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The generation of 3D tissue constructs with multiple cell types and matching mechanical properties remains a challenge in cardiac tissue engineering. Recently, 3D bioprinting has become a powerful tool to achieve these goals. Decellularized extracellular matrix (dECM) is a common scaffold material due to providing a native biochemical environment. Unfortunately, dECM’s low mechanical stability prevents usage for bioprinting applications alone. In this study, we developed bioinks composed of decellularized human heart ECM (dhECM) with either gelatin methacryloyl (GelMA) or GelMA-methacrylated hyaluronic acid (MeHA) hydrogels dual crosslinked with UV light and microbial transglutaminase (mTGase). We characterized the bioinks’ mechanical, rheological, swelling, printability, and biocompatibility properties. Composite GelMA–MeHA–dhECM (GME) hydrogels demonstrated improved mechanical properties by an order of magnitude compared to the GelMA–dhECM (GE) hydrogels. All hydrogels were extrudable and compatible with human induced pluripotent stem cell derived cardiomyocytes (iCMs) and human cardiac fibroblasts (hCFs). Tissue-like beating of the printed constructs with striated sarcomeric alpha-actinin and connexin 43 expression was observed. The order of magnitude difference between the elastic modulus of these hydrogel composites offers applications in in vitro modeling of the myocardial infarct boundary. Here, as a proof of concept, we created an infarct boundary region with control over the mechanical properties along with the cellular and macromolecular content through printing iCMs with GE bioink and hCFs with GME bioink.

Glial cells are essential for functionality of the nervous system. Growing evidence underscores the importance of astrocytes; however, analogous astroglia in peripheral organs are poorly understood. Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, we identify a neural crest–derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm. Nexus glia are labeled with gfap , glast , and glutamine synthetase , markers that typically denote astroglia cells. Further, analysis of single-cell sequencing datasets of human and murine hearts across ages reveals astrocyte-like cells, which we confirm through a multispecies approach. We show that cardiac nexus glia at the outflow tract are critical regulators of both the sympathetic and parasympathetic system. These data establish the crucial role of glia on cardiac homeostasis and provide a description of nexus glia in the PNS.

Glial cells are essential for functionality of the nervous system. Growing evidence underscores the importance of astrocytes; however, analogous astroglia in peripheral organs are poorly understood. Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, we identify a neural crest–derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm. Nexus glia are labeled with gfap, glast, and glutamine synthetase, markers that typically denote astroglia cells. Further, analysis of single-cell sequencing datasets of human and murine hearts across ages reveals astrocyte-like cells, which we confirm through a multispecies approach. We show that cardiac nexus glia at the outflow tract are critical regulators of both the sympathetic and parasympathetic system. These data establish the crucial role of glia on cardiac homeostasis and provide a description of nexus glia in the PNS. Do astrocyte-like cells exist in the peripheral nervous system? This study reveals that astrocyte-like cells termed cardiac nexus glia populate the heart, and that these cells are important for cardiac homeostasis, modulating heart rate and rhythm during development.

The extracellular matrix (ECM) is recognized as a key regulator of cell behavior, with its stiffness playing a crucial role in the progression of pathological conditions such as cancer and cardiovascular diseases. While extracellular vesicles (EVs) are essential mediators of intercellular communication, the influence of matrix stiffness on EV secretion remains poorly understood. This study investigates how substrate stiffness affects EV size and composition in mouse mammary and cardiac fibroblasts, the key stromal cell types in breast cancer and cardiac microenvironments. Importantly, we uncovered stiffness-tuned EV proteomic cargo, providing new insights into how mechanical cues can reprogram the signaling functions of fibroblast-derived vesicles. Our findings show that substrate stiffness significantly alters EV characteristics, with sizes increasing below stiffnesses of 20 kPa and decreasing on stiffer substrates. Mechanotransduction pathways involving p53 and thioredoxin were identified as regulators of these alterations, with thioredoxin dominating the modulation in mammary fibroblasts and p53 in cardiac fibroblasts. These results underscore the importance of ECM stiffness in modulating EV secretion and highlight candidate pathways influenced by ECM remodeling that may warrant further investigation for therapeutic relevance.

Myocardial infarction (MI) is the most prevalent among cardiovascular diseases (CVD), and its occurrence is highly associated with age. The elderly (>65-years-old) form the most vulnerable population for MI and post-MI healing is much more difficult in these patients. Unfortunately, there is a big gap in knowledge in cardiac aging, partially because of the lack of adequate models to study it. Young animal and cell models are still considered the gold standard and the age mismatch significantly limits the predictive abilities of the model systems. Relatedly, current regenerative and post-MI recovery therapies fall short to addressing the elderly. ECM and cell therapies are two common approaches to treating MI, and studies reported an inverse relationship between patient age and treatment success for both. There is a missing link in translating in vitro studies into the clinic and the age component appears to be the key factor. There is a need to develop age-appropriate cardiac models to study CVD and develop therapies without excluding the elderly, who will comprise most of the population soon. Towards this goal, this dissertation aims to bridge the knowledge gap by integrating insights from three key chapters. Chapter 2 highlights the individual and combined effects of cellular and cardiac extracellular matrix (ECM) aging and reveals the cell-ECM interplay using iPSC-derived cardiomyocytes (iCMs). The study demonstrates that young ECM promotes proliferation and drug responsiveness in young cells, inducing cell cycle re-entry and stress protection in aged cells. Adult ECM improves cardiac function, while aged ECM accelerates aging phenotypes and impairs cardiac function and stress defense machinery.Chapter 3 emphasizes the need for physiologically relevant models of aging hearts to better understand cardiac aging and to study ECM therapies. Transcriptomic and proteomic changes with human cardiac aging are investigated, and chronologically aged iCMs are shown to recapitulate age-related disease hallmarks. Using these cells, the effects of cell age on young ECM therapy are explored, revealing its potential for post-MI recovery, yet acknowledging the potential harm under normal conditions with no stress. The study demonstrates that the “one-size-fits-all” approach should not be followed in young ECM therapies, especially for the advanced aged groups. Chapter 4 explores the challenge of maturing iPSC-derived cardiomyocytes for effective cell-based therapies. The study investigates the use of adult human ECM to enhance iPSC cardiac differentiation and maturation. Functional maturation supported by mitochondrial network structure and metabolic maturation was verified in these ECM pretreated cells. Furthermore, critical glycoproteins and proteoglycans in adult ECM were identified that could potentially cause enhanced cardiac differentiation and maturation, as ECM denaturation didn’t nullify the observed benefits of the ECM pretreatment. Combining these chapters, this dissertation strives to provide a comprehensive understanding of cardiac aging and cell-ECM interactions, the impact of young ECM therapies on iPSC-derived cardiomyocytes, and the importance of age-appropriate models for studying and treating cardiovascular diseases, particularly in the context of myocardial infarction (MI). Additionally, a novel approach of using adult ECM in generating not just structurally, but functionally and metabolically mature cardiac cells was presented in this dissertation.Through this work, I aimed to contribute to the field of aging, providing age-dependent changes observed in mouse and human hearts as well as in lab grown cells. I specifically focused on the age-dependent cell-ECM interactions that are crucial in the development of more effective and targeted therapies for CVDs. Expanding on both ECM and cell therapies, I highlighted knowledge gaps and challenged the commonly accepted approaches, as needed. The information provided here will be valuable in furthering translational CVD studies and enhancing CVD therapy efficiencies, especially for the elderly.

The generation of 3D tissue constructs with multiple cell types and matching mechanical properties remains a challenge in cardiac tissue engineering. Recently, 3D bioprinting has become a powerful tool to achieve these goals. Decellularized extracellular matrix (dECM) is a common scaffold material due to providing a native biochemical environment. Unfortunately, low mechanical stability of dECM prevents usage for bioprinting applications alone. In this study, we developed bioinks composed of decellularized human heart ECM (dhECM) with either gelatin methacryloyl (GelMA) or GelMA- methacrylated hyaluronic acid (MeHA) hydrogels dual crosslinked with UV light and microbial Transglutaminase (mTGase). We characterized mechanical, rheological, swelling, printability and biocompatibility properties of the bioinks. Composite GelMA-MeHA-dhECM (GME) hydrogels demonstrated improved mechanical properties by an order of magnitude, compared to GelMA-dhECM (GE) hydrogels. All hydrogels were extrudable and compatible with human induced pluripotent stem cells derived cardiomyocytes (iCMs) and human cardiac fibroblasts (hCFs). Tissue-like beating of the printed constructs with striated sarcomeric alpha-actinin and Connexin 43 expression was observed. The order of magnitude difference between the elastic modulus of these hydrogel composites offers applications in in vitro modelling of the myocardial infarct boundary. Here, as a proof of concept, we created an infarct boundary region with control over mechanical properties along with cellular and macromolecular content through printing iCMs with GE bioink and hCFs with GME bioink. Competing Interest Statement The authors have declared no competing interest.

Abstract Cardiovascular diseases are the leading cause of death worldwide and their occurrence is highly associated with age. However, lack of knowledge in cardiac tissue aging is a major roadblock in devising novel therapies. Here, we studied the effects of cell and cardiac extracellular matrix (ECM) aging on the induced pluripotent stem cell (iPSC)-derived cardiomyocyte cell state, function, as well as response to myocardial infarction (MI)-mimicking stress conditions in vitro. Within 3-weeks, young ECM promoted proliferation and drug responsiveness in young cells, and induced cell cycle re-entry, and protection against stress in the aged cells. Adult ECM improved cardiac function, while aged ECM accelerated the aging phenotype, and impaired cardiac function and stress defense machinery of the cells. In summary, we have gained a comprehensive understanding of cardiac aging and highlighted the importance of cell-ECM interactions. This study is the first to investigate the individual effects of cellular and environmental aging and identify the biochemical changes that occur upon cardiac aging. Competing Interest Statement The authors have declared no competing interest.

Aging is the main risk factor for cardiovascular disease (CVD). As the world’s population ages rapidly and CVD rates rise, there is a growing need for physiologically relevant models of aging hearts to better understand cardiac aging. Translational research relies heavily on young animal models, however, these models correspond to early ages in human life, therefore cannot fully capture the pathophysiology of age-related CVD. Here, we chronologically aged human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and compared in vitro iCM aging to native human cardiac tissue aging. We showed that 14-month-old advanced aged iCMs had an aging profile similar to the aged human heart and recapitulated age-related disease hallmarks. We then used aged iCMs to study the effect of cell age on the young extracellular matrix (ECM) therapy, an emerging approach for myocardial infarction (MI) treatment and prevention. Young ECM decreased oxidative stress, improved survival, and post-MI beating in aged iCMs. In the absence of stress, young ECM improved beating and reversed aging-associated expressions in 3-month-old iCMs while causing the opposite effect on 14-month-old iCMs. The same young ECM treatment surprisingly increased SASP and impaired beating in advanced aged iCMs. Overall, we showed that young ECM therapy had a positive effect on post-MI recovery, however, cell age was determinant in the treatment outcomes without any stress conditions. Therefore, “one-size-fits-all” approaches to ECM treatments fail, and cardiac tissue engineered models with age-matched human iCMs are valuable in translational basic research for determining the appropriate treatment, particularly for the elderly.

Myocardial infarction can lead to the loss of billions of cardiomyocytes, and while cell-based therapies are a promising option, the immature nature of in vitro-generated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) is a significant roadblock to their development. Through the years, various approaches have emerged to improve iCM maturation, yet none could fully recapitulate the complexity of cardiac development and were not enough to achieve full cardiac maturity in vitro. Cardiac differentiation occurs at the early stages of development in a highly dynamic environment. Although significantly improved over the past two decades, small molecule-based iPSC differentiation protocols don’t go beyond producing high purity fetal iCMs. Recently adult extracellular matrix (ECM) was shown to retain tissue memory and has shown some success in driving tissue-specific differentiation in unspecified cells in various organ systems. Therefore, here, we first characterized the adult human heart left ventricle components. We then investigated the effect of adult human heart-derived ECM on iPSC cardiac differentiation and subsequent maturation. By preconditioning iPSCs with ECM, we tested whether creating a cardiac environment around iPSCs would drive them toward cardiac fate before small molecule mediated differentiation. Ultimately, we investigated ECM components that might be responsible for the observed effects. We identified critical glycoproteins and proteoglycans involved in early cardiac development in the adult heart ECM. Namely, adult ECM had extracellular galactin-1, fibronectin, fibrillins, and basement-membrane-specific heparan-sulfate proteoglycan (HSPG), which have been implicated in normal heart development and associated with various embryonic developmental processes. Relatedly, we showed that preconditioning iPSCs with adult ECM resulted in enhanced cardiac differentiation, yielding iCMs with higher functional maturity. Further investigation revealed that a more developed mitochondrial network and coverage as well as enhanced metabolic maturity and a shift towards a more energetic profile allowed the observed functional enhancement in ECM pretreated iCMs. These findings demonstrate the potential of using cardiac ECM for promoting iCM maturation and suggest a promising strategy for improving the development of iCM-based therapies and in vitro cardiac disease modeling and drug screening studies. Upon manipulating ECM, such as heat denaturation and sonication to eliminate protein components and release ECM bound vesicle contents, respectively, we concluded that the beneficial effects that we observed are not solely due to the ECM proteins, and might be related to the decorative units attached to them.