Umbilical Cord Pericytes Provide a Viable Alternative to Mesenchymal Stem Cells for Neonatal Vascular Engineering

Abstract

Reconstructive surgery of congenital heart disease (CHD) remains inadequate due to the inability of prosthetic grafts to match the somatic growth of pediatric patients. Functionalization of grafts with mesenchymal stem cells (MSCs) may provide a solution. However, MSCs represent a heterogeneous population characterized by wide diversity across different tissue sources. Here we investigated the suitability of umbilical cord pericytes (UCPs) in neonatal vascular engineering. Explant outgrowth followed by immunomagnetic sorting was used to isolate neural/glial antigen 2 (NG2)+/CD31− UCPs. Expanded NG2 UCPs showed consistent antigenic phenotype, including expression of mesenchymal and stemness markers, and high proliferation rate. They could be induced to a vascular smooth muscle cell-like phenotype after exposure to differentiation medium, as evidenced by the expression of transgelin and smooth muscle myosin heavy chain. Analysis of cell monolayers and conditioned medium revealed production of extracellular matrix proteins and the secretion of major angiocrine factors, which conferred UCPs with ability to promote endothelial cell migration and tube formation. Decellularized swine-derived grafts were functionalized using UCPs and cultured under static and dynamic flow conditions. UCPs were observed to integrate into the outer layer of the graft and modify the extracellular environment, resulting in improved elasticity and rupture strain in comparison with acellular grafts. These findings demonstrate that a homogeneous pericyte-like population can be efficiently isolated and expanded from human cords and integrated in acellular grafts currently used for repair of CHD. Functional assays suggest that NG2 UCPs may represent a viable option for neonatal tissue engineering applications.

Approaches for the isolation and long-term expansion of pericytes from human and animal tissues

“…Lastly, pericytes play critical roles in tissue restoration, promoting, and stabilizing reparative neovascularization, as reviewed in Navarro et al (30). Accordingly, regenerative therapies using exogenous pericytes have been preclinically tested (31)(32)(33)(34)(35)(36)(37). Pericytes were delivered either as dispersed preparations or as a part of a "tissue-engineered" product to models of myocardial infarction (MI) (33,36,37), congenital heart disease (34,35), limb ischemia (38), muscular dystrophy and skeletal muscle injuries (39)(40)(41)(42), blood-brain barrier disorders (43)(44)(45)(46)(47)(48), diabetic retinopathy (49)(50)(51), and kidney fibrosis (52).…”

“…Pre-warmed media is then added to cover the fragments completely. Explanted cultures are kept undisturbed for a few days until pericytes become visible as migrated cells from the tissue pieces ( 31 , 41 , 94 ). The advantages of this isolation technique are the increased yield of collected cells, low cost, and avoidance of the destructive stress caused by enzymes ( 95 ).…”

“…Enzymatic dissociation and microdissection are generally followed by separation of antigenically defined cell populations by FACS or immunomagnetic columns. This could be associated with using selective culture media during cell expansion ( 31 , 102 ).…”

“…The addition of Fetal Bovine Serum (FBS) is debated due to the reagent’s complexity ( 114 – 116 ). Additionally, pericytes from the heart ( 32 ), saphenous vein ( 117 ), and umbilical cord ( 31 ) were expanded in Endothelial Cell Growth Medium 2 (EGM-2), while bone marrow pericytes were cultured with α-MEM containing FBS ( 105 ). Antibiotic treatment with penicillin and streptomycin is usually added to the culture medium to reduce contamination.…”

“…Human umbilical cord pericytes can be isolated by enzymatic digestion and explant culture ( 31 ). Helmbold et al performed immunomagnetic sorting of cells using CD146-conjugated microbeads to separate the CD146 + population ( 137 ).…”