KLF2⁺ stemness maintains human mesenchymal stem cells in bone regeneration

Mesenchymal stem cells (MSCs), which are undifferentiated stem cells with the property of stemness and the potential to differentiate into multiple lineages, including osteoblasts, have attracted a great deal of attention in bone tissue engineering. Consistent with the heterogeneity of MSCs, various surface markers have been used. However, it is still unclear which markers of MSCs are best for cell amplification in vitro and later bone regeneration in vivo. Krüppel-like Factor 2 (KLF2) is an important indicator of the stemness of human MSCs (hMSCs) and as early vascularization is also critical for bone regeneration, we used KLF2 as a novel in vitro marker for MSCs and investigated the angiogenesis and osteogenesis between KLF2⁺ MSCs and endothelial cells (ECs). We found a synergistic interaction between hMSCs and human umbilical vein ECs (HUVECs) in that KLF2⁺ stemness-maintained hMSCs initially promoted the angiogenesis of HUVECs, which in turn more efficiently stimulated the osteogenesis of hMSCs. In fact, KLF2⁺ hMSCs secreted angiogenic factors initially, with some of the cells then differentiating into pericytes through the PDGF-BB/PDGFR-β signaling pathway, which improved blood vessel formation. The matured HUVECs in turn synergistically enhanced the osteogenesis of KLF2⁺ hMSCs through upregulated vascular endothelial growth factor. A three-dimensional coculture model using cell-laden gelatin methacrylate (GelMA) hydrogel further confirmed these results. This study provides insight into the stemness-directed synergistic interaction between hMSCs and HUVECs, and our results will have a profound impact on further strategies involving the application of KLF2⁺ hMSC/HUVEC-laden GelMA hydrogel in vascular network bioengineering and bone regeneration.