Bone Morphogenetic Protein-2 Accelerates Osteogenic Differentiation in Spheroid-Derived Mesenchymal Stem Cells

Spheroid culture systems more accurately recreate the in vivo microenvironment and are susceptible to factors that induce differentiation. In this study, we assessed whether bone morphogenetic protein (BMP)-2 induces enhanced osteogenic differentiation in spheroid-derived mesenchymal stem cells (MSCs). MSC spheroids were generated from human adipose tissue-derived MSCs using low-binding plates. Osteogenic differentiation of monolayer and spheroid-derived MSCs was induced by osteogenesis induction medium (OIM) with or without BMP-2. Increased alkaline phosphatase and Alizarin Red staining were observed in spheroid-derived MSCs treated with a mixture of OIM and BMP-2, compared with monolayer MSCs. Spheroid-derived MSCs had increased mRNA and protein expressions of osteogenic runt-related transcription factor 2 (Runx2) and osterix (OSX). The intranuclear expression of OSX was also observed in spheroid-derived MSCs treated with the mixture of OIM and BMP-2. In addition, spheroid-derived MSCs with BMP-2 treatment showed the upregulation of Smad5 mRNA and phosphorylated Smad1/5, suggesting that the Smad-BMP signaling pathway is enhanced in these cells. Our data indicate that the Smad-dependent BMP signaling pathway accelerates osteogenic differentiation in spheroid-derived MSCs, compared with monolayer MSCs.