"In vitro" engineering of human cryopreserved valve leaflets by replacing original cells with human mesenchymal stem cells

In spite of their limited durability, cryopreserved human homografts or glutaraldehyde-preserved porcine xenografts are still the mostly used substitutes in heart valve surgery. An innovative tool for autologous-like valve replacement could be represented by decellularization of native valves and subsequent "in vitro" repopulation with human bone marrow-mesenchymal stem cells (hBM-MSCs). Here, human cryopreserved pulmonary valve leaflets (hPVLs) underwent (i) decellularization with Triton X100 and sodium cholate (TRICOL) and nuclear fragment removal with endonuclease Benzonase®, (ii) disinfection protocol, (iii) coating with FBS and fibronectin, (iv) seeding with hBMMSCs (2x106 cells/cm2), and (v) statical culture for 30 days. The used hBM-MSCs had been priorly isolated by density-gradient centrifugation from young volunteer donors' bone marrow, selected for plastic adherence, expanded and characterized with immunofluorescence and FACS analysis. The processed hBM-MSC-seeded hPVLs were found to be lined by a monolayer of endothelium-like cells (positivity for vWF and CD31), and to contain fibroblast-like, myofibroblast-like, and smooth-muscle-cell-like cells (positivity for vimentin, platelet myosin, smooth muscle actin, SM22 and smooth muscle myosin), so roughly resembling native leaflets. Consistently, the presence of these cell phenotypes, collagen neosynthesis as well as good preservation of the preexistent ECM was revealed by ultrastructural analysis. However, persistence of original stem cell antigens (SSEA4, OCT3/4) was also assessed for some of the seeded cells. These results show that after cryopreservation, thawing and the decellularization treatments here used, hPVLs (i) are permissive for "in vitro" repopulation by hBM-MSCs and their proper differentiation and (ii) potentially could overcome the limited durability of current bioprostheses, since the most frequent transplant failure depends on calcification primed by the dead cells of donor valves.