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

Figure 6

Ca²⁺-based system has a synergic effect on adipogenic potential via negatively regulating the Wnt5a/β-catenin pathway in hUCB-MSCs. (a, b) MSCs-L were treated with Ca²⁺, and then a Wnt activator (100 ng/mL Wnt5a) or inhibitor (50 ng/mL Dkk-1) was added to the adipogenic medium for the initial 4 days of induction. (a) At day 14 of adipogenic induction, the cells were stained with Oil red O, and activity was quantified by counting the positively stained cells (scale bar = 50 μm, mean ± SD, , ). (b) Immunoblotting analysis was used to detect Wnt5a/β-catenin signaling and adipogenic markers in Ca²⁺-treated MSCs-L with Wnt5a or Dkk-1, with β-actin serving as a loading control. (c, d) Inhibition of β-catenin induced adipogenic differentiation in MSCs-L treated with Ca²⁺. MSCs-L were transfected with scramble siRNA (si Con) or β-catenin siRNA (si β-cat). (c) Cells were stained with Oil red O, and activity was quantified by counting the positively stained cells (scale bar = 50 μm, mean ± SD, , ). (d) The expression levels of Wnt5a/β-catenin signaling and adipogenic-related markers were measured using immunoblotting analysis. β-Actin was used at the loading control. (b, d) Expression levels were normalized to β-actin, with the expression levels in the control defined as 1 (right panel, mean ± SD, , , ). ns: not significant.

(a)

(b)

(c)

(d)