Successful Reconstruction of the Right Ventricular Outflow Tract by Implantation of Thymus Stem Cell Engineered Graft in Growing Swine

Visual Abstract

C ardiac tissue engineering holds great promise for definitive correction of congenital heart disease. By seeding cells on a biodegradable scaffold, this approach is aimed at developing a viable graft capable of growing and remodeling in parallel with the recipient's organ.
Once implanted in vivo, an ideal biofunctional graft is remodeled and eventually replaced by the host's own extracellular matrix (1). Several cell types have been used for cardiac tissue engineering, including mesenchymal stem cells (MSCs) (2), induced pluripotent stem cells (3), endothelial cells (ECs) (4), and pericytes (5). In particular, MSCs are a preferred choice because of their immune-privileged nature, multilineage differentiation potential, and ability to promote tissue healing by paracrine mechanisms (6,7). Furthermore, MSCs can be isolated from various tissues and organs, including bone marrow, umbilical cord, adipose tissue, and thymus (8,9). Infants who undergo palliative cardiac surgery for tetralogy of Fallot often have the thymus removed to facilitate access to the heart. Hence, the explanted gland represents a convenient source of autologous MSCs for use during definitive correction of congenital cardiac defects.
To date, cardiac tissue engineering has been applied principally to the reconstruction of pulmonary arteries or valves, whereas problems associated with the right ventricular outflow tract (RVOT) remain an unmet clinical need. In fact, surgical correction of RVOT obstruction is reportedly associated with the development of aneurysmal or akinetic regions and an arrhythmogenic substrate (10). These complications have long-term consequences for outcomes in patients with tetralogy of Fallot (11).
In this paper, we report a controlled, randomized study in growing female piglets, comparing the feasibility, safety, and efficacy of RVOT reconstruc-     where pixels were classified as representing either a     Figures 1A and 1B).
Furthermore, we confirmed the ability of T-MSCs to differentiate into mesenchymal lineages ( Figure 1C).  (Figure 2A). T-MSCs were viable within the scaffold at 2 weeks from seeding, with only a few dead cells being observed (4.0 AE 0.6% of total cell counts) ( Figure 2B). They were stratified on the   subject vs. seeded; p ¼ 0.052) ( Figure 3C). Similarly, the proportion of right ventricular (RV) fractional area change did not differ between groups and was within clinical reference range (32% to 60%) of normal RV function ( Table 2).

IMPLANTATION OF T-MSC-ENGINEERED GRAFTS
IMPROVES RVOT CONTRACTILITY COMPARED WITH UNSEEDED GRAFTS. Cardiac cardiac magnetic resonance assessments of RVOT motion and deformation were carried out before surgery and at termination, using unoperated adult pigs as control subjects for the latter time point ( Figure 3D). Basal RVOT-MS values did not differ between the study groups, thus excluding a chance of imbalance that may influence outcome, and were therefore cumulated (  Figure 3E).
The RVOT-MS increase observed in the seeded group was the consequence of a reduction of the systolic area, whereas diastolic area did not differ between the 2 groups ( Table 3).  CMs, and fibroblasts ( Figure 7). Some Ki67-positive ECs and fibroblast-like cells were observed in the seeded grafts explanted after 24 h, 1 week, and 2 weeks, whereas neither VSMCs nor CMs were positive for Ki67 ( Figure 7A).

DISCUSSION
Because of their similarities to humans and rapid growing rate, pigs are valuable animal models for preclinical assessment of cardiac tissue engineering (12,13). The present study confirms the feasibility and safety of RVOT reconstruction using a SIS-ECM graft in growing piglets. In addition, we report for the first time instability, re-entrant arrhythmias, and even sudden cardiac death (23,24). We did not perform electrophysiological studies; thus, we can only infer the absence of life-threatening arrhythmias from the fact that no fatality was observed. Another limitation of the study was the absence of a sham-operated group to assess unspecific background effect of surgery on the heart. However, the main comparator was the group of pigs with grafts that were not seeded with stem cells.
Most of the grafts currently used in cardiac surgery are either synthetic or fixed materials. These materials have not been proved to be particularly effective during long-term follow-up, leading to lack of contractility and mismatched mechanical properties, which might contribute to the risk for sudden death (25,26). Our feasibility, safety, and efficacy in vivo study has shown that seeding SIS-ECM with T-MSCs overcomes some of the known limitations of current prosthetic material. These results may pave the way to new modalities for effective surgical restoration of RV