EphB4 Promotes Osteogenesis of CTLA4-Modified Bone Marrow-Derived Mesenchymal Stem Cells Through Cross Talk with Wnt Pathway in Xenotransplantation

Background: Cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4-Ig)-modified bone marrow-derived mesenchymal stem cells (MSCs-CTLA4) have excellent osteogenic function in xenografts, but their mechanism of action remains to be elucidated. As bidirectional signaling between erythropoietin-producing hepatocyte receptors B4 (EphB4) and ephrinB2 is vital for bone remodeling, this study aimed to fully characterize the role of MSCs-CTLA4 in promoting bone regeneration in xenotransplantation through EphB4/ephrinB2 and their cross talk with the Wnt/beta-catenin pathway. Methods: MSCs-CTLA4 were investigated for their osteogenic capacity through xenotransplantation in vivo. MSCs-CTLA4 were treated with ephrinB2-FC or FC under conditions of osteogenic induction and cultured with or without immune activation conditions established by phytohemagglutinin and peripheral blood mononuclear cells in vitro. Osteogenesis markers and the Wnt pathway-related molecules such as EphB4, runt-related transcription factor 2 (Runx2), collagen 1 (COL1), osteocalcin (OCN), alkaline phosphatase (ALP), calcium nodus, β-catenin, phospho-glycogen synthase kinase 3-beta (p-GSK-3β)-Ser9, and glycogen synthase kinase 3-beta (GSK-3β) were detected. Results: MSCs-CTLA4-based xenografts show better osteogenic capacity compared with MSC-based xenografts. EphB4 expression was reduced in MSCs compared with MSCs-CTLA4 under immune activation conditions. In ephrinB2-FC-treated cells, levels of osteogenesis markers were increased compared with FC-treated cells. The activity of GSK-3 was inhibited and the expression of β-catenin in MSCs was increased by ephrinB2-FC treatment. Conclusions: CTLA4 modification maintains EphB4 expression in MSCs under immune activation conditions, and EphB4 cross talk with the Wnt pathway promotes osteogenic differentiation of MSCs-CTLA4.