We examined the myogenic response to infarction in neonatal and adult mice to determine the role of c-kit ⁺ cardiovascular precursor cells (CPC) that are known to be present in early heart ...development. Infarction of postnatal day 1–3 c-kit ᴮᴬC-EGFP mouse hearts induced the localized expansion of (c-kit)EGFP ⁺ cells within the infarct, expression of the c-kit and Nkx2.5 mRNA, myogenesis, and partial regeneration of the infarction, with (c-kit)EGFP ⁺ cells adopting myogenic and vascular fates. Conversely, infarction of adult mice resulted in a modest induction of (c-kit)EGFP ⁺ cells within the infarct, which did not express Nkx2.5 or undergo myogenic differentiation, but adopted a vascular fate within the infarction, indicating a lack of authentic CPC. Explantation of infarcted neonatal and adult heart tissue to scid mice, and adoptive transfer of labeled bone marrow, confirmed the cardiac source of myogenic (neonate) and angiogenic (neonate and adult) cells. FACS-purified (c-kit)EGFP ⁺/(αMHC)mCherry ⁻ (noncardiac) cells from microdissected infarcts within 6 h of infarction underwent cardiac differentiation, forming spontaneously beating myocytes in vitro; cre/LoxP fate mapping identified a noncardiac population of (c-kit)EGFP ⁺ myocytes within infarctions, indicating that the induction of undifferentiated precursors contributes to localized myogenesis. Thus, adult postinfarct myogenic failure is likely not due to a context-dependent restriction of precursor differentiation, and c-kit induction following injury of the adult heart does not define precursor status.
Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address ...these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.
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•CD73-EGFP labels multipotent stromal cells (MSCs) in various tissues•CD73-EGFP allows purification of MSCs from the bone compartment•CD73-EGFP also marks bone marrow sinusoidal cells enabling their purification•RNA-seq analysis reveals endothelial heterogeneity within the bone marrow
Breitbach et al. generated a CD73-EGFP reporter mouse that enabled identification and tracking of multipotent stromal cells in vivo. CD73-EGFP also labeled sinusoidal endothelial cells within the bone marrow, enabling the molecular characterization of this important endothelial component of the hematopoietic stem cell niche.
Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an ...adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.
Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts ...protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.
Directed differentiation of embryonic stem cells indicates that mesodermal lineages in the mammalian heart (cardiac, endothelial, and smooth muscle cells) develop from a common, multipotent ...cardiovascular precursor. To isolate and characterize the lineage potential of a resident pool of cardiovascular progenitor cells (CPcs), we developed BAC transgenic mice in which enhanced green fluorescent protein (EGFP) is placed under control of the c-kit locus (c-kitBAC-EGFP mice). Discrete c-kit-EGFP⁺ cells were observed at different stages of differentiation in embryonic hearts, increasing in number to a maximum at about postnatal day (PN) 2; thereafter, EGFP⁺ cells declined and were rarely observed in the adult heart. EGFP⁺ cells purified from PN 0-5 hearts were nestin⁺ and expanded in culture; 67% of cells were fluorescent after 9 days. Purified cells differentiated into endothelial, cardiac, and smooth muscle cells, and differentiation could be directed by specific growth factors. CPc-derived cardiac myocytes displayed rhythmic beating and action potentials characteristic of multiple cardiac cell types, similar to ES cell-derived cardiomyocytes. Single-cell dilution studies confirmed the potential of individual CPcs to form all 3 cardiovascular lineages. In adult hearts, cryoablation resulted in c-kit-EGFP⁺ expression, peaking 7 days postcryolesion. Expression occurred in endothelial and smooth muscle cells in the revascularizing infarct, and in terminally differentiated cardiomyocytes in the border zone surrounding the infarct. Thus, c-kit expression marks CPc in the neonatal heart that are capable of directed differentiation in vitro; however, c-kit expression in cardiomyocytes in the adult heart after injury does not identify cardiac myogenesis.
Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry ...could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45
cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.
Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that ...lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion hybrids, but almost exclusively in response to injuries or inflammation.
Mesenchymal stem cells (MSCs) are multipotent and considered to be of great potential for regenerative medicine. We could show recently (Breitbach, Kimura et al. 2018) that a subpopulation of MSCs as ...well as sinusoidal endothelial cells (sECs) in the bone marrow (BM) of CD73-EGFP reporter mice could be labeled in vivo.
We took advantage of this model to explore the plasticity and osteogenic potential of CD73-EGFP+ MSCs in vitro and their role in the regenerative response upon bone lesion in vivo. Herein we show that isolated CD73-EGFP+ MSCs displayed more pronounced stemness and stronger in vitro differentiation capacity into the osteogenic lineage compared to CD73-EGFP− MSCs. In a bone fracture model, endogenous BM-resident CD73-EGFP+ MSCs were found to migrate to the fracture site and differentiate into cartilage and bone cells. Our analysis also showed that CD73-EGFP+ sECs contributed to the neovascularization of the fracture site. In addition, grafting of CD73-EGFP+ MSCs into acute bone lesions revealed their capacity to differentiate into chondrocytes and osteocytes in vivo and their contribution to callus formation in the regeneration process of fracture healing. Thus, CD73+ MSCs display enhanced stemness and osteogenic differentiation potential in vitro and in vivo illustrating a prominent role of the CD73+ MSC subpopulation to promote fracture repair.
•CD73+ mesenchymal stem cells (MSCs) display pronounced proliferation potential.•CD73+ MSCs show high in vitro osteogenic differentiation capacity.•Endogenous bone marrow-resident CD73+ MSCs contribute to fracture healing.•Grafted CD73+ MSCs promote bone formation upon fracture repair.
Controversy exists in regard to the beneficial effects of transplanting cardiac or somatic progenitor cells upon myocardial injury. We have therefore investigated the functional short- and long-term ...consequences after intramyocardial transplantation of these cell types in a murine lesion model.
Myocardial infarction (MI) was induced in mice (n = 75), followed by the intramyocardial injection of 1-2×10(5) luciferase- and GFP-expressing embryonic cardiomyocytes (eCMs), skeletal myoblasts (SMs), mesenchymal stem cells (MSCs) or medium into the infarct. Non-treated healthy mice (n = 6) served as controls. Bioluminescence and fluorescence imaging confirmed the engraftment and survival of the cells up to seven weeks postoperatively. After two weeks MRI was performed, which showed that infarct volume was significantly decreased by eCMs only (14.8±2.2% MI+eCM vs. 26.7±1.6% MI). Left ventricular dilation was significantly decreased by transplantation of any cell type, but most efficiently by eCMs. Moreover, eCM treatment increased the ejection fraction and cardiac output significantly to 33.4±2.2% and 22.3±1.2 ml/min. In addition, this cell type exclusively and significantly increased the end-systolic wall thickness in the infarct center and borders and raised the wall thickening in the infarct borders. Repetitive echocardiography examinations at later time points confirmed that these beneficial effects were accompanied by better survival rates.
Cellular cardiomyoplasty employing contractile and electrically coupling embryonic cardiomyocytes (eCMs) into ischemic myocardium provoked significantly smaller infarcts with less adverse remodeling and improved cardiac function and long-term survival compared to transplantation of somatic cells (SMs and MSCs), thereby proving that a cardiomyocyte phenotype is important to restore myocardial function.
An Overview on Approaches for Coordination of Platoons Lesch, Veronika; Breitbach, Martin; Segata, Michele ...
IEEE transactions on intelligent transportation systems,
2022-Aug., 2022-8-00, Letnik:
23, Številka:
8
Journal Article
Recenzirano
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In the recent past, platooning evolved into an attractive cooperative driving technology, broadly discussed in research and practice. Vehicles in platoons use cooperative adaptive cruise control to ...drive at close distances to each other. Platooning (i) increases the capacity of the street by a factor of 2; (ii) reduces the fuel consumption and emissions by up to 20%; and (iii) has social implications as it increases driver comfort and safety. As platooning research progresses, platooning coordination becomes a major research focus. The coordination of platoons, including the assignment of vehicles to platoons, the management of inter- and intra-platoon interactions, and the coordination of interactions with other vehicles is an important step towards an effective usage of platooning in practice. Based on a literature review of 1,600 papers, this survey provides an overview of state of the art in platooning coordination research for both cars and trucks. In this paper, we present a novel taxonomy for platooning coordination and classify existing approaches. We use the results of the literature review to discuss challenges and outline avenues for future work such as multi-objectiveness and individualisation.
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