Osteoblast commitment and differentiation are controlled by multiple growth factors including members of the Notch signaling pathway. JAGGED1 is a cell surface ligand of the Notch pathway that is necessary for murine bone formation. The delivery of JAGGED1 to induce bone formation is complicated by its need to be presented in a bound form to allow for proper Notch receptor signaling. In this study, we investigate whether the sustained release of JAGGED1 stimulates human mesenchymal cells to commit to osteoblast cell fate using polyethylene glycol malemeide (PEG-MAL) hydrogel delivery system. Our data demonstrated that PEG-MAL hydrogel constructs are stable in culture for at least three weeks and maintain human mesenchymal cell viability with little cytotoxicity in vitro. JAGGED1 loaded on PEG-MAL hydrogel (JAGGED1-PEG-MAL) showed continuous release from the gel for up to three weeks, with induction of Notch signaling using a CHO cell line with a Notch1 reporter construct, and qPCR gene expression analysis in vitro. Importantly, JAGGED1-PEG-MAL hydrogel induced mesenchymal cells towards osteogenic differentiation based on increased Alkaline phosphatase activity and osteoblast genes expression including RUNX2, ALP, COL1, and BSP. These results thus indicated that JAGGED1 delivery in vitro using PEG-MAL hydrogel induced osteoblast commitment, suggesting that this may be a viable in vivo approach to bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 552–560, 2018.

Introduction: Administration of bone marrow-derived mesenchymal stem cells (MSCs) after myocardial infarction (MI) results in modest functional improvements. However; the effect of microenvironment changes after MI, such as elevated levels of oxidative stress on cardiogenic gene expression of MSCs, remains unclear. Methods: MSCs were isolated from the bone marrow of adult rats and treated for 1 week with H2O2 (0.1 to 100 μM) or 48 hours with glucose oxidase (GOX; 0 to 5 mU/ml) to mimic long-term pulsed or short-term continuous levels of H2O2, respectively. Results: In 100 μM H2O2 or 5 mU/ml GOX-treated MSCs, mRNA expression of selected endothelial genes (Flt1, vWF, PECAM1), and early cardiac marker (nkx2-5, αMHC) increased significantly, whereas early smooth muscle markers (smooth muscle α-actin and sm22α) and fibroblast marker vimentin decreased, as measured with real-time PCR. Interestingly, mRNA expression and activity of the cell-surface receptor Notch1 were significantly increased, as were its downstream targets, Hes5 and Hey1. Co-treatment of MSCs with 100 μM H2O2 and a γ-secretase inhibitor that prevents Notch signaling abrogated the increase in cardiac and endothelial genes, while augmenting the decrease in smooth muscle markers. Further, on GOX treatment, a significant increase in Wnt11, a downstream target of Notch1, was observed. Similar results were obtained with adult rat cardiac-derived progenitor cells. Conclusions: These data suggest that H2O2- or GOX-mediated oxidative stress upregulates Notch1 signaling, which promotes cardiogenic gene expression in adult stem/progenitor cells, possibly involving Wnt11. Modulating the balance between Notch activation and H2O2-mediated oxidative stress may lead to improved adult stem cell-based therapies for cardiac repair and regeneration.