Intracellular Calcium Determines the Adipogenic Differentiation Potential of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells via the Wnt5a/β-Catenin Signaling Pathway

Figure 2

Effect of the intracellular calcium level on adipogenic differentiation of hUCB-MSCs with high or low adipogenic differentiation potential. (a) Fluo4-AM was used to measure the intracellular calcium level in the control condition from MSCs-H (M-H) or MSCs-L (M-L). Upper: the Fluo4-AM expression levels of calcium were measured by flow cytometry. The populations shown indicate the Fluo4-AM staining profile (red circle) versus the isotype control staining profile, and the percentage of Fluo4-AM-positive cells is shown (mean ± SD, , ). Lower: the calcium levels were analyzed by fluorescence microscopy after Fluo4-AM staining (green). Nuclei were stained with 6-diamidino-2-phenylindole (DAPI; blue). The merged image is an overlay of the DAPI and Fluo4-AM images (scale bar = 50 μm, mean ± SD, , ). Confocal microscopy was used to determine the fluorescence intensity of the cytoplasmic Ca²⁺ level. (b, c) To assess the effect of Ca²⁺ or BAPTA-AM treatment on adipogenic differentiation, the cells cultured under each experimental condition were assessed for a differentiation period of 14 days. (b) Staining with Oil red O was significantly increased with Ca²⁺ treatment in MSCs-H and MSCs-L and was significantly decreased with BAPTA-AM treatment (mean ± SD, n = 3, ^∗∗p < 0.01, ^∗p < 0.05). Scale bar = 50 μm. (c) Adipogenic-related proteins (PPARγ and leptin) measured by immunoblotting, with β-actin serving as a loading control. Expression levels were normalized to β-actin, with the expression levels in the control defined as 1 (lower panel, mean ± SD, , , ). BAPTA: BAPTA-AM.

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