TGFβ1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton
Inhibition of actin polymerization promotes adipogenic differentiation but inhibits osteogenic differentiation in MSCs. MSCs underwent osteogenic or adipogenic differentiation by culturing cells in the appropriate medium for 7 days. Cells also underwent the indicated treatments. (a) Mineralized calcium deposition, as determined by alizarin red S staining in MSCs that were osteoinduced (OS), osteoinduced with TGFβ1 treatment two days prior to induction (OS + TGFβ1), osteoinduced with CYD treatment at the onset of induction (OS + CYD), or osteoinduced with both TGFβ1 and CYD treatment at the time points described above (OS + TGFβ1 + CYD). Lower panel: micrograph of stained wells. Upper panel: quantification of mineralized matrix formation under the indicated treatment conditions. Data are shown as the mean ± SD of three independent experiments (; ). (b) Gene expression of the osteogenic markers ALPL, RUNX2, and OCN, normalized to GAPDH, as determined by qRT-PCR. Cells were either not induced (NI) or induced under the conditions described in (a). Data are shown as the mean ± SD of three independent experiments (;, ). (c) Adipogenic differentiation of MSCs that were adipoinduced (AD), adipoinduced with TGFβ1 treatment 2 days prior to induction (AD + TGFβ1), adipoinduced with CYD treatment, initiated at the onset of induction (AD + CYD), or adipoinduced with both TGFβ1 and CYD treatment at the time points described above (AD + TGFβ1 + CYD). Lower panel: Oil red O staining of the indicated cells. Upper panel: Nile red quantification of oil content under the indicated conditions. (d) Gene expression of the adipogenic marker genes PPARG and LPL, determined by qRT-PCR and normalized to GAPDH, under the indicated treatment regimens. Data are shown as the mean ± SD of three independent experiments (, ). All controls were treated with vehicle only.
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