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Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19), with effective versus dysregulated responses influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the diverse genetic backgrounds of the Collaborative Cross founder strains crossed to K18-hACE2 transgenic mice that confers high susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 F1 progeny resulted in a spectrum of weight loss, survival, viral replication kinetics, histopathology, and cytokine profiles, some of which were sex-specific. Importantly, survival was associated with early type I interferon (IFN) expression and a phased proinflammatory response distinct from mice with severe disease. Thus, dynamics of inflammatory responses observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding antiviral immunity and evaluating countermeasures. ONE SENTENCE SUMMARY: Genetically diverse mice display a broad spectrum of clinically relevant responses to SARS-CoV-2 infection, reflecting variability in COVID-19 disease.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.One Sentence SummaryGenetically diverse mice model a spectrum of clinically relevant innate immune responses to SARS-CoV-2 infection.

Fibroblasts are diverse mesenchymal cells that participate in tissue homeostasis and disease by producing complex extracellular matrix and creating signaling niches through biophysical and biochemical cues. Transcriptionally and functionally heterogeneous across and within organs, fibroblasts encode regional positional information and maintain distinct cellular progeny. We summarize their development, lineages, functions, and contributions to fibrosis in four fibroblast-rich organs: skin, lung, skeletal muscle, and heart. We propose that fibroblasts are uniquely poised for tissue repair by easily reentering the cell cycle and exhibiting a reversible plasticity in phenotype and cell fate. These properties, when activated aberrantly, drive fibrotic disorders in humans.

Organ fibroblasts are essential components of homeostatic and diseased tissues. They participate in sculpting the extracellular matrix, sensing the microenvironment and communicating with other resident cells. Recent studies have revealed transcriptomic heterogeneity among fibroblasts within and between organs. To dissect the basis of inter-organ heterogeneity, we compare the gene expression of fibroblasts from different tissues (tail, skin, lung, liver, heart, kidney, gonads) and show that they display distinct positional and organ-specific transcriptome signatures that reflect their embryonic origins. We demonstrate that fibroblasts’ expression of genes typically attributed to the surrounding parenchyma is established in embryonic development and largely maintained in culture, bioengineered tissues, and ectopic transplants. Targeted knockdown of key organ-specific transcription factors affects fibroblasts functions, with modulation of genes related to fibrosis and inflammation. Our data open novel opportunities for the treatment of fibrotic diseases in a more precise, organ-specific manner.

Organ fibroblasts are essential components of homeostatic and diseased tissues. They participate in sculpting the extracellular matrix, sensing the microenvironment, and communicating with other resident cells. Recent studies have revealed transcriptomic heterogeneity among fibroblasts within and between organs. To dissect the basis of interorgan heterogeneity, we compare the gene expression of murine fibroblasts from different tissues (tail, skin, lung, liver, heart, kidney, and gonads) and show that they display distinct positional and organ-specific transcriptome signatures that reflect their embryonic origins. We demonstrate that expression of genes typically attributed to the surrounding parenchyma by fibroblasts is established in embryonic development and largely maintained in culture, bioengineered tissues and ectopic transplants. Targeted knockdown of key organ-specific transcription factors affects fibroblast functions, in particular genes involved in the modulation of fibrosis and inflammation. In conclusion, our data reveal that adult fibroblasts maintain an embryonic gene expression signature inherited from their organ of origin, thereby increasing our understanding of adult fibroblast heterogeneity. The knowledge of this tissue-specific gene signature may assist in targeting fibrotic diseases in a more precise, organ-specific manner.

Objective Congenital heart disease (CHD) is the most frequent birth defect worldwide. The number of adult patients with CHD, now referred to as ACHD, is increasing with improved surgical and treatment interventions. However the mechanisms whereby ACHD predisposes patients to heart dysfunction are still unclear. ACHD is strongly associated with metabolic syndrome, but how ACHD interacts with poor modern lifestyle choices and other comorbidities, such as hypertension, obesity, and diabetes, is mostly unknown. Methods We used a newly characterized mouse genetic model of ACHD to investigate the consequences and the mechanisms associated with combined obesity and ACHD predisposition. Metformin intervention was used to further evaluate potential therapeutic amelioration of cardiac dysfunction in this model. Results ACHD mice placed under metabolic stress (high fat diet) displayed decreased left ventricular ejection fraction. Comprehensive physiological, biochemical, and molecular analysis showed that ACHD hearts exhibited early changes in energy metabolism with increased glucose dependence as main cardiac energy source. These changes preceded cardiac dysfunction mediated by exposure to high fat diet and were associated with increased disease severity. Restoration of metabolic balance by metformin administration prevented the development of heart dysfunction in ACHD predisposed mice. Conclusions This study reveals that early metabolic impairment reinforces heart dysfunction in ACHD predisposed individuals and diet or pharmacological interventions can be used to modulate heart function and attenuate heart failure. Our study suggests that interactions between genetic and metabolic disturbances ultimately lead to the clinical presentation of heart failure in patients with ACHD. Early manipulation of energy metabolism may be an important avenue for intervention in ACHD patients to prevent or delay onset of heart failure and secondary comorbidities. These interactions raise the prospect for a translational reassessment of ACHD presentation in the clinic., Highlights • Adult congenital heart disease (ACHD) incidence is rapidly increasing. • Obesity triggers cardiac dysfunction in ACHD genetic predisposition mouse model. • Early metabolic and mitochondrial dysfunction was detected in ACHD hearts. • Energy modulation by metformin prevents progression of cardiac dysfunction in ACHD.

Supplemental Digital Content is available in the text., Background: Ischemic heart disease is a leading cause of heart failure and despite advanced therapeutic options, morbidity and mortality rates remain high. Although acute inflammation in response to myocardial cell death has been extensively studied, subsequent adaptive immune activity and anti-heart autoimmunity may also contribute to the development of heart failure. After ischemic injury to the myocardium, dendritic cells (DC) respond to cardiomyocyte necrosis, present cardiac antigen to T cells, and potentially initiate a persistent autoimmune response against the heart. Cross-priming DC have the ability to activate both CD4+ helper and CD8+ cytotoxic T cells in response to necrotic cells and may thus be crucial players in exacerbating autoimmunity targeting the heart. This study investigates a role for cross-priming DC in post–myocardial infarction immunopathology through presentation of self-antigen from necrotic cardiac cells to cytotoxic CD8+ T cells. Methods: We induced type 2 myocardial infarction–like ischemic injury in the heart by treatment with a single high dose of the β-adrenergic agonist isoproterenol. We characterized the DC population in the heart and mediastinal lymph nodes and analyzed long-term cardiac immunopathology and functional decline in wild type and Clec9a-depleted mice lacking DC cross-priming function. Results: A diverse DC population, including cross-priming DC, is present in the heart and activated after ischemic injury. Clec9a−/− mice deficient in DC cross-priming are protected from persistent immune-mediated myocardial damage and decline of cardiac function, likely because of dampened activation of cytotoxic CD8+ T cells. Conclusion: Activation of cytotoxic CD8+ T cells by cross-priming DC contributes to exacerbation of postischemic inflammatory damage of the myocardium and corresponding decline in cardiac function. Importantly, this provides novel therapeutic targets to prevent postischemic immunopathology and heart failure.

After myocardial infarct (MI), followed by ischemia and scar formation, interstitial cells play key roles in the adaptation to injury. Endothelial cells (ECs), for instance, can clonally expand, migrate into the infarct area and facilitate crucial functions promoting revascularization, reestablishment of oxygen supply and secretion of paracrine factors. Moreover, ECs can transiently undergo changes towards a mesenchymal phenotype (Endothelial-to-mesenchymal transition; EndMT). Whether this process contributes to long-term cardiac fibrosis or helps to facilitate post-ischemic vessel growth remains controversial. Here, we aim to delineate kinetics and characteristics of phenotypic changes in ECs with single cell RNA-sequencing (scRNA-seq). We performed a time course (homeostasis or 0 day (d), 1d, 3d, 5d, 7d, 14d, 28d post-MI) in mice and isolated the non-cardiomyocyte fraction for scRNA-seq (n=35,312 cells). Pecam1/Cdh5 double positive ECs showed expression of apoptosis, hypoxia and inflammation markers at 3d. Bioinformatic cell cycle analysis predicted high association with proliferative capacities at 3d, indicative of EC turnover post-MI. Metabolism, recently linked to regulate EndMT, was altered. We found genes of the glycolysis and the TCA-cycle pathway upregulated at 1d to 3d, and a decrease of fatty acid signaling genes. At 3d, mesenchymal markers Fn1, Vim, S100a4, Serpine1 transiently increased compared to homeostasis (>1.6-fold, p We additionally used Cdh5-CreERT2; R26-mT/mG mice followed by scRNA-seq to trace the fate of ECs. Bioinformatic analysis of GFP-positive ECs confirmed the gain in mesenchymal marker but revealed no full transition to the mesenchymal state at later timepoints. This suggests a transient mesenchymal activation of ECs rather than a complete lineage transition. We further induced EndMT with TGF-β2 in ECs in vitro and observed reversibility of the phenotype after withdrawal of the stimulus. After treatment, ECs upregulated various mesenchymal marker genes. Withdrawal of TGF-β2 at 3d or 7d, reverted expression to baseline levels. We further determined DNA methylation of EndMT gene loci to assess if TGF-β2 leads to a true fate change but did not observe changes after TGF-β2 stimulation and withdrawal. Taken together, our data suggests that ECs undergo a transient mesenchymal activation concomitant with a metabolic adaptation early after MI but do not acquire a long-term mesenchymal fate. This activation may facilitate EC migration and clonal expansion to regenerate the vascular network. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Center of Cardiovascular Research (DZHK), Deutsche Forschungsgemeinschaft (DFG) CRC1366 Project B4

Summary Interstitial cells have a crucial role in cardiac fibrosis and repair of the mammalian heart. Single-cell profiling using droplet-based technology has revolutionized the investigation of cell states and identities. Here, we present a protocol for the efficient isolation of high-quality live nucleated non-cardiomyocytes from adult murine heart, for unbiased single-cell RNA sequencing using 10× Chromium technology. This protocol has been applied to homeostatic and injured hearts from different mouse strains. For complete details on the use and execution of this protocol, please refer to Forte et al. (2020)., Graphical Abstract, Highlights • The protocol is optimized to isolate interstitial cells from adult murine hearts • Dead cell removal and live nucleated selection maximize cells capture for scRNA-seq • The protocol has been used for different mice strains, pre and post ischemic injury • The combination of DRAQ5 and PI is compatible with green reporter mice, Interstitial cells have a crucial role in cardiac fibrosis and repair of the mammalian heart. Single-cell profiling using droplet-based technology has revolutionized the investigation of cell states and identities. Here, we present a protocol for the efficient isolation of high-quality live nucleated non-cardiomyocytes from adult murine heart, for unbiased single-cell RNA sequencing using 10× Chromium technology. This protocol has been applied to homeostatic and injured hearts from different mouse strains.

Heart failure is the common final pathway of a range of conditions resulting in myocardial damage and a major cause of morbidity and mortality worldwide. Strategies to improve tissue repair and prevent heart failure thus remain an urgent clinical need. Recent studies have documented activation of the adaptive immune system in response to myocardial damage and have implicated anti-heart autoimmunity in the development of heart failure. In an attempt to target anti-heart autoimmune responses as new therapeutic avenue, the number of experimental studies using in vivo models of myocardial damage to study the ensuing immune response has surged.The beta-adrenergic agonist isoproterenol-hydrochloride has been used for its cardiac effects in a variety of different dosing and administration regimes. Most prominently, low doses (100mg/kg) induce cardiomyocyte necrosis and have been used to mimic acute myocardial necrotic lesions as seen in myocardial infarction (MI). However, despite significant resource and animal welfare advantages, concerns about off-target effects and clinical relevance have so far limited uptake in the cardiovascular research community.To assess suitability of the isoproterenol model for the analysis of chronic post-MI immunological readouts, we treated C57BL/6J mice with a single intra-peritoneal bolus injection of 160mg/kg isoproterenol. Our results confirm the presence of necrotic lesions in the myocardium with significant resemblance of the histopathology of Type 2 MI. Kidneys develop mild fibrosis secondary to early cardiac damage, while other organs remain unaffected. Most importantly, we showed that isoproterenol treatment causes myocardial inflammation and fibrosis, activation of T cells in the heart-draining mediastinal lymph nodes, deposition of mature antibodies in the myocardium and the presence of auto-antibodies against the heart in the serum 12 weeks after the initial injury.In summary, this simple and cost-effective experimental model with significant animal welfare benefits induces myocardial damage reminiscent of human Type 2 MI, which is followed by a persistent adaptive immune response against the heart. This makes it a suitable and high-throughput model to study pathological mechanisms of anti-heart autoimmunity as well as to test potential immunomodulatory therapeutic approaches.

Cardiac regeneration, that is, restoration of the original structure and function in a damaged heart, differs from tissue repair, in which collagen deposition and scar formation often lead to functional impairment. In both scenarios, the early-onset inflammatory response is essential to clear damaged cardiac cells and initiate organ repair, but the quality and extent of the immune response vary. Immune cells embedded in the damaged heart tissue sense and modulate inflammation through a dynamic interplay with stromal cells in the cardiac interstitium, which either leads to recapitulation of cardiac morphology by rebuilding functional scaffolds to support muscle regrowth in regenerative organisms or fails to resolve the inflammatory response and produces fibrotic scar tissue in adult mammals. Current investigation into the mechanistic basis of homeostasis and restoration of cardiac function has increasingly shifted focus away from stem cell-mediated cardiac repair towards a dynamic interplay of cells composing the less-studied interstitial compartment of the heart, offering unexpected insights into the immunoregulatory functions of cardiac interstitial components and the complex network of cell interactions that must be considered for clinical intervention in heart diseases.

Myocardial infarction (MI) is one of the main causes of mortality in Western Countries. The non-regenerative adult mammalian heart responds to injury with the formation of a scar. The outcome of repair is highly dependent on interstitial cells and on the delicate interplay between the inflammatory response and fibrosis, which leads to adverse ventricular remodeling and heart failure or cardiac rupture in the early phase post-injury. Our data show that inbred mice strains have different reliance to MI, suggesting differences in the cellular response to the injury. To profile the dynamic response of cardiac interstitial cells to MI and their embryological origin, we performed unbiased scRNAseq analysis of cardiac interstitial cells at homeostasis and 1, 3, 5, 7, 14, 28 days post-injury from transgenic mice on C57bl/6j background (B6), expressing ZsGreen under the control of the epicardial marker Wt1 ( Wt1 Cre ;RosaZsgreen f/+ ) . About 38,600 cells were captured using the 10xChromium technology and we distinguished 16 main cell populations that evolved over time. By high resolution sub-clustering of stromal cells, 7 main subpopulations were identified. Of these, one was endocardial derived and present at all time points, the others were epicardial derived (ZsGreen+) and included 3 populations of fibroblasts present at different frequencies at all time points, a transient population of immune-response activating fibroblasts (IR) prevailing at day1 and preceding the appearance of myofibroblasts between day 3 and 7 and matrifibrocytes prevalent at day 14 and day 28. To determine the role of cell composition in different reparative outcomes, we compared the data on the C57BL/6J background with 129S1/SvlmJ (129), the inbred strain presenting the highest frequency of cardiac rupture (70%) among the 9 tested. We observed increased number of activated IR and myofibroblasts in 129 3 days post-MI. 129 also showed higher level of collagens, chemokines, fibrin deposition and acute phase response genes. The hyperactivation of myofibroblasts was possibly mediated by the enhanced responsiveness to the Renin-Angiotensin System, currently one of the main targets of clinical therapies.

Recent technological advances have revolutionized the study of tissue biology and garnered a greater appreciation for tissue complexity. In order to understand cardiac development, heart tissue homeostasis, and the effects of stress and injury on the cardiovascular system, it is essential to characterize the heart at high cellular resolution. Single-cell profiling provides a more precise definition of tissue composition, cell differentiation trajectories, and intercellular communication, compared to classical bulk approaches. Here, we aim to review how recent single-cell multi-omic studies have changed our understanding of cell dynamics during cardiac development, and in the healthy and diseased adult myocardium.

Over the past two decades, several populations of cardiac stem cells have been described in the adult mammalian heart. For the most part, however, their lineage origins and in vivo functions remain largely unexplored. This Review summarizes what is known about different populations of embryonic and adult cardiac stem cells, including KIT+, PDGFRα+, ISL1+ and SCA1+ cells, side population cells, cardiospheres and epicardial cells. We discuss their developmental origins and defining characteristics, and consider their possible contribution to heart organogenesis and regeneration. We also summarize the origin and plasticity of cardiac fibroblasts and circulating endothelial progenitor cells, and consider what role these cells have in contributing to cardiac repair.

Summary Cardiac ischemia leads to the loss of myocardial tissue and the activation of a repair process that culminates in the formation of a scar whose structural characteristics dictate propensity to favorable healing or detrimental cardiac wall rupture. To elucidate the cellular processes underlying scar formation, here we perform unbiased single-cell mRNA sequencing of interstitial cells isolated from infarcted mouse hearts carrying a genetic tracer that labels epicardial-derived cells. Sixteen interstitial cell clusters are revealed, five of which were of epicardial origin. Focusing on stromal cells, we define 11 sub-clusters, including diverse cell states of epicardial- and endocardial-derived fibroblasts. Comparing transcript profiles from post-infarction hearts in C57BL/6J and 129S1/SvImJ inbred mice, which displays a marked divergence in the frequency of cardiac rupture, uncovers an early increase in activated myofibroblasts, enhanced collagen deposition, and persistent acute phase response in 129S1/SvImJ mouse hearts, defining a crucial time window of pathological remodeling that predicts disease outcome., Graphical Abstract, Highlights • Longitudinal transcriptional profiling of cardiac interstitial cells post-infarct • Identification of epicardial versus endocardial origin of cardiac stromal cells • A distinct early injury-response signature precedes appearance of myofibroblasts • Modulation of early fibrosis predicts cardiac rupture and pathological remodeling, Using single-cell transcriptional profiling of mouse hearts carrying a reporter for epicardial-derived cells, Forte et al. provide a dynamic view of cardiac interstitial responses across acute and chronic phases of remodeling post-infarction. Comparing responses on diverse genetic backgrounds reveals novel cellular and transcriptional features of cardiac rupture propensity.

SUMMARYThe interstitial and perivascular spaces of the mammalian heart contain a highly interactive tissue community essential for cardiac homeostasis, repair and regeneration. Mesenchymal cells (fibroblasts) are one of the most abundant cell types, playing key roles as sentinels, tissue architects, paracrine signaling hubs and lineage precursors, and are linked to heart disease through their roles in inflammation and fibrosis. Platelet-derived growth factors (PDGFs) are secreted by several cell types involved in cardiac injury and repair, and are recognized mitogens for cardiac fibroblasts and mesenchymal stem cells. However, their roles are complex and investigations of their impact on heart repair have produced contrasting outcomes, leaving therapeutic potential uncertain. Here, we use new approaches and tools, including single cell RNA sequencing, to explore cardiac fibroblast heterogeneity and how PDGF receptor α (PDGFRα) signaling impacts fibroblasts during heart repair. Short-term systemic delivery of PDGF-AB to mice from the time of myocardial infarction (MI) led to enhanced anatomical and functional recovery. Underpinning these benefits was a priming effect, in which PDGF-AB accelerated exit of fibroblasts from quiescence and induced a higher translational biosynthetic capacity in both fibroblasts and macrophages without triggering fibrosis. Our study highlights the significant biosynthetic heterogeneity and plasticity in cardiac fibroblast populations, and suggests a rationale for a novel therapeutic approach to cardiac injury involving controlled stimulation of fibroblast activation.

Mammalian hearts carry a number of primitive stem cell-like populations, although the magnitude of their contribution to tissue homeostasis and repair remains controversial. Recent CRE recombinase-based lineage tracing experiments suggest only a minor contribution to the formation of new cardiomyocytes from such cells, albeit one that might be augmented therapeutically. As the field explores clinical translation of cardiac stem cells, it will be important to understand the biology of these cells in great detail. In this review we document the various reported stem and progenitor cell populations in mammalian hearts and discuss the current state of knowledge on their origins and lineage capabilities.

The discovery and isolation of different resident cardiac progenitor cells (CPCs) a decade ago, as described by several research groups, stimulated the use of these cells for cardiac regeneration. Human CPCs are moving towards the clinic as one of the most promising cell types for cardiac repair, but the extent to which their molecular profiles vary as a result of donor heterogeneity or different isolation methods remain unclear. Defining a common molecular profile that defines CPC’s is therefore an important goal. Similarly, identifying robust and multilaboratory isolation and culture protocols that generate reproducible cell populations from genetically diverse donors is critical for their translational success. In this respect, we collected human auricle biopsy samples anonymously from 20 different adult patients that underwent bypass surgery and generated a total number of 33 different cardiac derived progenitor cell (CPC) lines (Table S1). Human CPCs were isolated according the original published protocol, based on c-kit 1 or Sca-1 2 expression or auricles were cut in 1 mm3 parts and cultured as explants to obtain Cardiospheres (CSps) 3 and Cardiosphere Derived Cells (CDCs) 4. CPCs were subsequently propagated in a panel of different media formulations, either in their originally described culture media or switched to media and culture coatings of the other CPC subsets (Figure S1, Table S7). When comparing individual CPC cell-lines, isolated with different methodologies, they shared a high degree of similarities and correlation in gene expression patterns (Fig. ​(Fig.1B).1B). By averaging expression profiles of individual CPC conditions, thereby reducing donor variability, similarities increased even more, ranging from 0.92 to 0.96 (Fig. ​(Fig.1C;1C; Table S2). These results suggest that individual donor differences were larger than influences of isolation and medium conditions. Moreover, the strongest correlations between the different CPC lines were observed when cells were isolated and cultured in the same conditions. Among the different CPCs, spheres-growing CSps showed the least correlation (0.91–0.96), while monolayer-growing CPCs shared higher correlations among them (0.96–0.98). We performed a moderated t-test to evaluate significant differentially expressed genes between the individual samples (Tables S3 and S4). Out of the 13,073 analysd genes, we found only few genes differentially expressed in 5 of 20 different monolayer-cultured CPC cell-lines comparisons. Only when the 3D-cultured CSps were compared with the other CPCs more differently expressed genes could be identified. Although only limited genes were different, we further explored if we could identify differences in gene patterns between the different CPC populations, based on selected genes important for stem cell-maintenance, their growth and biology. In particular, we evaluated genes involved in the regulation of different stem cell pathways like TGF-β, Wnt, NFkB, p53, JAK/STAT, Notch and Hedgehog (Fig. S2A), cell cycle (Fig. S2B), stem cell transcription factors (Fig. S2C), and growth factors, cytokines and chemokines (Fig. S2D). Detailed heat map analysis showed again; however, a very similar profile among all samples, with small differences mainly related to individual donors and not to different cell types or conditions (Fig. S2). Since CSps and monolayer growing CPCs have differently expressed patterns, we selected all the significantly differentially expressed genes that displayed a two fold or more difference and compared them with CDCs, and c-Kit and Sca-1+ CPCs monolayer-cultures (Table S6). Ingenuity pathway analysis identified a gene network in CSps that is enriched in genes encoding for growth factor production and signalling molecules involved in the development of cardiac muscle, vasculogenesis and angiogenesis (Fig. ​(Fig.2).2). Among them BMP-2, HGF, LIF, PTGS-2, VEGFA and PDGFRB are known to play an important role during cardiac development. Moreover, having a protective effect on a developing heart failure. Figure 1 Experimental design of the project (A) and hierarchical clustering of CPCs samples (B and C). Sca-1+ cells were isolated from human auricle biopsy and cultured in gelatin coated flask and Sca-1 medium (Sca GEL S-MED) (2) (d). After expansion cells were ... Figure 2 Ingenuity molecular networks analysis of the differentially expressed genes. Fold difference ≥2; p

Background Cardiac regenerative medicine is a rapidly evolving field, with promising future developments for effective personalized treatments. Several stem/progenitor cells are candidates for cardiac cell therapy, and emerging evidence suggests how multiple metabolic and biochemical pathways strictly regulate their fate and renewal. Scope of review In this review, we will explore a selection of areas of common interest for biology and biochemistry concerning stem/progenitor cells, and in particular cardiac progenitor cells. Numerous regulatory mechanisms have been identified that link stem cell signaling and functions to the modulation of metabolic pathways, and vice versa. Pharmacological treatments and culture requirements may be exploited to modulate stem cell pluripotency and self-renewal, possibly boosting their regenerative potential for cell therapy. Major conclusions Mitochondria and their many related metabolites and messengers, such as oxygen, ROS, calcium and glucose, have a crucial role in regulating stem cell fate and the balance of their functions, together with many metabolic enzymes. Furthermore, protein biochemistry and proteomics can provide precious clues on the definition of different progenitor cell populations, their physiology and their autocrine/paracrine regulatory/signaling networks. General significance Interdisciplinary approaches between biology and biochemistry can provide productive insights on stem/progenitor cells, allowing the development of novel strategies and protocols for effective cardiac cell therapy clinical translation. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Heart failure remains one of the main causes of morbidity and mortality in the Western world. Current therapies for myocardial infarction are mostly aimed at blocking the progression of the disease, preventing detrimental cardiac remodeling and potentiating the function of the surviving tissue. In the last decade, great interest has arisen from the possibility to regenerate lost tissue by using cells as a therapeutic tool. Different cell types have been tested in animal models, including bone marrow-derived cells, myoblasts, endogenous cardiac stem cells, embryonic cells and induced pluripotent stem cells. After the conflicting and often inconsistent results of the first clinical trials, a step backward needs to be performed, to understand the basic biological mechanisms underlying spontaneous and induced cardiac regeneration. Current studies aim at finding new strategies to enhance cellular homing, survival and differentiation in order to improve the overall outcome of cellular cardiomyoplasty.

Heart failure is one of the major causes of death in the Western world because cardiac muscle loss is largely irreversible and can lead to a relentless decline in cardiac function. Novel therapies are needed since the only therapy to effectively replace lost myocytes today is transplantation of the entire heart. The advent of embryonic and induced pluripotent stem cell (ESC/iPSC) technologies offers the unprecedented possibility of devising cell replacement therapies for numerous degenerative disorders. Not only are ESCs and iPSCs a plausible source of cardiomyocytes in vitro for transplantation, they are also useful tools to elucidate the biology of stem cells that reside in the adult heart and define signaling molecules that might enhance the limited regenerative capability of the adult human heart. Here, we review the extracellular factors that control stem cell cardiomyogenesis and describe new approaches that combine embryology with stem cell biology to discover drug-like small molecules that stimulate cardiogenesis and potentially contribute to the development of pharmaceutical strategies for heart muscle regeneration.

Colony-forming unit fibroblasts (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), have been identified in different tissues. CFU-Fs are multipotent progenitor cells, with a supportive paracrine and immunomodulatory function. They share the expression of surface antigens, are capable of self-renewal, and to differentiate into osteogenic, adipogenic and chondrogenic lineages in vitro. Our laboratory has isolated, for the first time, CFU-Fs from the adult murine heart. These cells are included within the Sca1+ PDGFRα+ CD31- fraction of interstitial cells, mainly associated with microvessels and the perivascular adventitial niche of larger vessels. Here we show that resident cCFU-Fs, are quiescent in homeostatic conditions, relatively more hypoxic and have a lower metabolic profile compared to other interstitial cells. Treatment with PDGF-AB ligand stimulates cCFU-Fs to exit the quiescent state, potentially making them more responsive to mitogenic and differentiative factors. In a mouse with a H2B-eGFP fusion gene knocked-in the Pdgfrα locus, cCFU-Fs lay in the GFPhigh population. After myocardial infarction, a GFPmed population appears, which has partially lost the expression of Sca1 and PDGFRα proteins, and consists largely of activated myofibroblasts. Likely derived from cCFU-Fs or related stromal fraction, GFPmed cells are significantly reduced by inhibiting PDGFRα signalling and increased by systemic PDGF-AB treatment. Despite augmenting the GFPmed myofibroblasts-like population, short-term PDGF-AB treatment is beneficial, leading to reduced scar, and increased vascular density and ejection fraction. A crucial factor in cardiac repair is the fine balance between pro-inflammatory (type1) and pro-reparative/pro-angiogenic (type2) immune responses, which can be regulated by MSCs. interestingly PDGF-AB treatment seems to be associated with an earlier resolution of the type1 response at 5 days after MI, compared to controls. We hypothesize that the beneficial effects of short term PDGF-AB treatment are due to early activation of cCFU-Fs and stromal cells from quiescence, leading to enhanced pro-angiogenic, anti-apoptotic and immunomodulatory impacts.

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target.

International audience; Autologous cardiac progenitor cells (CPCs) isolated as cardiospheres (CSps) represent a promising candidate for cardiac regenerative therapy. A better understanding of the origin and mechanisms underlying human CSps formation and maturation is undoubtedly required to enhance their cardiomyogenic potential. Epithelial-to-mesenchymal transition (EMT) is a key morphogenetic process that is implicated in the acquisition of stem cell-like properties in different adult tissues, and it is activated in the epicardium after ischemic injury to the heart. We investigated whether EMT is involved in the formation and differentiation of human CSps, revealing that an up-regulation of the expression of EMT-related genes accompanies CSps formation that is relative to primary explant-derived cells and CSp-derived cells grown in a monolayer. EMT and CSps formation is enhanced in the presence of transforming growth factor β1 (TGFβ1) and drastically blocked by the type I TGFβ-receptor inhibitor SB431452, indicating that TGFβ-dependent EMT is essential for the formation of these niche-like 3D-multicellular clusters. Since TGFβ is activated in the myocardium in response to injury, our data suggest that CSps formation mimics an adaptive mechanism that could potentially be enhanced to increase in vivo or ex vivo regenerative potential of adult CPCs.

The successful isolation and ex vivo expansion of resident cardiac stem/progenitor cells from human heart biopsies has allowed us to study their biological characteristics and their applications in therapeutic approaches for the repair of ischemic/infarcted heart, the preparation of tissue-engineered cardiac grafts and, possibly, the design of cellular kits for drug screening applications. From the first publication of the original method in 2004, several adjustments and slight changes have been introduced to optimize and adjust the procedure to the evolving experimental and translational needs. Moreover, due to the wide applicability of such a method (which is based on the exploitation of intrinsic functional properties of cells with regenerative properties that are present in most tissues), the key steps of this procedure have been used to derive several kinds of tissue-specific adult stem cells for preclinical or clinical purposes.In order to define the original procedure, complete with the up-to-date modifications introduced through the years, an exhaustive description of the current protocol is performed in this chapter, with particular attention in highlighting critical steps and troubleshoots. The procedure described here consists of modular steps, that could be employed to derive cells from any kind of tissue biopsy, and needs to be considered the gold standard of all the so-called "explant methods" or "cardiosphere methods," and it represents a milestone in the clinical translation of autologous cell therapy.