Cardiac Regenerative Therapy Research Articles

Extracellular Vesicles as a Cell-free Therapy for Cardiac Repair: a Systematic Review and Meta-analysis of Randomized Controlled Preclinical Trials in Animal Myocardial Infarction Models.

Stem cell therapy for cardiac regeneration has been gaining traction as a possible intervention for the reduction of the burden associated with MI and heart failure. However, stem cell therapies have several shortcomings, including poor engraftment, limited improvements in cardiac function, and possible teratogenicity. Recently, extracellular vesicles (EVs) from stem cell sources have been explored as a novel therapy to regenerate the injured myocardium in several animal MI trials. In this systematic review and meta-analysis, we investigate the use of stem cell-derived EVs for cardiac repair preclinical trials in animal MI models. Cochrane Library, Medline, Embase, PubMed, Scopus and Web of Science and grey literature (Canadian Agency for Drugs, Technologies in Health, and Google Scholar) were searched through August 20, 2020 and 37 articles were included in the final analysis. The overall effect size observed in EV-treated small animals after MI for ejection fraction (EF) was 10.85 [95%CI: 8.79, 12.90] and for fractional shortening (FS) was 7.19 [95%CI: 5.43, 8.96] compared to control-treated animals. The most abundant stem cell source used were mesenchymal stem cells which showed robust improvements in EF and FS (MD = 11.89 [95 % CI: 9.44, 14.34] and MD = 6.96 [95 % CI: 4.97, 8.96], respectively). Significant publication bias was detected for EF and FS outcomes. This study supports the use of EVs derived from stem cells as a novel therapy for cardiac repair after MI. Further investigation in larger animal studies may be necessary before clinical trials.PROSPERO registration number: CRD42019142218.

Cardiac stem cells: Current knowledge and future prospects.

Regenerative medicine is the field concerned with the repair and restoration of the integrity of damaged human tissues as well as whole organs. Since the inception of the field several decades ago, regenerative medicine therapies, namely stem cells, have received significant attention in preclinical studies and clinical trials. Apart from their known potential for differentiation into the various body cells, stem cells enhance the organ's intrinsic regenerative capacity by altering its environment, whether by exogenous injection or introducing their products that modulate endogenous stem cell function and fate for the sake of regeneration. Recently, research in cardiology has highlighted the evidence for the existence of cardiac stem and progenitor cells (CSCs/CPCs). The global burden of cardiovascular diseases’ morbidity and mortality has demanded an in-depth understanding of the biology of CSCs/CPCs aiming at improving the outcome for an innovative therapeutic strategy. This review will discuss the nature of each of the CSCs/CPCs, their environment, their interplay with other cells, and their metabolism. In addition, important issues are tackled concerning the potency of CSCs/CPCs in relation to their secretome for mediating the ability to influence other cells. Moreover, the review will throw the light on the clinical trials and the preclinical studies using CSCs/CPCs and combined therapy for cardiac regeneration. Finally, the novel role of nanotechnology in cardiac regeneration will be explored.

Resuscitating the Field of Cardiac Regeneration: Seeking Answers from Basic Biology.

Heart failure (HF) is one of the leading causes for hospital admissions worldwide. HF patients are classified based on the chronic changes in left ventricular ejection fraction (LVEF) as preserved (LVEF ≥ 50%), reduced (LVEF ≤ 40%), or mid-ranged (40% < LVEF < 50%) HFs. Treatments nowadays can prevent HFrEF progress, whereas only a few of the treatments have been proven to be effective in improving the survival of HFpEF. In this review, numerous mediators involved in the pathogenesis of HF are summarized. The regional upstream signaling and their diagnostic and therapeutic potential are also discussed. Additionally, the recent challenges and development in cardiac regenerative therapy that hold opportunities for future research and clinical translation are discussed.

The Present State and Future Perspectives of Cardiac Regenerative Therapy Using Human Pluripotent Stem Cells.

The number of patients with heart failure (HF) is increasing with aging in our society worldwide. Patients with HF who are resistant to medication and device therapy are candidates for heart transplantation (HT). However, the shortage of donor hearts is a serious issue. As an alternative to HT, cardiac regenerative therapy using human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, is expected to be realized. Differentiation of hPSCs into cardiomyocytes (CMs) is facilitated by mimicking normal heart development. To prevent tumorigenesis after transplantation, it is important to eliminate non-CMs, including residual hPSCs, and select only CMs. Among many CM selection systems, metabolic selection based on the differences in metabolism between CMs and non-CMs is favorable in terms of cost and efficacy. Large-scale culture systems have been developed because a large number of hPSC-derived CMs (hPSC-CMs) are required for transplantation in clinical settings. In large animal models, hPSC-CMs transplanted into the myocardium improved cardiac function in a myocardial infarction model. Although post-transplantation arrhythmia and immune rejection remain problems, their mechanisms and solutions are under investigation. In this manner, the problems of cardiac regenerative therapy are being solved individually. Thus, cardiac regenerative therapy with hPSC-CMs is expected to become a safe and effective treatment for HF in the near future. In this review, we describe previous studies related to hPSC-CMs and discuss the future perspectives of cardiac regenerative therapy using hPSC-CMs.

Abstract 10754: Disrupted N-Cadherin Expression Leads to Sarcomeric Disassembly and Cell Cycle Activation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: Induction of cardiomyocyte proliferation holds great promise for cardiac regenerative therapy. We showed previously that in the presence of Wnt signaling agonist, the removal of cell-cell contact between cardiomyocytes is the key to drive massive expansion of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the molecular mechanism regulating cell contact-mediated suppression of hiPSC-CM proliferation remains unknown. Hypothesis: We hypothesized that N-cadherin, the major adherens junction molecule expressed in cardiomyocytes, mediates contact inhibition of hiPSC-CM proliferation. Methods/Results: By qPCR and Western blotting, we showed comparable transcript and protein expression level of N-cadherin when hiPSC-CMs were cultured either densely (with cell contact) or sparsely (without cell contact), despite the observed N-cadherin localization at intercellular junction in densely cultured cells only. We disrupted N-cadherin expression with transient transfection of siRNA and found that the loss of N-cadherin expression resulted in 1.75-fold increase in Ki67 expression in densely but not sparsely cultured day 30 hiPSC-CM, suggesting that junction-localized N-cadherin may affect hiPSC-CM proliferation. Furthermore, we found that the loss of N-cadherin expression led to wide-spread sarcomeric disassembly. To examine whether sarcomeric disassembly alone is sufficient to stimulate cell cycle activity in densely cultured hiPSC-CMs, we knocked down α-actinin expression which led to sarcomere disruption and an increase in Ki67+ expression by 1.6-fold in day 30 hiPSC-CMs. Conclusions: Taken together, our results suggest that N-cadherin mediates the previously observed cell-cell contact inhibition of hiPSC-CM proliferation and the disruption of N-cadherin expression in densely cultured hiPSC-CMs leads to sarcomeric disassembly and hiPSC-CM proliferation. Furthermore, we showed that sarcomeric disassembly alone stimulates hiPSC-CM proliferation. These findings support the need for inducing cardiomyocyte dedifferentiation to achieve in situ cardiomyocyte proliferation for developing novel regenerative therapy to treat congenital and adult heart diseases in the future.

Transplantation of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Cardiac Regenerative Therapy.

Cardiovascular disease is the leading cause of death worldwide and bears an immense economic burden. Late-stage heart failure often requires total heart transplantation; however, due to donor shortages and lifelong immunosuppression, alternative cardiac regenerative therapies are in high demand. Human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells, have emerged as a viable source of human cardiomyocytes for transplantation. Recent developments in several mammalian models of cardiac injury have provided strong evidence of the therapeutic potential of hPSC-derived cardiomyocytes (hPSC-CM), showing their ability to electromechanically integrate with host cardiac tissue and promote functional recovery. In this review, we will discuss recent developments in hPSC-CM differentiation and transplantation strategies for delivery to the heart. We will highlight the mechanisms through which hPSC-CMs contribute to heart repair, review major challenges in successful transplantation of hPSC-CMs, and present solutions that are being explored to address these limitations. We end with a discussion of the clinical use of hPSC-CMs, including hurdles to clinical translation, current clinical trials, and future perspectives on hPSC-CM transplantation.

Maturation of Stem Cell-Derived Cardiomyocytes: Foe in Translation Medicine.

With the in-depth study of heart development, many human cardiomyocytes (CMs) have been generated in a laboratory environment. CMs derived from pluripotent stem cells (PSCs) have been widely used for a series of applications such as laboratory studies, drug toxicology screening, cardiac disease models, and as an unlimited resource for cell-based cardiac regeneration therapy. However, the low maturity of the induced CMs significantly impedes their applicability. Scientists have been committed to improving the maturation of CMs to achieve the purpose of heart regeneration in the past decades. In this review, we take CMs maturation as the main object of discussion, describe the characteristics of CMs maturation, summarize the key regulatory mechanism of regulating maturation and address the approaches to promote CMs maturation. The maturation of CM is gradually improving due to the incorporation of advanced technologies and is expected to continue.

Global RNA editing identification and characterization during human pluripotent-to-cardiomyocyte differentiation

SummaryRNA editing is widely involved in stem cell differentiation and development; however, RNA editing events during human cardiomyocyte differentiation have not yet been characterized and elucidated. Here, we identified genome-wide RNA editing sites and systemically characterized their genomic distribution during four stages of human cardiomyocyte differentiation. It was found that the expression level of ADAR1 affected the global number of adenosine to inosine (A-to-I) editing sites but not the editing degree. Next, we identified 43, 163, 544, and 141 RNA editing sites that contribute to changes in amino acid sequences, variation in alternative splicing, alterations in miRNA-target binding, and changes in gene expression, respectively. Generally, RNA editing showed a stage-specific pattern with 211 stage-shared editing sites. Interestingly, cardiac muscle contraction and heart-disease-related pathways were enriched by cardio-specific editing genes, emphasizing the connection between cardiomyocyte differentiation and heart diseases from the perspective of RNA editing. Finally, it was found that these RNA editing sites are also related to several congenital and noncongenital heart diseases. Together, our study provides a new perspective on cardiomyocyte differentiation and offers more opportunities to understand the mechanisms underlying cell fate determination, which can promote the development of cardiac regenerative medicine and therapies for human heart diseases.

Current status, challenges and perspectives of mesenchymal stem cell-based therapy for cardiac regeneration

Modern methods of treating heart failure are similar to the palliative care, since they mostly relieve the symptoms of the disease. The discovery of resident cardiac stem cells gave impetus to the development of “second generation” cell therapy, which quickly moved from animal research to clinical trials with critically ill patients. Many cardiac side population cells have been identified to have stem cells characteristics and some additional individual characteristics, both in vitro and in vivo. The results of clinical studies demonstrated that the stem cell treatment is safe, however, this type of cell-based therapy did not restore cardiac function. Its effects were limited to mildly improving left ventricular systolic pressure and reducing the scar area. Despite that, the promising nature of these therapeutic approaches for heart diseases have contributed to the development of next-generation cell therapy.

State-of-play for cellular therapies in cardiac repair and regeneration.

Cardiovascular disease is the primary cause of death around the world. For almost two decades, cell therapy has been proposed as a solution for heart disease. In this article, we report on the “state‐of‐play” of cellular therapies for cardiac repair and regeneration. We outline the progression of new ideas from the preclinical literature to ongoing clinical trials. Recent data supporting the mechanics and mechanisms of myogenic and paracrine therapies are evaluated in the context of long‐term cardiac engraftment. This discussion informs on promising new approaches to indicate future avenues for the field.

Recent Advances in Gene Therapy for Cardiac Tissue Regeneration.

Cardiovascular diseases (CVDs) are responsible for enormous socio-economic impact and the highest mortality globally. The standard of care for CVDs, which includes medications and surgical interventions, in most cases, can delay but not prevent the progression of disease. Gene therapy has been considered as a potential therapy to improve the outcomes of CVDs as it targets the molecular mechanisms implicated in heart failure. Cardiac reprogramming, therapeutic angiogenesis using growth factors, antioxidant, and anti-apoptotic therapies are the modalities of cardiac gene therapy that have led to promising results in preclinical studies. Despite the benefits observed in animal studies, the attempts to translate them to humans have been inconsistent so far. Low concentration of the gene product at the target site, incomplete understanding of the molecular pathways of the disease, selected gene delivery method, difference between animal models and humans among others are probable causes of the inconsistent results in clinics. In this review, we discuss the most recent applications of the aforementioned gene therapy strategies to improve cardiac tissue regeneration in preclinical and clinical studies as well as the challenges associated with them. In addition, we consider ongoing gene therapy clinical trials focused on cardiac regeneration in CVDs.

Direct in vivo reprogramming with non-viral sequential targeting nanoparticles promotes cardiac regeneration

microRNA-mediated direct cardiac reprogramming, directly converts fibroblasts into induced cardiomyocyte-like cells (iCMs), which holds great promise in cardiac regeneration therapy. However, effective approaches to deliver therapeutic microRNA into cardiac fibroblasts (CFs) to induce in vivo cardiac reprogramming remain to be explored. Herein, a non-viral biomimetic system to directly reprogram CFs for cardiac regeneration after myocardial injury was developed by coating FH peptide-modified neutrophil-mimicking membranes on mesoporous silicon nanoparticles (MSNs) loaded with microRNA1, 133, 208, and 499 (miR Combo). Through utilizing the natural inflammation-homing ability of neutrophil membrane protein and FH peptide's high affinity to tenascin-C (TN-C) produced by CFs, this nanoparticle could realize sequential targeting to CFs in the injured heart and precise intracellular delivery of miRCombo, which induced reprogramming resident CFs into iCMs. In a mouse model of myocardial ischemia/reperfusion injury, intravenous injection of the nanoparticles successfully delivered miRCombo into fibroblasts and led to efficient reprogramming, resulting in improved cardiac function and attenuated fibrosis. This delivery system is minimally invasive and bio-safe, providing a proof-of-concept for biomimetic and sequential targeting nanomedicine delivery system for microRNA-mediated reprogramming therapy in multiple diseases.

Applications of Omics Technologies in Regenerative Therapies for Cardiac Repair

Lately, cardio regenerative therapeutic approaches in clinic have received strong boost from promising research advances in stem cells. Researchers have shown applications of the first generation of clinical trials using cell-based therapies in the heart that have been performed with bone marrow and adipose tissue derived mesenchymal stem cells shortcomings in the first generation cells. Second generation cell therapies are considered superior and being considered towards the use of cardiac-committed cell populations. These include cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes. However, translating the research laboratory results along with pre-clinical data into effective clinical treatments is still thought to be challenging. This is due to the existing lack of knowledge on the regenerative mechanisms of action of these therapeutic products in addition to the stringent regulatory and safety concerns. This has prompted researchers to consider advanced analytical methods for characterization of such complex products and a deep understanding of their therapeutic effects at the cell and molecular level. This characterization is considered very critical to overcome the challenges and make these cells compatible with the advanced therapies. To this end, omics technologies including proteomics and glyco(proteo)mics that are based on state-of-the-art mass-spectrometry have shown tremendous promise to generate novel data on cell biology along with the required assessment of cell based-products that are applied in cardiac regeneration strategies. In this perspective, we have described proteomics and glyco(proteo)mics and discuss the impact of omics technologies on cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes biology cardiac regenerative therapies.

Mesenchymal stem cells derived from epicardial adipose tissue reverse cardiac remodeling in a rabbit model of myocardial infarction.

Myocardial infarction (MI) is one of the most important causes of death. MI-related tissue loss and cardiac remodeling may result in heart failure. Intramyocardial injection of mesenchymal stem cells derived from adipose tissues, in acute MI animal models, has shown promising regenerative capabilities. This study aimed to investigate the myocardial regenerative capacity of epicardial adipose tissue-derived mesenchymal stem cells (ADSCs) in a rabbit model of MI. A rabbit model of MI was performed in three groups: a sham-operated group, a control group, and a treatment group. MI was induced by coronary artery ligation via thoracotomy in the first operation. Four weeks after the first operation, intramyocardial injections of phosphate-buffered saline (PBS; control group) or ADSCs (10×106 in 100 μL; treatment group) were performed in the peri-infarct zone. Four weeks after the second operation, rabbits were sacrificed for further analysis. A significant increase in ejection fraction (p<0.0001) was detected in the treatment group, along with a significant increase in vascular density (p<0.001) and a significant decrease in infarct size (p<0.05) compared to the control group. Epicardial adipose tissue is a rich source of mesenchymal stem cells, which can differentiate into cardiomyocytes, as well as having neoangiogenic properties. Due to its potential to ameliorate chronic ischemic changes in the heart, it may be preferable in cardiac regenerative cell therapies.

Highly Efficient MicroRNA Delivery Using Functionalized Carbon Dots for Enhanced Conversion of Fibroblasts to Cardiomyocytes

IntroductionThe reprogramming of induced cardiomyocytes (iCMs) is of particular significance in regenerative medicine; however, it remains a great challenge to fabricate an efficient and safe gene delivery system to induce reprogramming of iCMs for therapeutic applications in heart injury. Here, we report branched polyethyleneimine (BP) coated nitrogen-enriched carbon dots (BP-NCDs) as highly efficient nanocarriers loaded with microRNAs-combo (BP-NCDs/MC) for cardiac reprogramming.MethodsThe BP-NCDs nanocarriers were prepared and characterized by several analytical techniques.ResultsThe BP-NCDs nanocarriers showed good microRNAs-combo binding affinity, negligible cytotoxicity, and long-term microRNAs expression. Importantly, BP-NCDs/MC nanocomplexes led to the efficient direct reprogramming of fibroblasts into iCMs without genomic integration and resulting in effective recovery of cardiac function after myocardial infarction (MI).ConclusionThis study offers a novel strategy to provide safe and effective microRNAs-delivery nanoplatforms based on carbon dots for promising cardiac regeneration and disease therapy.

Mesenchymal Stem Cells for Cardiac Regeneration: from Differentiation to Cell Delivery.

Mesenchymal stem cells (MSCs) are so far the most widely researched stem cells in clinics and used as an experimental cellular therapy module, particularly in cardiac regeneration and repair. Ever since the discovery of cardiomyogenesis induction in MSCs, a wide variety of differentiation protocols have been extensively used in preclinical models. However, pre differentiated MSC-derived cardiomyocytes have not been used in clinical trials; highlighting discrepancies and limitations in its use as a source of derived cardiomyocytes for transplantation to improve the damaged heart function. Therefore, this review article focuses on the strategies used to derive cardiomyocytes-like cells from MSCs isolated from three widely used tissue sources and their differentiation efficiencies. We have further discussed the role of MSCs in inducing angiogenesis as a cellular precursor to endothelial cells and its secretory aspects including exosomes. We have then discussed the strategies used for delivering cells in the damaged heart and how its retention plays a critical role in the overall outcome of the therapy. We have also conversed about the scope of the local and systemic modes of delivery of MSCs and the application of biomaterials to improve the overall delivery efficacy and function. We have finally discussed the advantages and limitations of cell delivery to the heart and the future scope of MSCs in cardiac regenerative therapy.

Characterization of encapsulated porcine cardiosphere-derived cells embedded in 3D alginate matrices

Myocardial infarction is caused by an interruption of coronary blood flow, leading to one of the main death causes worldwide. Current therapeutic approaches are palliative and not able to solve the loss of cardiac tissue. Cardiosphere derived cells (CDCs) reduce scarring, and increase viable myocardium, with safety and adequate biodistribution, but show a low rate engraftment and survival after implantation. In order to solve the low retention, we propose the encapsulation of CDCs within three-dimensional alginate-poly-L-lysine-alginate matrix as therapy for cardiac regeneration. In this work, we demonstrate the encapsulation of CDCs in alginate matrix, with no decrease in viability over a month, and showing the preservation of CDCs phenotype, differentiation potential, gene expression profile and growth factor release after encapsulation, moving a step forward to clinical translation of CDCs therapy in regeneration in heart failure.

Intramyocardial Transplantation of Human iPS Cell–Derived Cardiac Spheroids Improves Cardiac Function in Heart Failure Animals

The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study.

Application of Cell, Tissue, and Biomaterial Delivery in Cardiac Regenerative Therapy.

Cardiovascular diseases (CVD) are the leading cause of death around the world, being responsible for 31.8% of all deaths in 2017 (Roth, G. A. et al. The Lancet 2018, 392, 1736-1788). The leading cause of CVD is ischemic heart disease (IHD), which caused 8.1 million deaths in 2013 (Benjamin, E. J. et al. Circulation 2017, 135, e146-e603). IHD occurs when coronary arteries in the heart are narrowed or blocked, preventing the flow of oxygen and blood into the cardiac muscle, which could provoke acute myocardial infarction (AMI) and ultimately lead to heart failure and death. Cardiac regenerative therapy aims to repair and refunctionalize damaged heart tissue through the application of (1) intramyocardial cell delivery, (2) epicardial cardiac patch, and (3) acellular biomaterials. In this review, we aim to examine these current approaches and challenges in the cardiac regenerative therapy field.

Proarrhythmic Electrical Remodeling by Noncardiomyocytes at Interfaces With Cardiomyocytes Under Oxidative Stress.

Life-threatening ventricular arrhythmias, typically arising from interfaces between fibrosis and surviving cardiomyocytes, are feared sequelae of structurally remodeled hearts under oxidative stress. Incomplete understanding of the proarrhythmic electrical remodeling by fibrosis limits the development of novel antiarrhythmic strategies. To define the mechanistic determinants of the proarrhythmia in electrical crosstalk between cardiomyocytes and noncardiomyocytes, we developed a novel in vitro model of interface between neonatal rat ventricular cardiomyocytes (NRVMs) and controls [NRVMs or connexin43 (Cx43)-deficient HeLa cells] vs. Cx43+ noncardiomyocytes [aged rat ventricular myofibroblasts (ARVFs) or HeLaCx43 cells]. We performed high-speed voltage-sensitive optical imaging at baseline and following acute H2O2 exposure. In NRVM-NRVM and NRVM-HeLa controls, no arrhythmias occurred under either experimental condition. In the NRVM-ARVF and NRVM-HeLaCx43 groups, Cx43+ noncardiomyocytes enabled passive decremental propagation of electrical impulses and impaired NRVM activation and repolarization, thereby slowing conduction and prolonging action potential duration. Following H2O2 exposure, arrhythmia triggers, automaticity, and non-reentrant and reentrant arrhythmias emerged. This study reveals that myofibroblasts (which generate cardiac fibrosis) and other noncardiomyocytes can induce not only structural remodeling but also electrical remodeling and that electrical remodeling by noncardiomyocytes can be particularly arrhythmogenic in the presence of an oxidative burst. Synergistic electrical remodeling between H2O2 and noncardiomyocytes may account for the clinical arrhythmogenicity of myofibroblasts at fibrotic interfaces with cardiomyocytes in ischemic/non-ischemic cardiomyopathies. Understanding the enhanced arrhythmogenicity of synergistic electrical remodeling by H2O2 and noncardiomyocytes may guide novel safe-by-design antiarrhythmic strategies for next-generation iatrogenic interfaces between surviving native cardiomyocytes and exogenous stem cells or engineered tissues in cardiac regenerative therapies.