Presenting a potent and high-affinity integrin ligand on the surface of synthetic biomaterial scaffolds improves stem cell-biomaterial interactions for fetal tissue engineering.

Prenatal stem cell-based regenerative therapies have progressed substantially and have been demonstrated as effective treatment options for fetal diseases that were previously deemed untreatable. Due to immunoregulatory properties, self-renewal capacity, and multilineage potential, autologous human placental chorionic villus-derived mesenchymal stromal cells (CV-MSCs) are an attractive cell source for fetal regenerative therapies. However, as a general issue for MSC transplantation, the poor survival and engraftment is a major challenge of the application of MSCs. Particularly for the fetal transplantation of CV-MSCs in the naturally hypoxic fetal environment, improving the survival and engraftment of CV-MSCs is critically important. Hypoxic preconditioning (HP) is an effective priming approach to protect stem cells from ischemic damage. In this study, we developed an optimal HP protocol to enhance the survival and proangiogenic capacity of CV-MSCs for improving clinical outcomes in fetal applications. Total cell number, DNA quantification, nuclear area test, and cell viability test showed HP significantly protected CV-MSCs from ischemic damage. Flow cytometry analysis confirmed HP did not alter the immunophenotype of CV-MSCs. Caspase-3, MTS, and Western blot analysis showed HP significantly reduced the apoptosis of CV-MSCs under ischemic stimulus via the activation of the AKT signaling pathway that was related to cell survival. ELISA results showed HP significantly enhanced the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by CV-MSCs under an ischemic stimulus. We also found that the environmental nutrition level was critical for the release of brain-derived neurotrophic factor (BDNF). The angiogenesis assay results showed HP-primed CV-MSCs could significantly enhance endothelial cell (EC) proliferation, migration, and tube formation. Consequently, HP is a promising strategy to increase the tolerance of CV-MSCs to ischemia and improve their therapeutic efficacy in fetal clinical applications.

Endothelial cell (EC) transplantation via injectable collagen hydrogel has received much attention as a potential treatment for various vascular diseases. However, the therapeutic effect of transplanted ECs is limited by their poor viability, which partially occurs as a result of cellular apoptosis triggered by the insufficient cell-extracellular matrix (ECM) engagement. Integrin binding to the ECM is crucial for cell anchorage to the surrounding matrix, cell spreading and migration, and further activation of intracellular signaling pathways. Although collagen contains several different types of integrin binding sites, it still lacks sufficient specific binding sites for ECs. Previously, using one-bead one-compound (OBOC) combinatorial technology, we identified LXW7, an integrin αvβ3 ligand, which possessed a strong binding affinity to and enhanced functionality of ECs. In this study, to improve the EC-matrix interaction, we developed an approach to molecularly conjugate LXW7 to the collagen backbone, via a collagen binding peptide SILY, in order to increase EC specific integrin binding sites on the collagen hydrogel. Results showed that in the in vitro 2-dimensional (2D) culture model, the LXW7-treated collagen surface significantly improved EC attachment and survival and decreased caspase 3 activity in an ischemic-mimicking environment. In the in vitro 3dimensional (3D) culture model, LXW7-modified collagen hydrogel significantly improved EC spreading, proliferation, and survival. In a mouse subcutaneous implantation model, LXW7-modified collagen hydrogel improved the engraftment of transplanted ECs and supported ECs to form vascular network structures. Therefore, LXW7-functionalized collagen hydrogel has shown promising potential to improve vascularization in tissue regeneration and may be used as a novel tool for EC delivery and the treatment of vascular diseases.