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.

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.