Despite significant advances in interventional cardiology and cardiac surgery (1, 2), valvular repair and replacement procedures still cannot ensure long-term functionality and freedom from reoperation for pediatric patients with congenital heart defects (CHDs), largely because of deficits inherent to valve repair materials and prosthetic replacement valves themselves. In particular, the lack of a viable cell population associated with these interventions renders them incapable of somatic growth, repair, and functional adaptation in response to changing biochemical and biomechanical cues.
Heart valve tissue engineering (HVTE) holds the potential to resolve the currently unmet need for valvular repair and replacement tissue that can grow, self-repair, and remodel to support life-long performance. HVTE strategies can be broadly stratified into in vitro, in situ, and in vivo HVTE, with the former two being the most prevalent approaches (3, 4). In vitro HVTE strategies couple resorbable biomaterials and stromal cells with the aim of producing cellularised living replacement neo-tissue with the compositional, architectural and mechanical properties to function in vivo. While the biomaterial provides a structurally and mechanically robust scaffolding on which extracellular matrix (ECM) is deposited, the presence of a viable cell population in tissue engineered heart valves (TEHVs) ensures functional adaptation and remodelling of the ECM, analogous to the valvular interstitial cell population resident within the native valve (5). In situ TEHVs encompass bioengineered valve replacements that are decellularized prior to implantation and rely on infiltration of endogenous host cells to populate the construct in vivo. In situ strategies most often use decellularized in vitro TEHVs, and less frequently, acellular bioresorbable scaffolds or decellularized allogeneic or xenogeneic valves. Thus, all in vitro and a majority of in situ HVTE strategies are entirely contingent on the stromal cell source to synthesize structural ECM proteins and assemble tissue in vitro.
HVTE strategies have historically used mesenchymal stromal cells (MSCs) sourced from vascular, dermal, adipose and bone marrow tissues, as they resemble the fibroblast subpopulation of the native valve and can undergo myofibrogenesis to synthesize the three main constituent ECM proteins of the valve leaflet: collagen, elastin, and proteoglycans (6–9). In the context of pediatric HVTE, MSCs derived from conventional tissue sources are constrained by at least one of three limitations: (i) the invasive nature of cell isolation; (ii) a limited reserve of autologous MSCs; and (iii) immunogenicity associated with allogeneic MSCs. Thus, fetal MSCs derived from prenatal (amniotic fluid, amniotic membrane) and early postnatal (placenta, umbilical cord blood, umbilical cord matrix) tissues have been studied for their utility to pediatric HVTE (10–12).
Among fetal sources of MSCs, the umbilical cord is a promising candidate, as a rich source of autologous progenitor cells that can be harvested non-invasively from tissue that is typically discarded. Human umbilical cord perivascular cells (hUCPVCs) harvested from the perivascular region of the Wharton’s jelly (WJ) possess characteristic properties of MSCs (13–15) in accordance with the minimal criteria set by the International Society for Cell and Gene Therapy (ISCT) MSC committee (16) - hUCPVCs are plastic adherent, differentiate to osteogenic, adipogenic and chondrogenic lineages, and present appropriate cell surface markers (CD105+, CD73+, CD90+, CD45-, CD34-, CD14-, HLA-DR-) (14, 17–19). Additionally, hUCPVCs contain a high frequency of colony forming unit-fibroblasts (CFU-F) that maintain proliferative and multilineage differentiation potential (20). Their proven regenerative capacity in dermal wound healing (21), musculoskeletal repair (20), and acute myocardial infarction (14) make a compelling case for their use as autologous progenitor cells for pediatric HVTE. While in vitro cardiovascular tissue engineering strategies have taken advantage of umbilical cord derived MSCs to synthesize neo-tissue (22–28), these findings cannot be projected to hUCPVCs due to a lacking consensus in the literature with respect to both the anatomical descriptors of the specific regions from which umbilical cord MSCs were harvested and the methodological techniques used to harvest these cells. Furthermore, previous studies of umbilical cord derived MSCs for in vitro cardiovascular tissue engineering have been conducted in xenogenic serum-supplemented culture conditions, complicating their translational potential.
Clinical translation of TEHVs produced from the combination of biomaterial scaffolds and hUCPVCs ultimately necessitates reproducible in vitro culture conditions. However, supplementation of cell culture medium with undefined xenogenic serum poses a challenge to good manufacturing practices (GMP) as (i) xenogenic serum-containing media (SCM) carries an inherent risk of viral, bacterial, or prion disease transmission from the donor to culture adapted human MSCs (29, 30); (ii) the presence of xenogenic proteins in engineered constructs cultured with serum can provoke an immune response in a human recipient, undermining the use of autologous cells (31); and (iii) the undefined composition of serum and lot-to-lot variability resulting from differences in animal husbandry can induce variable responses in culture adapted cells (32, 33).
Here we report serum- and xeno-free culture of hUCPVCs for in vitro HVTE applications and benchmark them against human bone marrow derived MSCs (BMMSCs), the most pervasively used MSCs in regenerative applications. We show that a commercially available serum- and xeno-free culture medium supports the proliferative capacity of hUCPVCs and BMMSCs and establish serum- and xeno-free in vitro culture conditions to promote deposition of the ECM proteins critical to the structure and function of valve tissue (collagen, elastin, and proteoglycans). Finally, we confirm ECM synthesis by hUCPVCs on electrospun scaffold sheets in a preliminary study of engineered PV repair constructs. This study establishes hUCPVCs as a suitable cell source for HVTE, supported by their accessibility, rapid proliferation, and capacity to generate neo-tissue under serum- and xeno-free culture conditions.