Heart valve tissue engineering (HVTE) holds the potential to transform surgical management of the congenitally stenotic pulmonary valve (PV) in pediatric patients by producing a viable construct with the capacity for growth, repair, and remodeling. To date, HVTE strategies have not been able to produce a valve with long-term in vivo functionality, suggesting that bioengineered constructs do not sufficiently mimic the properties of the native PV. However, there is a paucity of pediatric PV characterization studies that provide standards against which pediatric tissue engineered heart valves (TEHVs) can be assessed. Furthermore, in vitro HVTE strategies rely on invasive cell sourcing methods not feasible for a pediatric population to obtain autologous progenitor cells. In the first part of this thesis, we conducted a comprehensive characterization of the native piglet PV as a model for the human pediatric PV. We used biaxial tensile testing to demonstrate the anisotropic and non-linear tensile behaviour of the piglet PV leaflet and defined material constants for a mathematical model to describe this behaviour for the first time in the pediatric leaflet. We quantified the constituent extracellular matrix (ECM) proteins (collagen, glycosaminoglycans, and elastin) of the native piglet leaflet and demonstrated their lamellar organization. Finally, we quantified the proportion of α-smooth muscle actin expressing valvular interstitial cells to quantitate the activated myofibroblast population within the piglet PV. In the second part of this thesis, we evaluated human umbilical cord perivascular cells (hUCPVCs) as an autologous progenitor cell source that can be non-invasively harvested from mesenchymal stromal cell (MSC)-rich tissues and cultured under serum- and xeno-free conditions for pediatric HVTE. To this end we evaluated the proliferative, clonogenic, multilineage differentiation, and ECM2

synthesis capacities of hUCPVCs in a commercial serum- and xeno-free culture medium (StemMACSTM) and benchmarked them to adult bone marrow-derived MSCs (BMMSCs). We showed that hUCPVCs had greater proliferative and clonogenic potential than BMMSCs in StemMACSTM (p<​0.05), without differentiation to osteogenic and adipogenic phenotypes associated with valve pathology. hUCPVCs synthesized significantly more total collagen, glycosaminoglycans, and elastin, the ECM constituents identified in the piglet PV, than BMMSCs. Importantly, hUCPVCs retained their ECM synthesizing capacity when cultured on polycarbonate polyurethane anisotropic electrospun scaffolds, a representative biomaterial for in vitro HVTE. The findings of this thesis characterize the fundamental properties of the native piglet to provide targets for tissue engineering pediatric PV and establish hUCPVCs as a readily available autologous neonatal cell source with in vitro ECM synthesizing capacity for pediatric HVTE.