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Mesothelial and epicardial cells give rise to various types of mesenchymal cells via epithelial (mesothelial)-to-mesenchymal transition during development. However, the genes controlling the differentiation and diversification of mesothelial/epicardial cells remain unclear. Here, we examined Wnt2b expression in the embryonic mesothelium and epicardium and performed lineage tracing of Wnt2b-expressing cells by using novel Wnt2b-2A-CreERT2 knock-in and LacZ-reporter mice. Wnt2b was expressed in mesothelial cells covering visceral organs, but the expression was restricted in their subpopulations. Wnt2b-expressing cells labeled at embryonic day (E) 10.5 were distributed to the mesothelium and mesenchyme in the lungs, abdominal wall, stomach, and spleen in Wnt2b 2A-CreERT2/+ ;R26R LacZ/+ mice at E13.0. Wnt2b was initially expressed in the proepicardial organ (PEO) at E9.5 and then in the epicardium after E10.0. Wnt2b-expressing PEO cells labeled at E9.5 differentiated into a small fraction of cardiac fibroblasts and preferentially localized at the left side of the postnatal heart. LacZ + epicardium-derived cells labeled at E10.5 differentiated into a small fraction of fibroblasts and smooth muscle cells in the postnatal heart. Taken together, our results reveal novel subpopulations of PEO and mesothelial/epicardial cells that are distinguishable by Wnt2b expression and elucidate the unique contribution of Wnt2b-expressing PEO and epicardial cells to the postnatal heart., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Decellularization is regarded as a xenogenic antigen-reduction technique because it effectively eliminates all cellular and nuclear components while mitigating any negative impact on the composition, biological functionality, and structural integrity of the remaining extracellular matrix. This study aimed to histologically evaluate native, freeze dried and chemically decellularized bovine pericardium membrane. Also, this study focused on preservation of extracellular matrix after decellularization. Bovine pericardium membrane was decellularized by freeze thaw cycle followed by freeze drying and 1% sodium dodecyl sulphate. Unprocessed pericardium was used as control. The effectiveness of Decellularization was assessed based on the reduction of histologically visible nuclei. Decellularization by freeze thaw cycle followed by freeze drying resulted in 17.84% reduction in nuclei content and decellularization by sodium dodecyl sulphate results in 92% reduction in nuclei content compare to control group. Picrosirius red staining for freeze dried group displayed loosely organised, thin collagen bundles that exhibit reddish-yellow birefringence and sodium dodecyl sulfate group revealed dense collagen bundles that are parallelly organised and compact, exhibiting reddish-yellow birefringence and showed good structural integrity. These results suggested that the sodium do decyl sulfate showed optimal decellularization results with better extracellular matrix preservation. It may be a suitable protocol for producing a suitable scaffold for periodontal tissue regeneration., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Epicardial epithelial-to-mesenchymal transition (EMT) plays a pivotal role in both heart development and injury response and involves dynamic cellular changes that are essential for cardiogenesis and myocardial repair. Specifically, epicardial EMT is a crucial process in which epicardial cells lose polarity, migrate into the myocardium, and differentiate into various cardiac cell types during development and repair. Importantly, following EMT, the epicardium becomes a source of paracrine factors that support cardiac growth at the last stages of cardiogenesis and contribute to cardiac remodeling after injury. As such, EMT seems to represent a fundamental step in cardiac repair. Nevertheless, endogenous EMT alone is insufficient to stimulate adequate repair. Redirecting and amplifying epicardial EMT pathways offers promising avenues for the development of innovative therapeutic strategies and treatment approaches for heart disease. In this review, we present a synthesis of recent literature highlighting the significance of epicardial EMT reactivation in adult heart disease patients., (© 2024. The Author(s).)

The epicardium, previously viewed as a passive outer layer around the heart, is now recognized as an essential component in development, regeneration, and repair. In this review, we explore the cellular and molecular makeup of the epicardium, highlighting its roles in heart regeneration and repair in zebrafish and salamanders, as well as its activation in young and adult postnatal mammals. We also examine the latest technologies used to study the function of epicardial cells for therapeutic interventions. Analysis of highly regenerative animal models shows that the epicardium is essential in regulating cardiomyocyte proliferation, transient fibrosis, and neovascularization. However, despite the epicardium's unique cellular programs to resolve cardiac damage, it remains unclear how to replicate these processes in nonregenerative mammalian organisms. During myocardial infarction, epicardial cells secrete signaling factors that modulate fibrotic, vascular, and inflammatory remodeling, which differentially enhance or inhibit cardiac repair. Recent transcriptomic studies have validated the cellular and molecular heterogeneity of the epicardium across various species and developmental stages, shedding further light on its function under pathological conditions. These studies have also provided insights into the function of regulatory epicardial-derived signaling molecules in various diseases, which could lead to new therapies and advances in reparative cardiovascular medicine. Moreover, insights gained from investigating epicardial cell function have initiated the development of novel techniques, including using human pluripotent stem cells and cardiac organoids to model reparative processes within the cardiovascular system. This growing understanding of epicardial function holds the potential for developing innovative therapeutic strategies aimed at addressing developmental heart disorders, enhancing regenerative therapies, and mitigating cardiovascular disease progression., Competing Interests: None.

During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The characterization of cell populations that reside in the outer layer of the heart has been hindered by difficulties in their isolation. Here, we present a protocol for isolation and single-nuclei multiomic analyses of the human fetal epicardium. We describe steps for microdissection, isolation, and enrichment of epicardial cells by mechanical dissociations and direct lysis. We then detail procedures for integrating transcriptome and chromatin accessibility datasets. This approach allows the analysis of diverse cell populations, marked by unique cis-regulatory elements. For complete details on the use and execution of this protocol, please refer to Travisano et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: This study aims to decellularized caprine pericardium tissue with varied non-ionic surfactant and anionic detergent concentrations., Methods: Protocol A consists of 1%, 0.5%, and 0.25% (w/v) sodium dodecyl sulphate (SDS). Protocol B uses 1%, 0.5%, and 0.25% (w/v) Triton X-100. Protocol C comprised 0.5% SDS + 0.5% Triton X-100, 0.5% + 0.25%, and 0.25% SDS + 0.5% Triton X-100., Results: Protocol B left a few countable cells in the pericardium tissue, but treatments A and C removed all cells. DNA quantification also demonstrated that protocol B had the most leftover DNA after decellularization. The pericardium tissue treated with an equal combination of anionic detergent and non-ionic surfactant preserves the matrix. However, changing the anionic detergent-non-ionic surfactant ratio disrupted the microstructure. Protocol A decreased pericardium tissue secant modulus (p < 0.05). Protocol B-treated pericardium tissue matched native tissue secant modulus and ultimate tensile stress. Protocol C strengthened pericardium tissue., Conclusion: The intact extracellular matrix and biomechanical properties like native tissues require optimal chemical doses and combinations., (© 2024. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Pericardial patches are currently used as reconstructive material in cardiac surgery for surgical treatment of cardiac septal defects. Autologous pericardial patches, either treated with glutaraldehyde or not, can be used as an alternative to synthetic materials or xenograft in congenital septal defects repair. The availability of an allogenic decellularized pericardium could reduce complication during and after surgery and could be a valid alternative. Decellularization of allogenic tissues aims at reducing the immunogenic reaction that might trigger inflammation and tissue calcification over time. The ideal graft for congenital heart disease repair should be biocompatible, mechanically resistant, non-immunogenic, and should have the ability to growth with the patients. The aim of the present study is the evaluation of the efficacy of a new decellularization protocol of homologous pericardium, even after cryopreservation. The technique has proven to be suitable as a tissue bank procedure and highly successful in the removal of cells and nucleic acids content, but also in the preservation of collagen and biomechanical properties of the human pericardium., (© 2023. The Author(s).)

The epicardium is integral to cardiac development and facilitates endogenous heart regeneration and repair. While miR-194-3p is associated with cellular migration and invasion, its impact on epicardial cells remains uncharted. In this work we use gain-of-function and loss-of-function methodologies to investigate the function of miR-194-3p in cardiac development. We culture embryonic epicardial cells in vitro and subject them to transforming growth factor β (TGF-β) treatment to induce epithelial-mesenchymal transition (EMT) and monitor miR-194-3p expression. In addition, the effects of miR-194-3p mimics and inhibitors on epicardial cell development and changes in EMT are investigated. To validate the binding targets of miR-194-3p and its ability to recover the target gene-phenotype, we produce a mutant vector p120-catenin -3'UTR-MUT. In epicardial cells, TGF-β-induced EMT results in a notable overexpression of miR-194-3p. The administration of miR-194-3p mimics promotes EMT, which is correlated with elevated levels of mesenchymal markers. Conversely, miR-194-3p inhibitor attenuates EMT. Further investigations reveal a negative correlation between miR-194-3p and p120-catenin, which influences β-catenin level in the cell adhesion pathway. The suppression of EMT caused by the miR-194-3p inhibitor is balanced by silencing of p120-catenin . In conclusion, miR-194-3p directly targets p120-catenin and modulates its expression, which in turn alters β-catenin expression, critically influencing the EMT process in the embryonic epicardial cells via the cell adhesion mechanism.

Macrophages conduct critical roles in heart repair, but the niche required to nurture and anchor them is poorly studied. Here, we investigated the macrophage niche in the regenerating heart. We analyzed cell-cell interactions through published single-cell RNA sequencing datasets and identified a strong interaction between fibroblast/epicardial (Fb/Epi) cells and macrophages. We further visualized the association of macrophages with Fb/Epi cells and the blockage of macrophage response without Fb/Epi cells in the regenerating zebrafish heart. Moreover, we found that ptx3a + epicardial cells associate with reparative macrophages, and their depletion resulted in fewer reparative macrophages. Further, we identified csf1a expression in ptx3a + cells and determined that pharmacological inhibition of the csf1a pathway or csf1a knockout blocked the reparative macrophage response. Moreover, we found that genetic overexpression of csf1a enhanced the reparative macrophage response with or without heart injury. Altogether, our studies illuminate a cardiac Fb/Epi niche, which mediates a beneficial macrophage response after heart injury., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system 1 . The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG + and IgA + plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs., (© 2023. The Author(s).)

Zebrafish heart regeneration is dependent on the activation of a regenerative programme in the cells surrounding the heart, known as the epicardium. A new paper in Development uses genome-wide transcriptomics and chromatin accessibility profiles to identify and validate candidate enhancers linked to genes induced during regeneration in epicardial cells. To hear more about the story, we caught up with first author Yingxi Cao and senior authors Professor Ken Poss from Duke University and Jingli Cao, Assistant Professor at Weill Cornell Medicine., (© 2022. Published by The Company of Biologists Ltd.)

The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, which are defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with five out of six elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in larval hearts. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration. This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Tissue engineering (TE) of long tracheal segments is conceptually appealing for patients with inoperable tracheal pathology. In tracheal TE, stem cells isolated from bone marrow or adipose tissue have been employed, but the ideal cell source has yet to be determined. When considering the origin of stem cells, cells isolated from a source embryonically related to the trachea may be more similar. In this study, we investigated the feasibility of isolating progenitor cells from pleura and pericard as an alternative cells source for tracheal tissue engineering. Porcine progenitor cells were isolated from pleura, pericard, trachea and adipose tissue and expanded in culture. Isolated cells were characterized by PCR, RNA sequencing, differentiation assays and cell survival assays and were compared to trachea and adipose-derived progenitor cells. Progenitor-like cells were successfully isolated and expanded from pericard and pleura as indicated by gene expression and functional analyses. Gene expression analysis and RNA sequencing showed a stem cell signature indicating multipotency, albeit that subtle differences between different cell sources were visible. Functional analysis revealed that these cells were able to differentiate towards chondrogenic, osteogenic and adipogenic lineages. Isolation of progenitor cells from pericard and pleura with stem cell features is feasible. Although functional differences with adipose-derived stem cells were limited, based on their gene expression, pericard- and pleura-derived stem cells may represent a superior autologous cell source for cell seeding in tracheal tissue engineering., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Extracellular matrix bioscaffolds can influence the cardiac microenvironment and modulate endogenous cellular mechanisms. These materials can optimize cardiac surgery for repair and reconstruction. We investigated the biocompatibility and bioinductivity of bovine pericardium fixed via dye-mediated photo-oxidation on human cardiac fibroblast activity. We compared a dye-mediated photo-oxidation fixed bioscaffold to glutaraldehyde-fixed and non-fixed bioscaffolds reported in contemporary literature in cardiac surgery. Human cardiac fibroblasts from consenting patients were seeded on to bioscaffold materials to assess the biocompatibility and bioinductivity. Human cardiac fibroblast gene expression, secretome, morphology and viability were studied. Dye-mediated photo-oxidation fixed acellular bovine pericardium preserves human cardiac fibroblast phenotype and viability; and potentiates a pro-vasculogenic paracrine response. Material tensile properties were compared with biomechanical testing. Dye-mediated photo-oxidation fixed acellular bovine pericardium had higher compliance compared to glutaraldehyde-fixed bioscaffold in response to tensile force. The biocompatibility, bioinductivity, and biomechanical properties of dye-mediated photo-oxidation fixed bovine pericardium demonstrate its feasibility as a bioscaffold for use in cardiac surgery. As a fixed yet bioinductive solution, this bioscaffold demonstrates enhanced compliance and retains bioinductive properties that may leverage endogenous reparative pathways. Dye-mediated photo-oxidation fixed bioscaffold warrants further investigation as a viable tool for cardiac repair and reconstruction.

Skeletal muscles represent 40% of body mass and its native regenerative capacity can be permanently lost after a traumatic injury, congenital diseases, or tumor ablation. The absence of physiological regeneration can hinder muscle repair preventing normal muscle tissue functions. To date, tissue engineering (TE) represents one promising option for treating muscle injuries and wasting. In particular, hydrogels derived from the decellularized extracellular matrix (dECM) are widely investigated in tissue engineering applications thanks to their essential role in guiding muscle regeneration. In this work, the myogenic potential of dECM substrate, obtained from decellularized bovine pericardium (Tissuegraft Srl), was evaluated in vitro using C2C12 murine muscle cells. To assess myotubes formation, the width, length, and fusion indexes were measured during the differentiation time course. Additionally, the ability of dECM to support myogenesis was assessed by measuring the expression of specific myogenic markers: α-smooth muscle actin (α-sma), myogenin, and myosin heavy chain (MHC). The results obtained suggest that the dECM niche was able to support and enhance the myogenic potential of C2C12 cells in comparison of those grown on a plastic standard surface. Thus, the use of extracellular matrix proteins, as biomaterial supports, could represent a promising therapeutic strategy for skeletal muscle tissue engineering.

In this study, we evaluated cardiomyogenic differentiation of electromechanically stimulated rat bone marrow-derived stem cells (rt-BMSCs) on an acellular bovine pericardium (aBP) and we looked at the functioning of this engineered patch in a rat myocardial infarct (MI) model. aBP was prepared using a detergent-based decellularization procedure followed by rt-BMSCs seeding, and electrical, mechanical, or electromechanical stimulations (3 millisecond pulses of 5 V cm -1 at 1 Hz, 5% stretching) to enhance cardiomyogenic differentiation. Furthermore, the electromechanically stimulated patch was applied to the MI region over 3 weeks. After this period, the retrieved patch and infarct region were evaluated for the presence of calcification, inflammatory reaction (CD68), patch to host tissue cell migration, and structural sarcomere protein expressions. In conjunction with any sign of calcification, a higher number of BrdU-labelled cells, and a low level of CD68 positive cells were observed in the infarct region under electromechanically stimulated conditions compared with static conditions. More importantly, MHC, SAC, Troponin T, and N-cad positive cells were observed in both infarct region, and retrieved engineered patch after 3 weeks. In a clear alignment with other results, our developed acellular patch promoted the expression of cardiomyogenic differentiation factors under electromechanical stimulation. Our engineered patch showed a successful integration with the host tissue followed by the cell migration to the infarct region., (© 2021 IOP Publishing Ltd.)

Rationale: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies., Aim: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research., Methods and Results: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor β3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs., Conclusion: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.

Previously, genetic lineage tracing based on the mesothelial marker Wt1, appeared to show that peritoneal mesothelial cells have a range of differentiative capacities and are the direct progenitors of vascular smooth muscle in the intestine. However, it was not clear whether this was a temporally limited process or continued throughout postnatal life. Here, using a conditional Wt1-based genetic lineage tracing approach, we demonstrate that the postnatal and adult peritoneum covering intestine, mesentery and body wall only maintained itself and failed to contribute to other visceral tissues. Pulse-chase experiments of up to 6 months revealed that Wt1-expressing cells remained confined to the peritoneum and failed to differentiate into cellular components of blood vessels or other tissues underlying the peritoneum. Our data confirmed that the Wt1-lineage system also labelled submesothelial cells. Ablation of Wt1 in adult mice did not result in changes to the intestinal wall architecture. In the heart, we observed that Wt1-expressing cells maintained the epicardium and contributed to coronary vessels in newborn and adult mice. Our results demonstrate that Wt1-expressing cells in the peritoneum have limited differentiation capacities, and that contribution of Wt1-expressing cells to cardiac vasculature is based on organ-specific mechanisms., (© 2021. The Author(s).)

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

Aims: Obesity, diabetes and cardiovascular disease are associated with COVID-19 risk and severity. Because epicardial adipose tissue (EAT) expresses ACE2, we wanted to identify the main factors associated with ACE2 levels and its cleavage enzyme, ADAM17, in epicardial fat., Materials and Methods: Epicardial and subcutaneous fat biopsies were obtained from 43 patients who underwent open-heart surgery. From 36 patients, biopsies were used for RNA expression analysis by real-time PCR of ACE1, ACE2 and ADAM17. From 8 patients, stromal vascular cells were submitted to adipogenesis or used for studying the treatment effects on gene expression levels. Soluble ACE2 was determined in supernatants by ELISA., Results: Epicardial fat biopsies expressed higher levels of ACE2 (1.53 [1.49-1.61] vs 1.51 [1.47-1.56] a.u., P < .05) and lower ADAM17 than subcutaneous fat (1.67 [1.65-1.70] vs 1.70 [1.66-1.74] a.u., P < .001). Both genes were increased in epicardial fat from patients with type 2 diabetes mellitus (T2DM) (1.62 [1.50-2.28] vs 1.52 [1.49-1.55] a.u., P = .05 for ACE2 and 1.68 [1.66-1.78] vs 1.66 [1.63-1.69] a.u., P < .05 for ADAM17). Logistic regression analysis determined that T2DM was the main associated factor with epicardial ACE2 levels (P < .01). The highest ACE2 levels were found on patients with diabetes and obesity. ACE1 and ACE2 levels were not upregulated by antidiabetic treatment (metformin, insulin or thiazolidinedione). Its cellular levels, which were higher in epicardial than in subcutaneous stromal cells (1.61 [1.55-1.63] vs 1 [1-1.34]), were not correlated with the soluble ACE2., Conclusion: Epicardial fat cells expressed higher levels of ACE2 in comparison with subcutaneous fat cells, which is enhanced by diabetes and obesity presence in patients with cardiovascular disease. Both might be risk factors for SARS-CoV-2 infection., (© 2020 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Adult mammalian heart is considered to be one of the least regenerative organs as it is not able to initiate endogenous regeneration in response to injury unlike in lower vertebrates and neonatal mammals. Evidence is now accumulating to suggest normal renewal and replacement of cardiomyocytes occurs even in middle-aged and old individuals. But underlying mechanisms leading to this are not yet clear. Do tissue-resident stem cells exist or somatic cells dedifferentiate leading to regeneration? Lot of attention is currently being focused on epicardium as it is involved in cardiac development, lodges multipotent progenitors and is a source of growth factors. Present study was undertaken to study the presence of stem cells in the pericardium. Intact adult mouse heart was subjected to partial enzymatic digestion to collect the pericardial cells dislodged from the surface. Pericardial cells suspension was processed to enrich the stem cells using our recently published protocol. Two populations of stem cells were successfully enriched from the pericardium of adult mouse heart along with distinct 'cardiospheres' with cytoplasmic continuity (formed by rapid proliferation and incomplete cytokinesis). These included very small embryonic-like stem cells (VSELs) and slightly bigger 'progenitors' cardiac stem cells (CSCs). Expression of pluripotent (Oct-4A, Sox-2, Nanog), primordial germ cells (Stella, Fragilis) and CSCs (Oct-4, Sca-1) specific transcripts was studied by RT-PCR. Stem cells expressed OCT-4, NANOG, SSEA-1, SCA-1 and c-KIT. c-KIT was expressed by cells of different sizes but only smaller CD45 - c-KIT + VSELs possess regenerative potential. Inadvertent loss of stem cells while processing for different experiments has led to misperceptions & controversies existing in the field of cardiac stem cells and requires urgent rectification. VSELs/CSCs have the potential to regenerate damaged cardiac tissue in the presence of paracrine support provided by the mesenchymal stromal cells.

The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This mapping provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

Genetic markers used to define discrete cell populations are seldom expressed exclusively in the population of interest and are, thus, unsuitable when evaluated individually, especially in the absence of spatial and morphological information. Here, we present fluorescence in situ hybridization for flow cytometry to allow simultaneous analysis of multiple marker genes at the single whole-cell level, exemplified by application to the embryonic epicardium. The protocol facilitates multiplexed quantification of gene and protein expression and temporal changes across specific cell populations. For complete details on the use and execution of this protocol, please refer to Lupu et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

We evaluated the content of active form of TGF-β1 in the intact and post-infarction heart and the effect of this factor on the properties of epicardial cells. During the acute stage after myocardial infarction, the production of TGF-β1 in the heart increased, which closely correlated with activation of epicardial cells (appearance of a pool of Wt1+ epicardial cells entering the epithelial-mesenchymal transition). The role of TGF-β1 as the factor of epicardial activation was confirmed by the results of in vitro experiments: addition of recombinant TGF-β1 to cultured epicardial cells led to enhanced expression of genes of epithelial-mesenchymal transition and phenotypic transformation of these cells leading to the appearance of cells with markers of smooth muscle cells and fibroblasts. Our findings suggest that the regulatory axis "TGF-β1-epicardium cells" can be considered as an important link of the post-infarction reparative process and adaptive response during heart remodeling after myocardial infarction and as the target for therapeutic interventions.

Due to its pronounced regenerative capacity, the zebrafish heart represents an advantageous model system for exploring the cellular and molecular mechanisms of cardiac regeneration. Upon injury, the epicardium, the outermost mesothelial tissue layer of vertebrate hearts, serves dual purposes in the regenerating heart as both a signaling center and a source for crucial cell types. Traditional in vivo genetic approaches to study heart regeneration can be time consuming and are not applicable to large-scale approaches and live surveillance of cellular behaviors. Here, we demonstrate ex vivo methods to culture, maintain, and study the regenerative responses of epicardial tissue in excised zebrafish hearts. Epicardial cell proliferation and migration are monitored in real time after uninjured or injured hearts are excised, washed, and cultured for up to 30 days. In addition to these techniques, we describe ex vivo genetic ablation of the epicardium, cell proliferation assays, partial ventricular explant culturing, and chemical screening.

The epicardium is a multipotent cell layer that is vital to myocardial development and regeneration. Epicardial cells contribute to cardiac fibroblast and smooth muscle populations of the heart and secrete paracrine factors that promote cardiomyocyte proliferation and angiogenesis. Despite a central role in cardiac biology, the mechanisms by which epicardial cells influence cardiac growth are largely unknown, and robust models of the epicardium are needed. Here, we review our protocol for differentiating induced pluripotent stem cells (iPSCs) into epicardial-like cells through temporal modulation of canonical Wnt signaling. iPSC-derived epicardial cells (iECs) resemble in vivo epicardial cells morphologically and display markers characteristic of the developing epicardium. We also review our protocol for differentiating iECs into fibroblasts and smooth muscle cells through treatment with bFGF and TGF-β1, respectively. iECs provide a platform for studying fundamental epicardial biology and can inform strategies for therapeutic heart regeneration.

After myocardial infarction (MI), epicardial cells reactivate their embryonic program, proliferate and migrate into the damaged tissue to differentiate into fibroblasts, endothelial cells and, if adequately stimulated, to cardiomyocytes. Targeting epicardium-derived stromal cells (EpiSC) by specific ligands might enable the direct imaging of EpiSCs after MI to better understand their biology, but also may permit the cell-specific delivery of small molecules to improve the post-MI healing process. Therefore, the aim of this study was to identify specific peptides by phage display screening to enable EpiSC specific cargo delivery by active targeting. To this end, we utilized a sequential panning of a phage library on cultured rat EpiSCs and then subtracted phage that nonspecifically bound blood immune cells. EpiSC specific phage were analyzed by deep sequencing and bioinformatics analysis to identify a total of 78 300 ± 31 900 different, EpiSC-specific, peptide insertion sequences. Flow cytometry of the five most highly abundant peptides (EP1, -2, -3, -7 or EP9) showed strong binding to EpiSCs but not to blood immune cells. The best binding properties were found for EP9 which was further studied by surface plasmon resonance (SPR). SPR revealed rapid and stable association of EpiSCs with EP9. As a negative control, THP-1 monocytes did not associate with EP9. Coupling of EP9 to perfluorocarbon nanoemulsions (PFCs) resulted in the efficient delivery of 19 F cargo to EpiSCs and enabled their visualization by 19 F MRI. Moreover, active targeting of EpiSCs by EP9-labelled PFCs was able to outcompete the strong phagocytic uptake of PFCs by circulating monocytes. In summary, we have identified a 7-mer peptide, (EP9) that binds to EpiSCs with high affinity and specificity. This peptide can be used to deliver small molecule cargos such as contrast agents to permit future in vivo tracking of EpiSCs by molecular imaging and to transfer small pharmaceutical molecules to modulate the biological activity of EpiSCs.

Brain natriuretic peptide (BNP) treatment increases heart function and decreases heart dilation after myocardial infarction (MI). Here, we investigated whether part of the cardioprotective effect of BNP in infarcted hearts related to improved neovascularisation. Infarcted mice were treated with saline or BNP for 10 days. BNP treatment increased vascularisation and the number of endothelial cells in all areas of infarcted hearts. Endothelial cell lineage tracing showed that BNP directly stimulated the proliferation of resident endothelial cells via NPR-A binding and p38 MAP kinase activation. BNP also stimulated the proliferation of WT1 + epicardium-derived cells but only in the hypoxic area of infarcted hearts. Our results demonstrated that these immature cells have a natural capacity to differentiate into endothelial cells in infarcted hearts. BNP treatment increased their proliferation but not their differentiation capacity. We identified new roles for BNP that hold potential for new therapeutic strategies to improve recovery and clinical outcome after MI., Competing Interests: NL, SR, CB, AB, TD, AC, MD, NR No competing interests declared, (© 2020, Li et al.)

Genetic lineage tracing has been widely used for studying in vivo cell fate plasticity during embryogenesis, tissue homeostasis, and disease development. Recent applications with multiple site-specific recombinases have been used in complex and sophisticated genetic fate mapping studies. However, the previous multicolor reporters for dual recombinases had limitations of precise in situ quantification of cell number, which is mainly due to the intermingling of cells in condensed tissues. Here, we generated a dual recombinase-mediated nuclear-localized GFP and tdTomato reporter line, which enables clear, simultaneous quantification of two distinct cell lineages in vivo. Combining this dual genetic reporter with Tbx18-Cre and Cdh5-Dre lines, which genetically trace epicardial and endothelial cells, respectively, we obtained high-resolution images for the anatomic distribution of the descendants of these two distinct cell lineages in the valve mesenchyme during development, remodeling, and maturation stages. This new dual genetic reporter is expected to facilitate fate tracing of two cell lineages and their objective quantification in vivo., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: This study sought to evaluate the histologic and mechanical properties of autologous in vivo tissue-engineered vascular grafts (in vivo TEVGs) used for pediatric heart surgery., Description: Molds of in vivo TEVGs made of silicone drain tubes were embedded into subcutaneous spaces in 2 boys during their first operation and were used as patch materials to treat pulmonary artery stenosis during the second operation. The remaining pieces of the patches were evaluated histologically and mechanically., Evaluation: In vivo TEVGs had very smooth luminal surfaces, and their walls mainly comprised collagen fibers and small numbers of fibroblasts. Mean wall thickness was 200 μm, mean suture retention strength was 2.26 N, and burst pressure was 3057 mm Hg., Conclusions: Human in vivo TEVGs mainly comprise collagen fibers, and their mechanical properties prove them safe for pulmonary arterioplasty. Therefore, human in vivo TEVGs may be promising alternatives to autologous pericardium for pediatric cardiovascular surgical procedures that often require multistage operations., (Copyright © 2020 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Decellularization involves removal of cellular material from tissue which results in a scaffold material consisting of only the extra cellular matrix (ECM). The effect of each individual decellularizing detergent on the final ECM scaffold and how that may differ from the combined use of these detergents is currently a gap in decellularization methodologies. This study evaluates the hypothesis that a synergistic effect exists when commonly used decellularization detergents are combined. This was evaluated with regard to decellularization efficiency, tissue strength, and collagen structure. Bovine pericardium was decellularized using a combination of 0.5% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate (SDC) and 1% TritonX-100, and compared to the use of each detergent individually. The combined detergent decellularization protocol showed effective decellularization (p = .004), with minimal effects on tissue strength (p = .21) and structure (p = .21). Use of detergents individually, resulted in detrimental effects on tissue structure and integrity or ineffective decellularization. This study shows a synergistic relationship between SDS, SDC and TritonX-100 when combined at specific concentrations. The use of detergents in combination instead of individually appears to be superior, as it results in less ECM damage and improved decellularization effectivity., (© 2020 Wiley Periodicals, Inc.)

Epicardial cells are cardiac progenitors that give rise to the majority of cardiac fibroblasts, coronary smooth muscle cells, and pericytes during development. An integral phase of epicardial fate transition is epithelial-to-mesenchymal transition (EMT) that confers motility. We uncover an essential role for the protein arginine methyltransferase 1 (PRMT1) in epicardial invasion and differentiation. Using scRNA-seq, we show that epicardial-specific deletion of Prmt1 reduced matrix and ribosomal gene expression in epicardial-derived cell lineages. PRMT1 regulates splicing of Mdm4, which is a key controller of p53 stability. Loss of PRMT1 leads to accumulation of p53 that enhances Slug degradation and blocks EMT. During heart development, the PRMT1-p53 pathway is required for epicardial invasion and formation of epicardial-derived lineages: cardiac fibroblasts, coronary smooth muscle cells, and pericytes. Consequently, this pathway modulates ventricular morphogenesis and coronary vessel formation. Altogether, our study reveals molecular mechanisms involving the PRMT1-p53 pathway and establish its roles in heart development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cardiovascular development is a complex developmental process in which multiple cell lineages are involved, namely the deployment of first and second heart fields. Beside the contribution of these cardiogenic fields, extracardiac inputs to the developing heart are provided by the migrating cardiac neural crest cells and the proepicardial derived cells. The proepicardium (PE) is a transitory cauliflower-like structure located between the cardiac and hepatic primordia. The PE is constituted by an internal mesenchymal component surrounded by an external epithelial lining. With development, cells derived from the proepicardium migrate to the neighboring embryonic heart and progressive cover the most external surface, leading to the formation of the embryonic epicardium. Experimental evidence in chicken have nicely demonstrated that epicardial derived cells can distinctly contribute to fibroblasts, endothelial and smooth muscle cells. Surprisingly, isolation of the developing PE anlage and ex vivo culturing spontaneously lead to differentiation into beating cardiomyocytes, a process that is enhanced by Bmp but halted by Fgf administration. In this study we provide a comprehensive characterization of the developmental expression profile of multiple microRNAs during epicardial development in chicken. Subsequently, we identified that miR-125, miR-146, miR-195 and miR-223 selectively enhance cardiomyogenesis both in the PE/ST explants as well as in the embryonic epicardium, a Smurf1- and Foxp1-driven process. In addition we identified three novel long non-coding RNAs with enhanced expression in the PE/ST, that are complementary regulated by Bmp and Fgf administration and well as by microRNAs that selectively promote cardiomyogenesis, supporting a pivotal role of these long non coding RNAs in microRNA-mediated cardiomyogenesis of the PE/ST cells.

The embryonic epicardium, originating from the proepicardial organ (PEO), provides a source of multipotent progenitors for cardiac lineages, including pericytes, fibroblasts, and vascular smooth muscle cells. Maximizing the regenerative capacity of the adult epicardium depends on recapitulating embryonic cell fates. The potential of the epicardium to contribute coronary endothelium is unclear, due to conflicting Cre-based lineage trace data. Controversy also surrounds when epicardial cell fate becomes restricted. Here, we systematically investigate expression of five widely used epicardial markers, Wt1, Tcf21, Tbx18, Sema3d, and Scx, over the course of development. We show overlap of markers in all PEO and epicardial cells until E13.5, and find no evidence for discrete proepicardial sub-compartments that might contribute coronary endothelium via the epicardial layer. Our findings clarify a number of prevailing discrepancies and support the notion that epicardium-derived cell fate, to form fibroblasts or mural cells, is specified after epithelial-mesenchymal transition, not pre-determined within the PEO., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The embryonic epicardium generates a population of epicardial-derived mesenchymal cells (EPDC) whose contribution to the coronary endothelium is minor or, according to some reports, negligible. We have compared four murine cell-tracing models related to the EPDC in order to elucidate this contribution. Cre recombinase was expressed under control of the promoters of the Wilms' tumor suppressor (Wt1), the cardiac troponin (cTnT), and the GATA5 genes, activating expression of the R26R EYFP reporter. We have also used the G2 enhancer of the GATA4 gene as a driver due to its activation in the proepicardium. Recombination was found in most of the epicardium/EPDC in all cases. The contribution of these lineages to the cardiac endothelium was analyzed using confocal microscopy and flow cytometry. G2-GATA4 lineage cells are the most frequent in the endothelium, probably due to the recruitment of circulating endothelial progenitors. The contribution of the WT1 cell lineage increases along gestation due to further endothelial expression of WT1. GATA5 and cTnT lineages represent 4% of the cardiac endothelial cells throughout the gestation, probably standing for the actual EPDC contribution to the coronary endothelium. These results suggest caution when using a sole cell-tracing model to study the fate of the EPDC., (© 2020 Federation of American Societies for Experimental Biology.)

Background: Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in animal models. This study sought to scale-up a 2-cm 2 preclinical construct into a human-size advanced therapy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation., Methods: The PeriCord is a clinical-size (12-16 cm 2 ) decellularised pericardial matrix colonised with human viable Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative population doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for DNA content, matrix stiffness, pore size, and absence of microbiological growth., Findings: PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the non-infarcted area. At implantation, the 16-cm 2 pericardial scaffold contained 12·5 × 10 6 viable WJ-MSCs (85·4% cell viability; <0·51 endotoxin units (EU)/mL). Intraoperative PeriCord delivery was expeditious, and secured with surgical glue. The post-operative course showed non-adverse reaction to the PeriCord, without requiring host immunosuppression. The three-month clinical follow-up was uneventful, and three-month cardiac magnetic resonance imaging showed ~9% reduction in scar mass in the treated area., Interpretation: This preliminary report describes the development of a scalable clinical-size allogeneic PeriCord cardiac bioimplant, and its first-in-human implantation., Funding: La Marató de TV3 Foundation, Government of Catalonia, Catalan Society of Cardiology, "La Caixa" Banking Foundation, Spanish Ministry of Science, Innovation and Universities, Institute of Health Carlos III, and the European Regional Development Fund., Competing Interests: Declarations of Competing Interest There are no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The epicardium is essential during cardiac development, homeostasis, and repair, and yet fundamental insights into its underlying cell biology, notably epicardium formation, lineage heterogeneity, and functional cross-talk with other cell types in the heart, are currently lacking. In this study, we investigated epicardial heterogeneity and the functional diversity of discrete epicardial subpopulations in the developing zebrafish heart. Single-cell RNA sequencing uncovered three epicardial subpopulations with specific genetic programs and distinctive spatial distribution. Perturbation of unique gene signatures uncovered specific functions associated with each subpopulation and established epicardial roles in cell adhesion, migration, and chemotaxis as a mechanism for recruitment of leukocytes into the heart. Understanding which mechanisms epicardial cells employ to establish a functional epicardium and how they communicate with other cardiovascular cell types during development will bring us closer to repairing cellular relationships that are disrupted during cardiovascular disease., Competing Interests: Declaration of Interests P.R.R. is co-founder and equity holder in OxStem Cardio, an Oxford University spinout that seeks to exploit therapeutic strategies stimulating endogenous repair in cardiovascular regenerative medicine., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The mammalian heart contains multiple cell types that appear progressively during embryonic development. Advance in determining cardiac lineage diversification has often been limited by the unreliability of genetic tracers. Here we combine clonal analysis, genetic lineage tracing, tissue transplantation, and mutant characterization to investigate the lineage relationships between epicardium, arterial mesothelial cells (AMCs), and the coronary vasculature. We report a contribution of the second heart field (SHF) to a vasculogenic niche composed of AMCs and sub-mesothelial cells at the base of the pulmonary artery. Sub-mesothelial cells from this niche differentiate into lymphatic endothelial cells and, in close association with AMC-derived cells, contribute to and are essential for the development of ventral cardiac lymphatics. In addition, regionalized epicardial/mesothelial retinoic acid signaling regulates lymphangiogenesis, contributing to the niche properties. These results uncover a SHF vasculogenic contribution to coronary lymphatic development through a local niche at the base of the great arteries., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The epicardium, the outermost tissue layer that envelops all vertebrate hearts, plays a crucial role in cardiac development and regeneration and has been implicated in potential strategies for cardiac repair. The heterogenous cell population that composes the epicardium originates primarily from a transient embryonic cell cluster known as the proepicardial organ (PE). Characterized by its high cellular plasticity, the epicardium contributes to both heart development and regeneration in two critical ways: as a source of progenitor cells and as a critical signaling hub. Despite this knowledge, there are many unanswered questions in the field of epicardial biology, the resolution of which will advance the understanding of cardiac development and repair. We review current knowledge in cross-species epicardial involvement, specifically in relation to lineage specification and differentiation during cardiac development., (Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved.)

Very Small Embryonic-Like (VSEL) stem cells are a proposed pluripotent population, residing in adult tissues. VSELs have been described in multiple tissues including bone marrow, cord blood, and gonads. They exhibit multiple characteristics of embryonic stem cells including the ability to differentiate into cellular lineages of all three germ layers, including cardiomyocytes and vascular endothelial cells. However, their presence in adult solid organs such as heart in humans has not been established. VSELs are valuable source of stem cells for tissue regeneration and replacement of cells for turnover and usual wear-and-tear. The purpose of our study was to explore the existence of human VSELs (huVSELs) in human heart tissue and examine the changes in their prevalence with aging and cardiac disease. Human heart tissue, collected from healthy and ischemic heart disease subjects was examined for the prevalence of VSELS, defined as CD45-/CD133+/SSEA4+. Both epicardial and endocardial tissues were examined comparing VSEL numbers across different age groups. Our data confirm the existence of huVSELs in adult hearts with decreasing prevalence during aging. This is the first evidence of huVSELs in adult cardiac tissue. Cardiac huVSELs could be further explored in future studies to characterize their primitive potential and therapeutic potential in regenerative studies.

The heart is lined by a single layer of mesothelial cells called the epicardium that provides important cellular contributions for embryonic heart formation. The epicardium harbors a population of progenitor cells that undergo epithelial-to-mesenchymal transition displaying characteristic conversion of planar epithelial cells into multipolar and invasive mesenchymal cells before differentiating into nonmyocyte cardiac lineages, such as vascular smooth muscle cells, pericytes, and fibroblasts. The epicardium is also a source of paracrine cues that are essential for fetal cardiac growth, coronary vessel patterning, and regenerative heart repair. Although the epicardium becomes dormant after birth, cardiac injury reactivates developmental gene programs that stimulate epithelial-to-mesenchymal transition; however, it is not clear how the epicardium contributes to disease progression or repair in the adult. In this review, we will summarize the molecular mechanisms that control epicardium-derived progenitor cell migration, and the functional contributions of the epicardium to heart formation and cardiomyopathy. Future perspectives will be presented to highlight emerging therapeutic strategies aimed at harnessing the regenerative potential of the fetal epicardium for cardiac repair.

The formation of pericardial adipose tissue (PAT) and its regulatory function in cardiac inflammation are not well understood. We investigated the potential role of the ubiquitous ATP/UTP nucleotide receptor P2Y 2 in the PAT by using P2Y 2 -null mice. We observed that P2Y 2 -null mice displayed a lower mass of PAT and a reduced density of its fat-associated lymphoid clusters (FALCs) and, more particularly, B cells. Loss of P2Y 2 receptor in pericardial preadipocytes decreased their adipogenic differentiation and maturation abilities in vitro. Gene profiling identified P2Y 2 target genes in PAT linked to immunomodulation. These data led to the identification of an increase of M2c anti-inflammatory macrophages correlated with increased apoptosis of B lymphocytes in P2Y 2 -null pericardial fat. In addition, follicular helper T cells, which contribute to B cell expansion in germinal centers, were dramatically decreased. The effect of P2Y 2 loss was also investigated after ischemia-mediated expansion of FALCs in a model of myocardial infarct. Loss of P2Y 2 led to reduced expansion of B and neutrophil populations in these clusters, whereas density of M2c anti-inflammatory macrophages was increased. Our study defines the P2Y 2 nucleotide receptor as a regulator of the formation and inflammatory status of pericardial fat. The P2Y 2 receptor could represent a therapeutic target in the regulation of PAT function before and during cardiac ischemia.

Objectives: Our goal was to present histopathological findings of human explants of a tissue-engineered bovine pericardium CardioCel (Admedus Regen Pty Ltd, Malaga, WA, Australia) used for heart valve repair in patients with congenital and acquired heart valve disease., Methods: Sixty patients underwent heart valve repair from May 2014 to November 2018 using CardioCel as a substitute for valve tissue. We identified 9 cases in which the CardioCel patch was explanted following valve repair and available for histomorphological analyses. CardioCel explants were evaluated histologically using haematoxylin and oeosin, Elastica van Gieson and immunohistochemical stains., Results: The indications for explantation were related to the CardioCel patch in 6 patients. Median time between the implantation and explantation was 242 (range 3-1247) days. We demonstrated a characteristic remodelling pattern with superficial coating of the tissue-engineered bovine pericardium by granulation tissue composed of histiocytes, few lymphocytes and fibrin. We had 2 cases with a multifocal nodular disruption, fragmentation and sclerosis of the decellularized collagen matrix with focal calcification after 795 and 1247 days in situ., Conclusions: Our data suggest that the tissue-engineered CardioCel patch is initially tolerated in the valvular position in the majority of patients. However, we also experienced graft failures that showed degeneration with fragmentation of the collagen matrix and even 2 cases with focal calcification evident from the histopathological analysis. Further analyses of mid- and long-term performance are mandatory., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

The murine developing epicardium heterogeneously expresses the transcription factors TCF21 and WT1. Here, we show that this cell heterogeneity is conserved in human epicardium, regulated by BNC1 and associated with cell fate and function. Single cell RNA sequencing of epicardium derived from human pluripotent stem cells (hPSC-epi) revealed that distinct epicardial subpopulations are defined by high levels of expression for the transcription factors BNC1 or TCF21. WT1 + cells are included in the BNC1 + population, which was confirmed in human foetal hearts. THY1 emerged as a membrane marker of the TCF21 population. We show that THY1 + cells can differentiate into cardiac fibroblasts (CFs) and smooth muscle cells (SMCs), whereas THY1 - cells were predominantly restricted to SMCs. Knocking down BNC1 during the establishment of the epicardial populations resulted in a homogeneous, predominantly TCF21 high population. Network inference methods using transcriptomic data from the different cell lineages derived from the hPSC-epi delivered a core transcriptional network organised around WT1, TCF21 and BNC1. This study unveils a list of epicardial regulators and is a step towards engineering subpopulations of epicardial cells with selective biological activities., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: The composition and function of the adipose tissue covering the heart are poorly known. In this study, we have investigated the epicardial adipose tissue (EAT) covering the cardiac ventricular muscle and the EAT covering the left anterior descending artery (LAD) on the human heart, to identify their resident stem cell functional activity., Methods: EAT covering the cardiac ventricular muscle was isolated from the apex (avoiding areas irrigated by major vessels) of the heart (ventricular myocardium adipose tissue (VMAT)) and from the area covering the epicardial arterial sulcus of the LAD (PVAT) in human hearts excised during heart transplant surgery. Adipose stem cells (ASCs) from both adipose tissue depots were immediately isolated and phenotypically characterized by flow cytometry. The different behavior of these ASCs and their released secretome microvesicles (MVs) were investigated by molecular and cellular analysis., Results: ASCs from both VMAT (mASCs) and the PVAT (pASCs) were characterized by the expression of CD105, CD44, CD29, CD90, and CD73. The angiogenic-related genes VEGFA, COL18A1, and TF, as well as the miRNA126-3p and miRNA145-5p, were analyzed in both ASC types. Both ASCs were functionally able to form tube-like structures in three-dimensional basement membrane substrates. Interestingly, pASCs showed a higher level of expression of VEGFA and reduced level of COL18A1 than mASCs. Furthermore, MVs released by mASCs significantly induced human microvascular endothelial cell migration., Conclusion: Our study indicates for the first time that the resident ASCs in human epicardial adipose tissue display a depot-specific angiogenic function. Additionally, we have demonstrated that resident stem cells are able to regulate microvascular endothelial cell function by the release of MVs.

Despite the importance of understanding how variability across induced pluripotent stem cell (iPSC) lines due to non-genetic factors (clone and passage) influences their differentiation outcome, large-scale studies capable of addressing this question have not yet been conducted. Here, we differentiated 191 iPSC lines to generate iPSC-derived cardiovascular progenitor cells (iPSC-CVPCs). We observed cellular heterogeneity across the iPSC-CVPC samples due to varying fractions of two cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Comparing the transcriptomes of CM-fated and EPDC-fated iPSCs, we discovered that 91 signature genes and X chromosome dosage differences are associated with these two distinct cardiac developmental trajectories. In an independent set of 39 iPSCs differentiated into CMs, we confirmed that sex and transcriptional differences affect cardiac-fate outcome. Our study provides novel insights into how iPSC transcriptional and X chromosome gene dosage differences influence their response to differentiation stimuli and, hence, cardiac cell fate., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute myocardial infarction (MI) provokes an inflammatory response in the heart that removes damaged tissues to facilitate tissue repair/regeneration. However, overactive and prolonged inflammation compromises healing, which may be counteracted by antiinflammatory mechanisms. A key regulatory factor in an inflammatory response is the antiinflammatory cytokine IL-10, which can be produced by a number of immune cells, including subsets of B lymphocytes. Here, we investigated IL-10-producing B cells in pericardial adipose tissues (PATs) and their role in the healing process following acute MI in mice. We found that IL-10-producing B cells were enriched in PATs compared to other adipose depots throughout the body, with the majority of them bearing a surface phenotype consistent with CD5 + B-1a cells (CD5 + B cells). These cells were detected early in life, maintained a steady presence during adulthood, and resided in fat-associated lymphoid clusters. The cytokine IL-33 and the chemokine CXCL13 were preferentially expressed in PATs and contributed to the enrichment of IL-10-producing CD5 + B cells. Following acute MI, the pool of CD5 + B cells was expanded in PATs. These cells accumulated in the infarcted heart during the resolution of MI-induced inflammation. B cell-specific deletion of IL-10 worsened cardiac function, exacerbated myocardial injury, and delayed resolution of inflammation following acute MI. These results revealed enrichment of IL-10-producing B cells in PATs and a significant contribution of these cells to the antiinflammatory processes that terminate MI-induced inflammation. Together, these findings have identified IL-10-producing B cells as therapeutic targets to improve the outcome of MI., Competing Interests: The authors declare no competing interest.

The epicardium plays a crucial role in embryonic heart development and adult heart repair; however, the molecular events underlying its maturation remain unknown. Wt1 , one of the main markers of the embryonic epicardium, is essential for epicardial development and function. Here, we analyse the transcriptomic profile of epicardial-enriched cells at different stages of development and from control and epicardial-specific Wt1 knockout ( Wt1KO ) mice. Transcriptomic and cell morphology analyses of epicardial cells from epicardial-specific Wt1KO mice revealed a defect in the maturation process of the mutant epicardium, including sustained upregulation of Bmp4 expression and the inability of mutant epicardial cells to transition into a mature squamous phenotype. We identified Bmp4 as a transcriptional target of Wt1, thus providing a molecular basis for the retention of the cuboidal cell shape observed in the Wt1KO epicardium. Accordingly, inhibition of the Bmp4 signalling pathway both ex vivo and in vivo rescued the cuboidal phenotype of the mutant epicardium. Our findings indicate the importance of the cuboidal-to-squamous transition in epicardial maturation, a process regulated by Wt1., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)