal stem cells (hUC-MSCs) transfected with GATA binding protein 4 (GATA-4) and myocyte enhancer factor 2C (MEF2C), separately and in combination, in comparison to the control cells (scale bar: 50 μm); B: Bar diagrams showing quantification of positive cells using ImageJ software. Also shown is the comparison between the individual and combination groups. Results are expressed as the mean ± SE (n = 5). Differences between groups are considered statistically significant where ^aP < 0.05 and ^cP < 0.001. GATA-4: GATA binding protein 4; MEF2C: Myocyte enhancer factor 2C; CTnT: Cardiac troponin T; NKX2.5: NK2 homeobox 5; NS: Not significant.