Evaluating Human Umbilical Cord Perivascular Cells as an Alternative to Bone-Marrow Derived Mesenchymal Stromal Cells for Heart Valve Tissue Engineering

Nejad, Shouka Parvin 1, 2; Simmons, Craig A. 1, 2, 3

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada; 2. Ted Rogers Centre for Heart Research, Toronto, Canada; 3. Department of Mechanical & Industrial Engineering, University of Toronto, Toronto, Canada

Introduction: Bone marrow-derived MSCs (bm-MSCs) are a prevalent cell source used in heart valve tissue engineering (HVTE) as they resemble the fibroblast subpopulation of valvular interstitial cells. However, bm-MSCs are constrained by a limited reserve of autologous cells, the possibility of immune rejection of donor MSCs, and the invasive nature of cell isolation. Furthermore, existing HVTE cell culture practices require xenogenic serum, complicating subsequent clinical translatability. A promising alternative are human umbilical cord perivascular cells (hUCPVCs), which are readily isolated at birth and can be expanded under serum-free (SF), xeno-free conditions for subsequent regenerative applications. However, the growth kinetics, multilineage differentiation capacity, and extracellular matrix (ECM) producing capacity of hUCPVCs and bm-MSCs in SF culture have not yet been explored. Methods: To determine cell growth kinetics, hUCPVCs and bm-MSCs were expanded in serum-supplemented (SS) and SF culture and counted after 8 days using a hemocytometer. Colony forming unit fibroblast (CFU-F) frequency of hUCPVCs and bm-MSCs expanded in SF media was determined by crystal violet staining. The multilineage differentiation capacity of SF expanded hUCPVCs and bm-MSCs were assessed by alizarin red and oil red-O staining after induction with osteogenic and adipogenic media. ECM production by SF expanded cells in basal media (BM) and media supplemented with 50 uM ascorbic acid (AA) was quantified by chloramine-T/Ehrlich’s reagent assay (collagen), dimethylmethylene blue dye-binding assay (sulfated glycosaminoglycans (s-GAGs)), and the Biocolor Fastin Elastin assay kit (elastin). ECM production was normalized to DNA content quantified using the Hoescht 33258 dye binding assay. Results: hUCPVCs demonstrated superior proliferation to bm-MSCs, with a shorter average population doubling time under both SF (36.78±0.45 vs. 154.08±18.67 hours, p=0.0002) and SS conditions (40.25±0.34 vs. 171.56±31.11 hours, p<0.0001). After 2 weeks in culture, the CFU-F frequency of hUCPVCs was significantly higher (26±7%) than that of bm-MSCs (1±1%) in SF culture (p<0.01) . While bm-MSCs differentiated to adipocytes and osteoblasts after induction, hUCPVCs retained their initial phenotype. In SF culture, hUCPVCs synthesized significantly more collagen (BM: 6.47±1.25 ug hydroxyproline/ug DNA vs. not detected (ND); AA: 6.68±1.46 vs. 1.66±0.43 ug hydroxyproline/ug DNA, p<0.0001), elastin (BM: 213.45±28.21 vs. 176.14±1.81 ug elastin/ug DNA; AA: 196.95±6.00 vs. 65.52±33.00 ug elastin/ug DNA, p<0.0001), and s-GAGs (BM: 15.23±3.01 ug s-GAG/ug DNA vs ND; AA: 15.93±5.10 ug s-GAG/ug DNA vs ND) than bm-MSCs in BM and/or media supplemented with AA. Conclusion: hUCPVCs are a superior alternative to bm-MSCs for HVTE due to their enhanced proliferation, CFU-F frequency, lack of adipogenic and osteogenic differentiation, and ECM-synthesis capacity under xeno-free, SF culture conditions.