Amniotic Fluid Stem Cell Derived Endothelial Cells As a Vascular Source for Tissue Engineering

A major limitation in tissue engineering strategies for correction of birth defects is the inability to provide a significant source of oxygen, nutrient, and waste transport in an avascular scaffold. Successful vascularization requires developing a reliable method to generate vascular cells and designing a scaffold capable of supporting vessel formation. Broad potential for differentiation, high proliferation rates, and autologous availability for neonatal patients make amniotic fluid-derived stem cells (AFSC) well suited for use in regenerative medicine strategies for neonates. AFSC-derived endothelial cells (AFSC-EC) express key proteins and functional phenotypes associated with endothelial cells. Fibrin-based hydrogels were shown to stimulate AFSC-derived network formation in vitro but were limited by rapid degradation. Incorporation of poly(ethylene glycol) (PEG) provided mechanical stability (65%±9% weight retention vs 0% for fibrin-only gels at day 14) while retaining key benefits of fibrin-based scaffolds – quick formation (10±3sec), biocompatibility (88%±5% viability), and vasculogenic stimulation. To determine the contribution of both AFSC-EC, our proposed vascular cell source, and AFSC, our proposed perivascular cell source, we compared them to established sources of these cell types – human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), respectively. Co-cultures were seeded at a 4:1 ratio of endothelial-to-perivascular cells, and gels were incubated at 37°C for 2 weeks. Mechanical testing was performed using a stress-controlled rheometer (G’=95±10Pa), and cell-seeded hydrogels were assessed based on cell morphology. Network formation was analyzed based on key parameters such as vessel thickness, length, and area, as well as the degree of branching. There was no statistical difference between individual cultures of AFSC-EC and HUVEC in regard to these parameters, suggesting the vasculogenic potential of AFSC-EC; however, the development of robust vessels required the presence of both an endothelial and a perivascular cell source and was seen in AFSC co-cultures (70%±20% vessel length, 90%±10% vessel area, and 105%±10% vessel thickness compared to HUVEC/MSC). At a fixed seeding density, the co-culture of AFSC with AFSC-EC resulted in a synergistic effect on network parameters similar to MSC (150% vessel length, 147% vessel area, 150% vessel thickness, and 155% branching). These results suggest AFSC-EC and AFSC have significant vasculogenic and perivasculogenic potential, respectively, and are suited for in vivo evaluation.