Expression dynamics of integrin α2, α3, and αV upon osteogenic differentiation of human mesenchymal stem cells

BackgroundThe differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts (OBs) is a prerequisite for bone formation. However, little is known about the definitive surface markers for OBs during osteogenesis.MethodsTo study the surface markers on OBs, we generated and used monoclonal antibodies (MAbs) against surface molecules on transforming growth factor-beta 1 (TGF-beta 1)-treated cancer cells. The generated MAbs were further selected toward expression changes on hMSCs cultured with TGF-beta 1/bone morphogenetic protein-2 (BMP-2) or osteogenic differentiation medium (ODM) by flow cytometry. Immunoprecipitation and mass spectrometry were performed to identify target antigens of selected MAbs. Expression changes of the target antigens were evaluated in hMSCs, human periodontal ligament cells (hPDLCs), and human dental pulp cells (hDPCs) during osteogenic and adipogenic differentiation by quantitative polymerase chain reaction (qPCR) and flow cytometry. hMSCs were also sorted by the MAbs using magnetic-activated cell sorting system, and osteogenic potential of sorted cells was evaluated via Alizarin Red S (ARS) staining and qPCR.ResultsThe binding reactivity of MR14-E5, one of the MAbs, was downregulated in hMSCs with ODM while the binding reactivity of ER7-A7, ER7-A8, and MR1-B1 MAbs was upregulated. Mass spectrometry and overexpression identified that MR14-E5, ER7-A7/ER7-A8, and MR1-B1 recognized integrin alpha 2, alpha 3, and alpha V, respectively. Upon osteogenic differentiation of hMSCs, the expression of integrin alpha 2 was drastically downregulated, but the expression of integrin alpha 3 and alpha V was upregulated in accordance with upregulation of osteogenic markers. Expression of integrin alpha 3 and alpha V was also upregulated in hPDLCs and hDPCs during osteogenic differentiation. Cell sorting showed that integrin alpha V-high hMSCs have a greater osteogenic potential than integrin alpha V-low hMSCs upon the osteogenic differentiation of hMSCs. Cell sorting further revealed that the surface expression of integrin alpha V is more dramatically induced even in integrin alpha V-low hMSCs.ConclusionThese findings suggest that integrin alpha 3 and alpha V induction is a good indicator of OB differentiation. These findings also shed insight into the expression dynamics of integrins upon osteogenic differentiation of hMSCs and provide the reason why different integrin ligands are required for OB differentiation of hMSCs.