Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart

Development of the Pulmonary Vein and the Systemic Venous Sinus: An Interactive 3D Overview

Knowledge of the normal formation of the heart is crucial for the understanding of cardiac pathologies and congenital malformations. The understanding of early cardiac development, however, is complicated because it is inseparably associated with other developmental processes such as embryonic folding, formation of the coelomic cavity, and vascular development. Because of this, it is necessary to integrate morphological and experimental analyses. Morphological insights, however, are limited by the difficulty in communication of complex 3D-processes. Most controversies, in consequence, result from differences in interpretation, rather than observation. An example of such a continuing debate is the development of the pulmonary vein and the systemic venous sinus, or “sinus venosus”. To facilitate understanding, we present a 3D study of the developing venous pole in the chicken embryo, showing our results in a novel interactive fashion, which permits the reader to form an independent opinion. We clarify how the pulmonary vein separates from a greater vascular plexus within the splanchnic mesoderm. The systemic venous sinus, in contrast, develops at the junction between the splanchnic and somatic mesoderm. We discuss our model with respect to normal formation of the heart, congenital cardiac malformations, and the phylogeny of the venous tributaries

ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

Three-dimensional and molecular analysis of the developing human heart

Aleksandr Sizarov beschrijft op eiwit- en RNA-niveau de expressiepatronen van transcriptiefactoren en andere eiwitten gedurende de ontwikkeling van het menselijke hart. De resultaten worden op een nieuwe interactieve wijze in driedimensionale modellen gepresenteerd. Zijn morfologische en moleculaire beschrijving van groei en differentiatie van het menselijke hart is in overeenstemming met de huidige kennis verkregen uit proefdieronderzoek. Het vormt dus een stevige basis voor vervolgstudies naar de pathogenese van aangeboren hartafwijkingen

Molecular Analysis of Patterning of Conduction Tissues in the Developing Human Heart

Background-Recent studies in experimental animals have revealed some molecular mechanisms underlying the differentiation of the myocardium making up the conduction system. To date, lack of gene expression data for the developing human conduction system has precluded valid extrapolations from experimental studies to the human situation. Methods and Results-We performed immunohistochemical analyses of the expression of key transcription factors, such as ISL1, TBX3, TBX18, and NKX2-5, ion channel HCN4, and connexins in the human embryonic heart. We supplemented our molecular analyses with 3-dimensional reconstructions of myocardial TBX3 expression. TBX3 is expressed in the developing conduction system and in the right venous valve, atrioventricular ring bundles, and retro-aortic nodal region. TBX3-positive myocardium, with exception of the top of the ventricular septum, is devoid of fast-conducting connexin40 and connexin43 and hence identifies slowly conducting pathways. In the early embryonic heart, we found wide expression of the pacemaker channel HCN4 at the venous pole, including the atrial chambers. HCN4 expression becomes confined during later developmental stages to the components of the conduction system. Patterns of expression of transcription factors, known from experimental studies to regulate the development of the sinus node and atrioventricular conduction system, are similar in the human and mouse developing hearts. Conclusions-Our findings point to the comparability of mechanisms governing the development of the cardiac conduction patterning in human and mouse, which provide a molecular basis for understanding the functioning of the human developing heart before formation of a discrete conduction system. (Circ Arrhythm Electrophysiol. 2011;4:532-542.)Stem cells & developmental biolog

Building foundations for transcatheter intervascular anastomoses: 3D anatomy of the great vessels in large experimental animals

To provide comprehensive illustrations of anatomy of the relevant vessels in large experimental animals in an interactive format as preparation for developing an effective and safe transcatheter technique of aortopulmonary and bidirectional cavopulmonary intervascular anastomoses. Computed tomographic angiographic studies in two calves and two sheep were used to prepare 3D reconstructions of the aorta, pulmonary arteries, and caval and pulmonary veins. Based on these reconstructions, computer simulations of the creation of stent-enhanced aortopulmonary and bidirectional cavopulmonary anastomoses were made. We observed the following major anatomical features: (i) caudal course of the main pulmonary artery and its branches with the proximal right pulmonary artery located immediately caudal to the aortic arch, and with the central left pulmonary artery lying at a substantial distance from the descending aorta; and (ii) the distal right pulmonary artery is located dorsal to the right atrium and inferior caval vein at a substantial distance from the superior caval vein. Animations showed creation of transcatheter analogues of Waterston's and Potts' aortopulmonary shunts through placement of a covered spool-shaped stent, and the transcatheter creation of bidirectional Glenn's cavopulmonary anastomosis, by placement of a long covered trumpet-shaped stent. There are considerable differences in vascular anatomy between large experimental animals and humans. Given the need to elaborate new transcatheter techniques for intervascular anastomoses in suitable animal models before application to human, it is crucial to take these anatomical differences into account during testing and optimization of the proposed procedure

Molecular Analysis of Patterning of Conduction Tissues in the Developing Human Heart

Background-Recent studies in experimental animals have revealed some molecular mechanisms underlying the differentiation of the myocardium making up the conduction system. To date, lack of gene expression data for the developing human conduction system has precluded valid extrapolations from experimental studies to the human situation. Methods and Results-We performed immunohistochemical analyses of the expression of key transcription factors, such as ISL1, TBX3, TBX18, and NKX2-5, ion channel HCN4, and connexins in the human embryonic heart. We supplemented our molecular analyses with 3-dimensional reconstructions of myocardial TBX3 expression. TBX3 is expressed in the developing conduction system and in the right venous valve, atrioventricular ring bundles, and retro-aortic nodal region. TBX3-positive myocardium, with exception of the top of the ventricular septum, is devoid of fast-conducting connexin40 and connexin43 and hence identifies slowly conducting pathways. In the early embryonic heart, we found wide expression of the pacemaker channel HCN4 at the venous pole, including the atrial chambers. HCN4 expression becomes confined during later developmental stages to the components of the conduction system. Patterns of expression of transcription factors, known from experimental studies to regulate the development of the sinus node and atrioventricular conduction system, are similar in the human and mouse developing hearts. Conclusions-Our findings point to the comparability of mechanisms governing the development of the cardiac conduction patterning in human and mouse, which provide a molecular basis for understanding the functioning of the human developing heart before formation of a discrete conduction system. (Circ Arrhythm Electrophysiol. 2011;4:532-542.)Stem cells & developmental biolog

Three-dimensional and molecular analysis of the arterial pole of the developing human heart

Labeling experiments in chicken and mouse embryos have revealed important roles for different cell lineages in the development of the cardiac arterial pole. These data can only fully be exploited when integrated into the continuously changing morphological context and compared with the patterns of gene expression. As yet, studies on the formation of separate ventricular outlets and arterial trunks in the human heart are exclusively based on histologically stained sections. So as to expand these studies, we performed immunohistochemical analyses of serially sectioned human embryos, along with three-dimensional reconstructions. The development of the cardiac arterial pole involves several parallel and independent processes of formation and fusion of outflow tract cushions, remodeling of the aortic sac and closure of an initial aortopulmonary foramen through formation of a transient aortopulmonary septum. Expression patterns of the transcription factors ISL1, SOX9 and AP2a show that, in addition to fusion of the SOX9-positive endocardial cushions, intrapericardial protrusion of pharyngeal mesenchyme derived from the neural crest contributes to the separation of the developing ascending aorta from the pulmonary trunk. The non-adjacent walls of the intrapericardial arterial trunks are formed through addition of ISL1-positive cells to the distal outflow tract, while the facing parts of the walls form from the protruding mesenchyme. The morphogenetic steps, along with the gene expression patterns reported in this study, are comparable to those observed in the mouse. They confirm the involvement of mesenchymal tissues derived from endocardium, mesoderm and migrating neural crest cells in the process of initial septation of the distal part of the outflow tract, and its subsequent separation into discrete intrapericardial arterial trunks. Key words: aortic arches; heart development; human embryo; outflow trac