Direct differentiation of rat skin fibroblasts into cardiomyocytes.

Myocardial infarction (MI) is one of the major cardiovascular diseases caused by diminished supply of nutrients and oxygen to the heart due to obstruction of the coronary artery. Different treatment options are available for cardiac diseases, however, they do not completely repair the damage. Therefore, reprogramming terminally differentiated fibroblasts using transcription factors is a promising strategy to differentiate them into cardiac like cells in vitro and to increase functional cardiomyocytes and reduce fibrotic scar in vivo. In this study, skin fibroblasts were selected for reprogramming because they serve as a convenient source for the autologous cell therapy. Fibroblasts were isolated from skin of rat pups, propagated, and directly reprogrammed towards cardiac lineage. For reprogramming, two different approaches were adopted, i.e., cells were transfected with: (1) combination of cardiac transcription factors; GATA4, MEF2c, Nkx2.5 (GMN), and (2) combination of cardiac transcription factors; GATA4, MEF2c, Nkx2.5, and iPSC factors; Oct4, Klf4, Sox2 and cMyc (GMNO). After 72 h of transfection, cells were analyzed for the expression of cardiac markers at the mRNA and protein levels. For in vivo study, rat MI models were developed by ligating the left anterior descending coronary artery and the reprogrammed cells were transplanted in the infarcted heart. qPCR results showed that the reprogrammed cells exhibited significant upregulation of cardiac genes. Immunocytochemistry analysis further confirmed cardiomyogenic differentiation of the reprogrammed cells. For the assessment of cardiac function, animals were analyzed via echocardiography after 2 and 4 weeks of cell transplantation. Echocardiographic results showed that the hearts transplanted with the reprogrammed cells improved ejection fraction, fractional shortening, left ventricular internal systolic and diastolic dimensions, and end systolic and diastolic volumes. After 4 weeks of cell transplantation, heart tissues were harvested and processed for histology. The histological analysis showed that the reprogrammed cells improved wall thickness of left ventricle and reduced fibrosis significantly as compared to the control. It is concluded from the study that novel combination of cardiac transcription factors directly reprogrammed skin fibroblasts and differentiated them into cardiomyocytes. These differentiated cells showed cardiomyogenic characters in vitro, and reduced fibrosis and improved cardiac function in vivo. Furthermore, direct reprogramming of fibroblasts transfected with cardiac transcription factors showed better regeneration of the injured myocardium and improved cardiac function as compared to the indirect approach in which combination of cardiac and iPSC factors were used. The study after further optimization could be used as a better strategy for cell-based therapeutic approaches for cardiovascular diseases.

Lin28a cardiomyocyte-specific modified mRNA translation system induces cardiomyocyte cell division and cardiac repair.

The mammalian heart has a limited regenerative capacity. Previous work suggested the heart can regenerate during development and immediately after birth by inducing cardiomyocyte (CM) proliferation; however, this capacity is lost seven days after birth. modRNA gene delivery, the same technology used successfully in the two mRNA vaccines against SARS-CoV-2, can prompt cardiac regeneration, cardiovascular regeneration and cardiac protection. We recently established a novel CM-specific modRNA translational system (SMRTs) that allows modRNA translation only in CMs. We demonstrated that this system delivers potent intracellular genes (e.g., cell cyclepromoting Pkm2), which are beneficial when expressed in one cell type (i.e., CMs) but not others (non-CMs). Here, we identify Lin28a as an important regulator of the CM cell cycle. We show that Lin28a is expressed in CMs during development and immediately after birth, but not during adulthood. We describe that specific delivery of Lin28a into CM, using CM SMRTs, enables CM cell division and proliferation. Further, we determine that this proliferation leads to cardiac repair and better outcome post MI. Moreover, we identify the molecular pathway of Lin28a in CMs. We also demonstrate that Lin28a suppress Let-7 which is vital for CM proliferation, partially due to its suppressive role on cMYC, HMGA2 and K-RAS.

Transient formation of collaterals contributes to the restoration of the arterial tree during cardiac regeneration in neonatal mice.

Revascularization of ischemic myocardium following cardiac damage is an important step in cardiac regeneration. However, the mechanism of arteriogenesis has not been well described during cardiac regeneration. Here we investigated coronary artery remodeling and collateral growth during cardiac regeneration. Neonatal MI was induced by ligature of the left descending artery (LAD) in postnatal day (P) 1 or P7 pups from the Cx40-GFP mouse line and the arterial tree was reconstructed in 3D from images of cleared hearts collected at 1, 2, 4, 7 and 14 days after infarction. We show a rapid remodeling of the left coronary arterial tree induced by neonatal MI and the formation of numerous collateral arteries, which are transient in regenerating hearts after MI at P1 and persistent in non-regenerating hearts after MI at P7. This difference is accompanied by restoration of a perfused or a non-perfused LAD following MI at P1 or P7 respectively. Interestingly, collaterals ameliorate cardiac perfusion and drive LAD repair, and lineage tracing analysis demonstrates that the restoration of the LAD occurs by remodeling of pre-existing arterial cells independently of whether they originate in large arteries or arterioles. These results demonstrate that the restoration of the LAD artery during cardiac regeneration occurs by pruning as the rapidly forming collaterals that support perfusion of the disconnected lower LAD subsequently disappear on restoration of a unique LAD. These results highlight a rapid phase of arterial remodeling that plays an important role in vascular repair during cardiac regeneration.

Direct reprogramming as a route to cardiac repair.

Ischemic heart disease is the leading cause of morbidity, mortality, and healthcare expenditure worldwide due to an inability of the heart to regenerate following injury. Thus, novel heart failure therapies aimed at promoting cardiomyocyte regeneration are desperately needed. In recent years, direct reprogramming of resident cardiac fibroblasts to induced cardiac-like myocytes (iCMs) has emerged as a promising therapeutic strategy to repurpose the fibrotic response of the injured heart toward a functional myocardium. Direct cardiac reprogramming was initially achieved through the overexpression of the transcription factors (TFs) Gata4, Mef2c, and Tbx5 (GMT). However, this combination of TFs and other subsequent cocktails demonstrated limited success in reprogramming adult human and mouse fibroblasts, constraining the clinical translation of this therapy. Over the past decade, significant effort has been dedicated to optimizing reprogramming cocktails comprised of cardiac TFs, epigenetic factors, microRNAs, or small molecules to yield efficient cardiac cell fate conversion. Yet, efficient reprogramming of adult human fibroblasts remains a significant challenge. Underlying mechanisms identified to accelerate this process have been centered on epigenetic remodeling at cardiac gene regulatory regions. Further studies to achieve a refined understanding and directed means of overcoming epigenetic barriers are merited to more rapidly translate these promising therapies to the clinic.

To Repair a Broken Heart: Stem Cells in Ischemic Heart Disease.

Despite improvements in contemporary medical and surgical therapies, cardiovascular disease (CVD) remains a significant cause of worldwide morbidity and mortality; more specifically, ischemic heart disease (IHD) may affect individuals as young as 20 years old. Typically managed with guideline-directed medical therapy, interventional or surgical methods, the incurred cardiomyocyte loss is not always completely reversible; however, recent research into various stem cell (SC) populations has highlighted their potential for the treatment and perhaps regeneration of injured cardiac tissue, either directly through cellular replacement or indirectly through local paracrine effects. Different stem cell (SC) types have been employed in studies of infarcted myocardium, both in animal models of myocardial infarction (MI) as well as in clinical studies of MI patients, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), Muse cells, multipotent stem cells such as bone marrow-derived cells, mesenchymal stem cells (MSCs) and cardiac stem and progenitor cells (CSC/CPCs). These have been delivered as is, in the form of cell therapies, or have been used to generate tissue-engineered (TE) constructs with variable results. In this text, we sought to perform a narrative review of experimental and clinical studies employing various stem cells (SC) for the treatment of infarcted myocardium within the last two decades, with an emphasis on therapies administered through thoracic incision or through percutaneous coronary interventions (PCI), to elucidate possible mechanisms of action and therapeutic effects of such cell therapies when employed in a surgical or interventional manner.

Toward the latest advancements in cardiac regeneration using induced pluripotent stem cells (iPSCs) technology: approaches and challenges.

A novel approach to treating heart failures was developed with the introduction of iPSC technology. Knowledge in regenerative medicine, developmental biology, and the identification of illnesses at the cellular level has exploded since the discovery of iPSCs. One of the most frequent causes of mortality associated with cardiovascular disease is the loss of cardiomyocytes (CMs), followed by heart failure. A possible treatment for heart failure involves restoring cardiac function and replacing damaged tissue with healthy, regenerated CMs. Significant strides in stem cell biology during the last ten years have transformed the in vitro study of human illness and enhanced our knowledge of the molecular pathways underlying human disease, regenerative medicine, and drug development. We seek to examine iPSC advancements in disease modeling, drug discovery, iPSC-Based cell treatments, and purification methods in this article.

Programmed spontaneously beating cardiomyocytes in regenerative cardiology.

Stem cells have gained attention as a promising therapeutic approach for damaged myocardium, and there have been efforts to develop a protocol for regenerating cardiomyocytes (CMs). Certain cells have showed a greater aptitude for yielding beating CMs, such as induced pluripotent stem cells, embryonic stem cells, adipose-derived stromal vascular fraction cells and extended pluripotent stem cells. The approach for generating CMs from stem cells differs across studies, although there is evidence that Wnt signaling, chemical additives, electrical stimulation, co-culture, biomaterials and transcription factors triggers CM differentiation. Upregulation of Gata4, Mef2c and Tbx5 transcription factors has been correlated with successfully induced CMs, although Mef2c may potentially play a more prominent role in the generation of the beating phenotype, specifically. Regenerative research provides a possible candidate for cardiac repair; however, it is important to identify factors that influence their differentiation. Altogether, the spontaneously beating CMs would be monumental for regenerative research for cardiac repair.

Latest progress of self-healing hydrogels in cardiac tissue engineering.

Cardiovascular diseases represent a significant public health challenge and are responsible for more than 4 million deaths annually in Europe alone (45% of all deaths). Among these, coronary-related heart diseases are a leading cause of mortality, accounting for 20% of all deaths. Cardiac tissue engineering has emerged as a promising strategy to address the limitations encountered after myocardial infarction. This approach aims to improve regulation of the inflammatory and cell proliferation phases, thereby reducing scar tissue formation and restoring cardiac function. In cardiac tissue engineering, biomaterials serve as hosts for cells and therapeutics, supporting cardiac restoration by mimicking the native cardiac environment. Various bioengineered systems, such as 3D scaffolds, injectable hydrogels, and patches play crucial roles in cardiac tissue repair. In this context, self-healing hydrogels are particularly suitable substitutes, as they can restore structural integrity when damaged. This structural healing represents a paradigm shift in therapeutic interventions, offering a more native-like environment compared to static, non-healable hydrogels. Herein, we sharply review the most recent advances in self-healing hydrogels in cardiac tissue engineering and their potential to transform cardiovascular healthcare.

The potential mechanisms and treatment effects of stem cell-derived exosomes in cardiac reengineering.

Exosomes are extracellular vesicles of diverse compositions that are secreted by numerous cell types. Exosomes contain significant bioactive components, including lipids, proteins, mRNA, and miRNA. Exosomes play an important role in regulating cellular signaling and trafficking under both normal physiological and pathological circumstances. A multitude of factors, including thermal stress, ribosomal stress, endoplasmic reticulum stress, and oxidative stress influence the concentrations of exosomal mRNA, miRNA, proteins, and lipids. It has been stated that exosomes derived from stem cells (SCs) modulate a range of stresses by preventing or fostering cell balance. Exosomes derived from SCs facilitate recovery by facilitating cross-cellular communication via the transmission of information in the form of proteins, lipids, and other components. For this reason, exosomes are used as biomarkers to diagnose a wide variety of diseases. The focus of this review is the bioengineering of artificial exosomal cargoes. This process encompasses the control and transportation of particular exosomal cargoes, including but not limited to small molecules, recombinant proteins, immune modulators, and therapeutic medications. Therapeutic approaches of this nature have the potential to deliver therapeutic medications precisely to the intended site for the cure of a variety of disorders. Notably, our attention has been directed towards the therapeutic implementations of exosomes derived from SCs in the cure of cardiovascular ailments, including but not limited to ischemic heart disease, myocardial infarction, sepsis, heart failure, cardiomyopathy, and cardiac fibrosis. In general, researchers employ two methodologies when it comes to exosomal bioengineering. This review aims to explain the function of exosomes derived from SCs in the regulation of stress and present a novel therapeutic approach for cardiovascular disorders.

Mechanisms regulating vascular and lymphatic regeneration in the heart after myocardial infarction.

Myocardial infarction, caused by a thrombus or coronary vascular occlusion, leads to irreversible ischaemic injury. Advances in early reperfusion strategies have significantly reduced short-term mortality after myocardial infarction. However, survivors have an increased risk of developing heart failure, which confers a high risk of death at 1 year. The capacity of the injured neonatal mammalian heart to regenerate has stimulated extensive research into whether recapitulation of developmental regeneration programmes may be beneficial in adult cardiovascular disease. Restoration of functional blood and lymphatic vascular networks in the infarct and border regions via neovascularisation and lymphangiogenesis, respectively, is a key requirement to facilitate myocardial regeneration. An improved understanding of the endogenous mechanisms regulating coronary vascular and lymphatic expansion and function in development and in adult patients after myocardial infarction may inform future therapeutic strategies and improve translation from pre-clinical studies. In this review, we explore the underpinning research and key findings in the field of cardiovascular regeneration, with a focus on neovascularisation and lymphangiogenesis, and discuss the outcomes of therapeutic strategies employed to date. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Recent Advances in Pathology: the 2023 Annual Review Issue of The Journal of Pathology.

The 2023 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 12 invited reviews on topics of current interest in pathology. This year, our subjects include immuno-oncology and computational pathology approaches for diagnostic and research applications in human disease. Reviews on the tissue microenvironment include the effects of apoptotic cell-derived exosomes, how understanding the tumour microenvironment predicts prognosis, and the growing appreciation of the diverse functions of fibroblast subtypes in health and disease. We also include up-to-date reviews of modern aspects of the molecular basis of malignancies, and our final review covers new knowledge of vascular and lymphatic regeneration in cardiac disease. All of the reviews contained in this issue are written by expert groups of authors selected to discuss the recent progress in their particular fields and all articles are freely available online (https://pathsocjournals.onlinelibrary.wiley.com/journal/10969896). © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stem cell therapy for cardiac regeneration: past, present, and future.

Cardiac disorders remain the leading cause of mortality worldwide. Current clinical strategies, including drug therapy, surgical interventions, and organ transplantation offer limited benefits to patients without regenerating the damaged myocardium. Over the past decade, stem cell therapy has generated a keen interest owing to its unique self-renewal and immune privileged characteristics. Furthermore, the ability of stem cells to differentiate into specialized cell types, has made them a popular therapeutic tool against various diseases. This comprehensive review provides an overview of therapeutic potential of different types of stem cells in reference to cardiovascular diseases. Furthermore, it sheds light on the advantages and limitations associated with each cell type. An in-depth analysis of the challenges associated with stem cell research and the hurdles for its clinical translation and their possible solutions have also been elaborated upon. It examines the controversies surrounding embryonic stem cells and the emergence of alternative approaches, such as the use of induced pluripotent stem cells for cardiac therapeutic applications. Overall, this review serves as a valuable resource for researchers, clinicians, and policymakers involved in the field of regenerative medicine, guiding the development of safe and effective stem cell-based therapies to revolutionize patient care.

Cardiac Metabolism.

The heart is composed of a heterogeneous mixture of cellular components perfectly intermingled and able to integrate common environmental signals to ensure proper cardiac function and performance. Metabolism defines a cell context-dependent signature that plays a critical role in survival, proliferation, or differentiation, being a recognized master piece of organ biology, modulating homeostasis, disease progression, and adaptation to tissue damage. The heart is a highly demanding organ, and adult cardiomyocytes require large amount of energy to fulfill adequate contractility. However, functioning under oxidative mitochondrial metabolism is accompanied with a concomitant elevation of harmful reactive oxygen species that indeed contributes to the progression of several cardiovascular pathologies and hampers the regenerative capacity of the mammalian heart. Cardiac metabolism is dynamic along embryonic development and substantially changes as cardiomyocytes mature and differentiate within the first days after birth. During early stages of cardiogenesis, anaerobic glycolysis is the main energetic program, while a progressive switch toward oxidative phosphorylation is a hallmark of myocardium differentiation. In response to cardiac injury, different signaling pathways participate in a metabolic rewiring to reactivate embryonic bioenergetic programs or the utilization of alternative substrates, reflecting the flexibility of heart metabolism and its central role in organ adaptation to external factors. Despite the well-established metabolic pattern of fetal, neonatal, and adult cardiomyocytes, our knowledge about the bioenergetics of other cardiac populations like endothelial cells, cardiac fibroblasts, or immune cells is limited. Considering the close intercellular communication and the influence of nonautonomous cues during heart development and after cardiac damage, it will be fundamental to better understand the metabolic programs in different cardiac cells in order to develop novel interventional opportunities based on metabolic rewiring to prevent heart failure and improve the limited regenerative capacity of the mammalian heart.

Neural Crest.

This chapter discusses the role of cardiac neural crest cells in the formation of the septum that divides the cardiac arterial pole into separate systemic and pulmonary arteries. Further, cardiac neural crest cells directly support the normal development and patterning of derivatives of the caudal pharyngeal arches, including the great arteries, thymus, thyroid, and parathyroids. Recently, cardiac neural crest cells have also been shown to indirectly influence the development of the secondary heart field, another derivative of the caudal pharynx, by modulating signaling in the pharynx. The contribution and function of the cardiac neural crest cells has been learned in avian models; most of the genes associated with cardiac neural crest function have been identified using mouse models. Together these studies show that the neural crest cells may not only critical for normal cardiovascular development but also may be involved secondarily because they represent a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Cardiac neural crest cells span from the caudal pharynx into the outflow tract, and therefore may be susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations resulting from genetic and/or environmental insults necessarily requires better understanding the role of cardiac neural crest cells in cardiac development.

Musings on intrinsic cardiomyocyte cell cycle activity and myocardial regeneration.

Although the myocardial renewal rate in the adult mammalian heart is quite low, recent studies have identified genetic variants which can impact the degree of cardiomyocyte cell cycle reentry. Here we use the compound interest law to model the level of regenerative growth over time in mice exhibiting different rates of cardiomyocyte cell cycle reentry following myocardial injury. The modeling suggests that the limited ability of S-phase adult cardiomyocytes to progress through cytokinesis, rather than the ability to reenter the cell cycle per se, is a major contributor to the low levels of intrinsic regenerative growth in the adult myocardium.

The characteristics of proliferative cardiomyocytes in mammals.

Better understanding of the mechanisms regulating the proliferation of pre-existing cardiomyocyte (CM) should lead to better options for regenerating injured myocardium. The absence of a perfect research model to definitively identify newly formed mammalian CMs is lacking. However, methodologies are being developed to identify and enrich proliferative CMs. These methods take advantages of the different proliferative states of CMs during postnatal development, before and after injury in the neonatal heart. New approaches use CMs labeled in lineage tracing animals or single cell technique-based CM clusters. This review aims to provide a timely update on the characteristics of the proliferative CMs, including their structural, functional, genetic, epigenetic and metabolic characteristics versus non-proliferative CMs. A better understanding of the characteristics of proliferative CMs should lead to the mechanisms for inducing endogenous CMs to self-renew, which is a promising therapeutic strategy to treat cardiac diseases that cause CM death in humans.

Reprogramming of cardiac cell fate as a therapeutic strategy for ischemic heart disease.

Direct reprogramming of resident cardiac fibroblasts to induced cardiomyocytes is an attractive therapeutic strategy to restore function and remuscularize the injured heart. The cardiac transcription factors Gata4, Mef2c, and Tbx5 have been the mainstay of direct cardiac reprogramming strategies for the past decade. Yet, recent discoveries have identified alternative epigenetic factors capable of reprogramming human cells in the absence of these canonical factors. Further, single-cell genomics evaluating cellular maturation and epigenetics in the setting of injury and heart failure models following reprogramming have continued to inform the mechanistic underpinnings of this process and point toward future areas of discovery for the field. These discoveries and others covered in this review have provided complementary approaches that further enhance the effectiveness of reprogramming as a means of promoting cardiac regeneration following myocardial infarction and heart failure.

Cardiac regeneration - Past advancements, current challenges, and future directions.

Cardiovascular disease is the leading cause of mortality and morbidity worldwide. Despite improvements in the standard of care for patients with heart diseases, including innovation in pharmacotherapy and surgical interventions, none have yet been proven effective to prevent the progression to heart failure. Cardiac transplantation is the last resort for patients with severe heart failure, but donor shortages remain a roadblock. Cardiac regenerative strategies include cell-based therapeutics, gene therapy, direct reprogramming of non-cardiac cells, acellular biologics, and tissue engineering methods to restore damaged hearts. Significant advancements have been made over the past several decades within each of these fields. This review focuses on the advancements of: 1) cell-based cardiac regenerative therapies, 2) the use of noncoding RNA to induce endogenous cell proliferation, and 3) application of bioengineering methods to promote retention and integration of engrafted cells. Different cell sources have been investigated, including adult stem cells derived from bone marrow and adipose cells, cardiosphere-derived cells, skeletal myoblasts, and pluripotent stem cells. In addition to cell-based transplantation approaches, there have been accumulating interest over the past decade in inducing endogenous CM proliferation for heart regeneration, particularly with the use of noncoding RNAs such as miRNAs and lncRNAs. Bioengineering applications have focused on combining cell-transplantation approaches with fabrication of a porous, vascularized scaffold using biomaterials and advanced bio-fabrication techniques that may offer enhanced retention of transplanted cells, with the hope that these cells would better engraft with host tissue to improve cardiac function. This review summarizes the present status and future challenges of cardiac regenerative therapies.

Cardiac injection of USSC boosts remuscularization of the infarcted heart by shaping the T-cell response.

Regenerating the injured heart remains one of the most vexing challenges in cardiovascular medicine. Cell therapy has shown potential for treatment of myocardial infarction, but low cell retention so far has limited its success. Here we show that intramyocardial injection of highly apoptosis-resistant unrestricted somatic stem cells (USSC) into infarcted rat hearts resulted in an unprecedented thickening of the left ventricular wall with cTnT+/BrdU+ cardiomyocytes that was paralleled by progressively restored ejection fraction. USSC induced significant T-cell enrichment in ischemic tissue with enhanced expression of T-cell related cytokines. Inhibition of T-cell activation by anti-CD28 monoclonal antibody, fully abolished the regenerative response which was restored by adoptive T-cell transfer. Secretome analysis of USSC and lineage tracing studies suggest that USSC secrete paracrine factors over an extended period of time which boosts a T-cell driven endogenous regenerative response mainly from adult cardiomyocytes.

Non-coding RNAs in cardiac regeneration: Mechanism of action and therapeutic potential.

In the past two decades, thousands of non-coding RNAs (ncRNAs) have been discovered, annotated, and characterized in nearly every tissue under both physiological and pathological conditions. Here, we will focus on the role of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) in ischemic heart disease (IHD), which remains the leading cause of morbidity and mortality in humans-resulting in 8.9 million deaths annually. Cardiomyocyte (CM) proliferation, differentiation, and survival in addition to neovascularization of injured tissues and the prevention of fibrosis are commonly regarded as critically important for the recovery of the heart following myocardial infarction (MI). An abundance of evidence has been accumulated to show ncRNAs participate in cardiac recovery after MI. Because miRNAs are important regulators of cardiac regeneration, the therapeutic potential of at least five of these molecules has been assessed in large animal models of human IHD. In particular, miRNA-based interventions based on miR-132 and miR-92a inhibition in related diseases have displayed favorable outcomes that have provided the impetus for miRNA-based clinical trials for IHD. At the same time, the functional roles of lncRNAs and circRNAs in cardiac regeneration are also being explored. In the present review, we will summarize the latest ncRNA studies aimed at reversing damage to the ischemic heart and discuss the therapeutic potential of targeting miRNAs, lncRNAs, and circRNAs to stimulate cardiac regeneration.