BackgroundLiving grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts.Methods and ResultsCD34pos cells, negative for the endothelial markers CD31 and CD146, were identified by immunohistochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34pos/CD31neg cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kitpos cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft.ConclusionsThese findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.

Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace threedimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.

Summary B chromosomes are centric chromosomal fragments present in thousands of eukaryotic genomes. Because most B chromosomes are non-essential, they can be lost without consequence. In order to persist, however, some B chromosomes can impose strong forms of intra-genomic conflict. An extreme case is the paternal sex ratio (PSR) B chromosome in the jewel wasp Nasonia vitripennis. Transmitted solely via the sperm, PSR 'imprints' the paternal chromatin so that it is destroyed during the first mitosis of the embryo. Owing to the haplo-diploid reproduction of N. vitripennis, PSR-induced loss of the paternal chromatin converts embryos that should become females into PSR-transmitting males. This conversion is key to the persistence of PSR, although the underlying mechanisms are largely unexplored. We assessed how PSR affects the paternal chromatin and then investigated how PSR is transmitted efficiently at the cellular level. We found that PSR does not affect progression of the paternal chromatin through the cell cycle but, instead, alters its normal Histone H3 phosphorylation and loading of the Condensin complex. PSR localizes to the outer periphery of the paternal nucleus, a position that we propose is crucial for it to escape from the defective paternal set. In sperm, PSR consistently localizes to the extreme anterior tip of the elongated nucleus, while the normal wasp chromosomes localize broadly across the nucleus. Thus, PSR may alter or bypass normal nuclear organizational processes to achieve its position. These findings provide new insights into how selfish genetic elements can impact chromatin-based processes for their survival.

Current vascular replacement grafts used in congenital heart defect corrective surgery have poor longevity and growth potential. Recipient patients often require multiple reoperations. Tissue engineering has the promise to produce a graft with the potential to grow, remodel and repair. Here we aimed at developing an amnion-based scaffold suitable for cardiovascular tissue engineering applications and in vivo usage. The developed human amnion-based scaffold was made by an enzymatic decellularization process followed by freeze-drying as a single or multi-layered structure. These structures were compared to native amnion for seeded cell viability and biomechanical properties then tested for in vivo biocompatibility. Our results demonstrated that while native amnion tissue supported little cell growth, the decellularized-amnion allowed cell engraftment and proliferation cell survival. Additionally, preservation of the scaffold by freeze-drying as a single layer, allowed further improved cell engraftment and cell growth. Multi-layering the freeze-dried amnion-scaffolds resulted in a similar cell growth potential of the single layered construct but superior mechanical strength. The multi-layered construct showed in vitro biocompatibility with endothelial cells, smooth muscle cells, cardiac myocytes, and thymus and cord-blood-derived MSCs. When implanted in a piglet model of left pulmonary artery grafting, the multi-layered construct showed its in vivo suitability and biocompatibility for vascular repair as demonstrated by the development of newly formed endothelium in the intima, a smooth muscle cell-rich medial layer and an adventitia containing new vasa vasorum. endothelial cell layer in the inner side of the graft and a smooth muscle layer in the outer side. In conclusion, our developed amnion-derived scaffold represents an off-the-shelf biocompatible structure that can be seeded with the patient's own MSCs to produce an autologous vascular graft.

Mesenchymal stem cells (MSCs) are attractive tools for regenerative medicine because of their multidifferentiation potential and immunomodulation capacity. In congenital heart defect surgical correction, replacement grafts lacking growth potential are commonly used. Tissue engineering promises to overcome the limitations of these grafts. In this study, we hypothesized that human thymus-derived MSCs are a suitable tool to tissue engineer a living vascular graft with good integration and patency once implanted in vivo. Human thymus-derived MSCs (hT-MSCs) were identified by the expression of MSC markers and mesenchymal differentiation potential. When cultured onto natural scaffold to produce tissue-engineered graft, hT-MSCs exhibited great proliferation potential and the ability to secrete their own extracellular matrix. In addition, when implanted in vivo in a piglet model of left pulmonary grafting, the engineered graft exhibited good integration within the host tissue, indicating potential suitability for corrective cardiovascular surgery. The optimized xeno-free, good manufacturing practices-compliant culture system proved to be optimum for large-scale expansion of hT-MSCs and production of tissue-engineered cardiovascular grafts, without compromising the quality of cells. This study demonstrated the feasibility of engineering clinical-grade living autologous replacement grafts using hT-MSCs and proved the compatibility of these grafts for in vivo implantation in a left pulmonary artery position.

Lack of growth potential of available grafts represents a bottleneck in the correction of congenital heart defects. Here we used a swine small intestinal submucosa (SIS) graft functionalized with mesenchymal stem cell (MSC)-derived vascular smooth muscle cells (VSMCs), for replacement of the pulmonary artery in piglets. MSCs were expanded from human umbilical cord blood or new-born swine peripheral blood, seeded onto decellularized SIS grafts and conditioned in a bioreactor to differentiate into VSMCs. Results indicate the equivalence of generating grafts engineered with human or swine MSC-derived VSMCs. Next, we conducted a randomized, controlled study in piglets (12–15 kg), which had the left pulmonary artery reconstructed with swine VSMC-engineered or acellular conduit grafts. Piglets recovered well from surgery, with no casualty and similar growth rate in either group. After 6 months, grafted arteries had larger circumference in the cellular group (28.3 ± 2.3 vs 18.3 ± 2.1 mm, P < 0.001), but without evidence of aneurism formation. Immunohistochemistry showed engineered grafts were composed of homogeneous endothelium covered by multi-layered muscular media, whereas the acellular grafts exhibited a patchy endothelial cell layer and a thinner muscular layer. Results show the feasibility and efficacy of pulmonary artery reconstruction using clinically available grafts engineered with allogeneic VSMCs in growing swine.

The materials available for the Right Ventricular Outflow Tract (RVOT) reconstruction in patients with Tetralogy of Fallot (TOF)/pulmonary atresia come with the severe limitation of long-term degeneration and lack of growth potential, causing right ventricular dysfunction, aneurysm formation and arrhythmias, thus necessitating several high-risk reoperations throughout patients' life.In this study, we evaluated the capacity of Mesenchymal Stem Cells (MSCs) derived from the Wharton's Jelly (WJ-MSCs), the gelatinous inner portion of the umbilical cord, to grow and recellularize an extracellular matrix (ECM) graft in our optimised xeno-free, good manufacturing practice-compliant culture system. WJ-MSCs were phenotypically and functionally characterised by flow cytometry and multi-lineage differentiation capacity, respectively. The typical MSCs immunophenotype and functional characteristics were retained in our xeno-free culture system, as well as the capacity to grow and engraft onto a naturally occurring scaffold. Wharton's Jelly MSCs, from both human and swine source, showed excellent capacity to recellularize ECM graft producing a living cell-seeded construct.In addition, we have provided an in vivo proof of concept of feasibility of the cellularised conduit, engineered with swine Wharton's Jelly MSCs, to be used in a novel porcine model of main pulmonary artery reconstruction, where it showed good integration within the host tissue.Our study indicates that the addition of WJ-MSCs to the ECM scaffold can upgrade the material, converting it into a living tissue, with the potential to grow, repair and remodel the RVOT. These results could potentially represent a paradigm shift in paediatric cardiac intervention towards new modalities for effective and personalised surgical restoration of pulmonary artery and RVOT function in TOF/pulmonary atresia patients.

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