Ischemic heart disease is one of the leading causes of heart failure and death worldwide. The loss of cardiomyocytes following a myocardial infarction drives the remodeling process, which, in most cases, ultimately leads to heart failure. Since the available treatment options only slow down the remodeling process without tackling the causes of heart failure onset (i.e., cardiomyocyte loss and inability of the remaining cardiomyocytes to enter the cell cycle and regenerate the heart), in the last two decades, cardiovascular research focused on finding alternative solutions to regenerate the heart. So far, the investigated approaches include a variety of methods aiming at manipulation of non-coding RNAs, such as long non-coding RNA (lncRNA), circular RNA (circRNA), and microRNA (miRNA), and growth factors to enable the cardiomyocytes to re-enter the cell cycle, direct reprogramming of fibroblasts into cardiomyocytes (CM), and CM replacement therapy, all of them with the main goal to replace the loss of cardiomyocytes and restore the heart function. The development of reprogramming protocols from somatic cells to induced pluripotent stem cells (iPSCs) by Yamanaka and Takahashi, along with advancements in differentiation protocols to generate almost pure populations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), has fostered optimism in cardiac regenerative medicine. Despite these advancements, critical concerns arose regarding the survival and retention of the engrafted cells, arrhythmogenicity, and immune response. Over time, much effort has been put into enhancing iPSC-CM therapy with different methods, ranging from anti-apoptotic small molecule-based approaches to tissue engineering. In this review, we discuss the evolution of cardiac cell therapy, highlighting recent advancements and the remaining challenges that must be overcome to translate this promising approach into clinical practice.

Since its first use in 1922, insulin therapy has transformed diabetes from a fatal disease to a manageable condition. However, long-term insulin injections lead to significant complications. β-cell replacement, derived from either a limited number of deceased donors or embryonic stem cells, offers an encouraging alternative. While these procedures allow patients to be insulin-independent, they still require systemic immunosuppressants to prevent graft rejection, which poses immunological challenges. Direct reprogramming holds considerable promise as a method for generating β-cells from various sources, enabling autologous therapies that mitigate the risk of immune rejection and eliminate the need to harvest cells from embryos. This review provides an overview of the latest advances in direct reprogramming strategies, with a focus on key transcriptional regulators that drive phenotypic conversion and maintenance of various cell types into β-like cells.

Cell plasticity enables the dynamic changes in cell identities necessary for normal development and tissue repair. Induced cell reprogramming, which leverages this plasticity, holds great promise for regenerative medicine and personalized therapies. However, the success of cell reprogramming is often impeded by various molecular barriers, such as epigenetic marks, cell senescence, and the activation of alternative or refractory routes. In this review, we examine the cell reprogramming events that occur within or between germ layers and adult stem cell lineages and propose that the overall similarity in the pre-existing chromatin accessibility landscape is a major determinant of reprogramming efficiency from one cell type to another. A better understanding of the regulation and control of chromatin accessibility should facilitate the development of new methods and strategies to improve cell reprogramming efficiency and advance translational research.

Recent advances in delivery systems for transcription factors (TFs) have opened new therapeutic opportunities in regenerative medicine, cancer therapy, and genetic disorders. However, effective TF delivery still faces substantial obstacles, including limited cellular uptake, inefficient nuclear translocation, low cargo stability, and insufficient target specificity. Furthermore, artificial TFs have enabled targeted modulation of gene expression, further expanding their therapeutic potential. This review comprehensively discusses current progress in TF delivery methodologies, including direct TF protein delivery using cell-penetrating peptides, and extracellular vesicles, as well as TF gene delivery approaches utilizing both lipid-based nanoparticles and viral strategies. Notably, engineered nanoparticles have emerged as promising platforms due to their precise control over TF delivery, improved specificity, and minimized off-target effects. Despite these significant advancements, major hurdles in delivery efficiency, cargo stability, and overall safety persist. Overcoming these obstacles will be essential to accelerate the clinical translation of TF-based therapeutics for a broad spectrum of diseases.

Cardiomyocytes derived from pluripotent stem cells (PSCs) hold great promise in heart damage repair in vivo and drug screening in vitro. However, PSC-derived cardiomyocytes exhibit immature structural and functional properties, which hinder their widespread application. To address this challenge, we designed bimetallic gold-platinum nanoparticles (Au@Pt NPs) endowed with intrinsic oxidase-like, peroxidase-like, and catalase-like activities and high electrical conductivity for promoting cardiomyocyte maturation.

Mouse embryonic stem cell (ESC)-derived and neonatal mouse cardiomyocytes were used to evaluate the effects of Au@Pt NPs on cardiomyocyte maturation. The expression and alignment of cardiomyocyte myofibril proteins were analyzed by qRT-PCR, western blot, and immunofluorescence staining. Cellular functionality was analyzed by the multi-electrode array.

By adding Au@Pt NPs at different stages of cardiac differentiation of mouse ESCs, we found that treatment with Au@Pt NPs at the late stage could promote the maturation of differentiated cardiomyocytes, evidenced by increased expression of mature myofibril protein isoforms, more aligned myofibrils, and enhanced sarcomere length. Additionally, Au@Pt NPs can enhance the expression of mature sarcomere components, increase sarcomere length, and significantly boost beating amplitude and conduction velocity in neonatal mouse cardiomyocytes. Furthermore, Au@Pt NPs promoted cell cycle arrest, increased intracellular reactive oxygen species levels, and promoted contractility by inducing the ERK1/2 signaling pathway.

Our results indicate that the bimetallic Au@Pt NPs with intrinsic oxidase-like, peroxidase-like, and catalase-like activities and high electrical conductivity could promote the maturation of ESCs-derived and neonatal mouse cardiomyocytes, providing a promising approach for cardiomyocyte maturation and cell therapy for cardiovascular disease.

The creation of induced pluripotent stem cells (iPSCs) has enabled scientists to explore the function, mechanisms, and differentiation processes of many types of cells. One of the fastest and most efficient approaches is transcription factor (TF) over-expression. However, finding the right combination of TFs to over-express to differentiate iPSCs directly into other cell types is a difficult task. Here, we describe a machine-learning (ML) pipeline, called CellCartographer, that uses chromatin accessibility and transcriptomics data to design multiplex TF pooled-screening experiments for cell-type conversions that then may be iteratively refined. We validate this method by differentiating iPSCs into twelve cell types at low efficiency in preliminary screens and iteratively refine our TF combinations to achieve high-efficiency differentiation for six of these cell types in <6 days. Finally, we functionally characterize iPSC-derived cytotoxic T cells (iCytoTs), regulatory T cells (iTregs), type II astrocytes (iAstIIs), and hepatocytes (iHeps) to validate functionally accurate differentiation.

MXenes, a two-dimensional transition metal carbide and nitride, have shown significant potential in various biological applications. In particular, the distinct properties of MXenes─including their functionalizable surface, biocompatibility, and conductive characteristics, make them highly promising materials for advancing biomedical technologies. Here, we report that MXene, under specific electromagnetic field (EMF) conditions, effectively promotes the direct lineage reprogramming of induced dopaminergic (iDA) neurons both in vitro and in vivo. Remarkably, we found that electromagnetized MXene leads to specific activation of histone acetylation during the induced dopaminergic neuronal reprogramming process and efficiently alleviates symptoms in a mouse model of Parkinson's disease (PD). Moreover, MXene-mediated electromagnetic stimulation effectively promotes the direct reprogramming of human iDA neurons from skin fibroblasts. Therefore, our study highlights MXene's application in cell reprogramming, offering promising advancements in regenerative medicine through improved efficiency and reliability.

This article presents a distributed pinning control strategy aimed at achieving global stabilization of Markovian jump Boolean control networks. The strategy relies on network matrix information to choose controlled nodes and adopts the algebraic state space representation approach for designing pinning controllers. Initially, a sufficient criterion is established to verify the global stability of a given Markovian jump Boolean network (MJBN) with probability one at a specific state within finite time. To stabilize an unstable MJBN at a predetermined state, the selection of pinned nodes involves removing the minimal number of entries, ensuring that the network matrix transforms into a strictly lower (or upper) triangular form. For each pinned node, two types of state feedback controllers are developed: 1) mode-dependent and 2) mode-independent, with a focus on designing a minimally updating controller. The choice of controller type is determined by the feasibility condition of the mode-dependent pinning controller, which is articulated through the solvability of matrix equations. Finally, the theoretical results are illustrated by studying the T cell large granular lymphocyte survival signaling network consisting of 54 genes and 6 stimuli.

The limited capacity of adult mammalian cardiomyocytes to undergo cell division and proliferation is one of the key factors contributing to heart failure. In newborn mice, cardiac proliferation occurs during a brief window, but this proliferative capacity diminishes by 7 days after birth. Current studies on cardiac regeneration focused on elucidating changes in regulatory factors within the heart before and after this proliferative window, aiming to determine whether potential association between these factors and cell cycle arrest in cardiomyocytes. Facilitating the re-entry of cardiomyocytes into the cell cycle or reversing their exit from it represents a critical strategy for cardiac regeneration. This paper provides an overview of the role of cell cycle arrest in cardiac regeneration, briefly describes cardiomyocyte proliferation and cardiac regeneration, and systematically summarizes the regulation of the cell cycle arrest in cardiomyocytes, and the potential metabolic mechanisms underlying cardiomyocyte cycle arrest. Additionally, we highlight the development of cardiovascular disease drugs targeting cardiomyocyte cell cycle regulation and their status in clinical treatment. Our goal is to outline strategies for promoting cardiac regeneration and repair following cardiac injury, while also pointing toward future research directions that may offer new technologies and prospects for treating cardiovascular diseases, such as myocardial infarction, arrhythmia and heart failure.

The sparse and stochastic nature of conversion has obscured our understanding of how transcription factors (TFs) drive cells to new identities. To overcome this limit, we develop a tailored, high-efficiency conversion system that increases the direct conversion of fibroblasts to motor neurons 100-fold. By tailoring the cocktail to a minimal set of transcripts, we reduce extrinsic variation, allowing us to examine how proliferation and TFs synergistically drive conversion. We show that cell state-as set by proliferation history-defines how cells interpret the levels of TFs. Controlling for proliferation history and titrating each TF, we find that conversion correlates with levels of the pioneer TF Ngn2. By isolating cells by both their proliferation history and Ngn2 levels, we demonstrate that levels of Ngn2 expression alone are insufficient to predict conversion rates. Rather, proliferation history and TF levels combine to drive direct conversion. Finally, increasing the proliferation rate of adult human fibroblasts generates morphologically mature induced human motor neurons at high rates.

Direct cardiac reprogramming has emerged as a promising therapeutic strategy to remuscularize injured myocardium. This approach converts non-contractile fibroblasts to induced cardiomyocytes (iCMs) that spontaneously contract, yet the intrinsic metabolic requirements driving cardiac reprogramming are not fully understood. Using single-cell metabolic flux estimation and flux balance analysis, we characterized the metabolic heterogeneity of iCMs and identified fatty acid oxidation (FAO) as a critical factor in iCM conversion. Both pharmacological and genetic inhibition of FAO impairs iCM generation. We further identified stearoyl-coenzyme A desaturase 1 (SCD1) as a metabolic switch that suppresses iCM reprogramming. Mechanistically, Scd1 knockdown activates PGC1α and PPARβ signaling, enhancing FAO-related gene expression and mitochondrial biogenesis, thereby improving reprogramming efficacy. Pharmacological manipulations targeting SCD1, PGC1α, and the PPARβ signaling axis further improved iCM generation and mitochondrial function. Our findings collectively highlight FAO as a key determinant of iCM fate and offer new therapeutic avenues for advancing reprogramming strategies.

Control of gene expression is commonly mediated by distinct combinations of transcription factors (TFs). This cooperative action allows the integration of multiple biological signals at regulatory elements, resulting in highly specific gene expression patterns. It is unclear whether combinatorial binding is also necessary to bring together TFs with distinct biochemical functions, which collaborate to effectively recruit and activate RNA polymerase II. Using a cardiac differentiation model, we find that the largely ubiquitous homeodomain proteins MEIS act as actuators, fully activating transcriptional programs selected by lineage-restricted TFs. Combinatorial binding of MEIS with lineage-enriched TFs, GATA, and HOX, provides selectivity, guiding MEIS to function at cardiac-specific enhancers. In turn, MEIS TFs promote the accumulation of the methyltransferase KMT2D to initiate lineage-specific enhancer commissioning. MEIS combinatorial binding dynamics, dictated by the changing dosage of its partners, drive cells into progressive stages of differentiation. Our results uncover tissue-specific transcriptional activation as the result of ubiquitous actuator TFs harnessing general transcriptional activator at tissue-specific enhancers, to which they are directed by binding with lineage- and domain-specific TFs.

Degeneration of the cochlear spiral ganglion neurons (SGNs) is one of the major causes of sensorineural hearing loss and significantly impacts the outcomes of cochlear implantation. Functional regeneration of SGNs holds great promise for treating sensorineural hearing loss. In this study, we systematically screened 33 transcriptional regulators implicated in neuronal and SGN fate. Using gene expression array and principal component analyses, we identified a sequential combination of Ascl1, Pou4f1 and Myt1l (APM) in promoting functional reprogramming of SGNs. The neurons induced by APM expressed mature neuronal and SGN lineage-specific markers, displayed mature SGN-like electrophysiological characteristics and exhibited single-cell transcriptomes resembling the endogenous SGNs. Thus, transcription factors APM may serve as novel candidates for direct reprogramming of SGNs and hearing recovery due to SGN damages.

Cardiac sarcomere assembly is a highly orchestrated process requiring integration between intracellular contractile components and extracellular adhesions. While α-actinin-2 (ACTN2) is well known for its structural role at Z-discs, the function of the "non-muscle" paralog α-actinin-1 (ACTN1) in cardiomyocytes remains unclear. Using human induced pluripotent stem cell-derived cardiac myocytes (hiCMs), we demonstrate that ACTN1 is essential for sarcomere assembly. siRNA-mediated depletion of ACTN1 disrupted Z-line formation and impaired sarcomere organization, defects that were rescued by exogenous ACTN1 but not ACTN2, revealing non-redundant functions. Unlike ACTN2, ACTN1 localized predominantly to focal adhesions and was required for adhesion maturation, as evidenced by reduced adhesion size and number following ACTN1 depletion. Live-cell imaging of vinculin dynamics showed decreased stability of adhesion-associated vinculin in ACTN1-deficient cells, whereas paxillin dynamics were unaffected. These results suggest that ACTN1 stabilizes focal adhesions to promote effective force transmission during sarcomere assembly.

Cell reprogramming represents a powerful approach to achieve the conversion cells of one type into cells of another type of interest, which has substantially changed the landscape in the field of developmental biology, regenerative medicine, disease modeling, drug discovery and cancer immunotherapy. Cell reprogramming is a complex and ordered process that involves the coordination of transcriptional, epigenetic, translational and metabolic changes. Over the past two decades, a range of questions regarding the facilitators/barriers, the trajectories, and the mechanisms of cell reprogramming have been extensively investigated. This review summarizes the recent advances in cell reprogramming mediated by transcription factors or chemical molecules, followed by elaborating on the important roles of biophysical cues in cell reprogramming. Additionally, this review will detail our current understanding of the mechanisms that govern cell reprogramming, including the involvement of the recently discovered biomolecular condensates. Finally, the review discusses the broad applications and future directions of cell reprogramming in developmental biology, disease modeling, drug development, regenerative/rejuvenation therapy, and cancer immunotherapy.

Cardiovascular disease (CVD) remains the leading cause of death globally, with myocardial infarction (MI) being a major contributor. The current therapeutic approaches are limited in effectively regenerating damaged cardiac tissue. Up-to-date strategies for heart regeneration/reconstitution aim at cardiac remodeling through repairing the damaged tissue with an external cell source or by stimulating the existing cells to proliferate and repopulate the compromised area. Cell reprogramming is addressed to this challenge as a promising solution, converting fibroblasts and other cell types into functional cardiomyocytes, either by reverting cells to a pluripotent state or by directly switching cell lineage. Several strategies such as gene editing and the application of miRNA and small molecules have been explored for their potential to enhance cardiac regeneration. Those strategies take advantage of cell plasticity by introducing reprogramming factors that regress cell maturity in vitro, allowing for their later differentiation and thus endorsing cell transplantation, or promote in situ cell proliferation, leveraged by scaffolds embedded with pro-regenerative factors promoting efficient heart restoration. Despite notable advancements, important challenges persist, including low reprogramming efficiency, cell maturation limitations, and safety concerns in clinical applications. Nonetheless, integrating these innovative approaches offers a promising alternative for restoring cardiac function and reducing the dependency on full heart transplants.

Cardiac fibroblasts (CFs) are the essential cell type for heart morphogenesis and homeostasis. In addition to maintaining the structural integrity of the heart tissue, muscle fibroblasts are involved in complex signaling cascades that regulate cardiomyocyte proliferation, migration, and maturation. While CFs serve as the primary source of extracellular matrix proteins (ECM), tissue repair, and paracrine signaling, they are also responsible for adverse pathological changes associated with cardiovascular disease. Following activation, fibroblasts produce excessive ECM components that ultimately lead to fibrosis and cardiac dysfunction. Decades of research have led to a much deeper understanding of the role of CFs in cardiogenesis. Recent studies using the single-cell genomic approach have focused on advancing the role of CFs in cellular interactions, and the mechanistic implications involved during cardiovascular development and disease. Arguably, the unique role of fibroblasts in development, tissue repair, and disease progression categorizes them into the friend or foe category. This brief review summarizes the current understanding of cardiac fibroblast biology and discusses the key findings in the context of development and pathophysiological conditions.

The extracellular matrix (ECM) plays a crucial role in providing structural integrity and regulating cell communication essential for organ development, homeostasis, and regeneration, including hearts. Evidence indicates that disruptions in the spatiotemporal expression or alterations in ECM components lead to cardiac malformations, including a wide range of congenital heart diseases (CHDs). Furthermore, research on injured hearts across various vertebrate species, some of which show effective regeneration while others experience irreversible fibrosis, underscores the significance of ECM molecules in cardiac regeneration. This review presents an overview of heart development and the dynamics of ECM during cardiac morphogenesis, beginning with the formation of the contractile heart tube and advancing to the development of distinct chambers separated by valves to facilitate unidirectional blood flow. Furthermore, we discuss research emphasizing the multifaceted roles of secreted molecules in mediating fibrosis and regeneration following myocardial injury.

As heart transplantation continues to be the gold standard therapy for end-stage heart failure, the imbalance between the supply of hearts, and the demand for them, continues to get worse. In the US alone, with less than 4,000 hearts suitable for transplant and over 100,000 potential recipients, this therapy is only available to a very few. The use of hearts Donated after Circulatory Death (DCD) and Donation after Brain Death (DBD) using ex vivo machine perfusion (EVMP) is a promising approach that has already increased the availability of suitable organs for heart transplantation. EVMP offers the promise of enabling the expansion of the overall number of heart transplants and lower rates of early graft dysfunction. These are realized through (1) safe extension of the time between procurement and transplantation and (2) ex vivo assessment of preserved hearts. Notably, ex vivo perfusion has facilitated the donation of DCD hearts and improved the success of transplantation. Nevertheless, DCD hearts suffer from serious preharvest ischemia/reperfusion injury (IRI). Despite these developments, only 40% of hearts offered for transplantation can be utilized. These devices do offer an opportunity to evaluate donor hearts for transplantation, resuscitate organs previously deemed unsuitable for transplantation, and provide a platform for the development of novel therapeutics to limit cardiac injury. Bone Morphogenetic Protein (BMP) signaling is a new target which holds the potential for ameliorating myocardial IRI. Recent studies have demonstrated that BMP signaling has a significant role in blocking the deleterious effects of injury to the heart. We have designed novel small peptide BMP mimetics that act via activin receptor-like kinase (ALK3), a type I BMP receptor. They are capable of (1) inhibiting inflammation and apoptosis, (2) blocking/reversing the epithelial-mesenchymal transition (EMT) and fibrosis, and (3) promoting tissue regeneration. In this review, we explore the promise that novel therapeutics, including these BMP mimetics, offer for the protection of hearts against myocardial injury during ex vivo transportation for cardiac transplantation. This protection represents a significant advance and a promising ex vivo therapeutic approach to expanding the donor pool by increasing the number of transplantable hearts.

Heart disease, particularly resulting from myocardial infarction (MI), continues to be a leading cause of mortality, largely due to the limited regenerative capacity of the human heart. Current therapeutic approaches seek to generate new cardiomyocytes from alternative sources. Direct cardiac reprogramming, which converts fibroblasts into induced cardiomyocytes (iCMs), offers a promising alternative by enabling in situ cardiac regeneration and minimizing tumorigenesis concerns. Here we review recent advancements in the understanding of transcriptional and epigenetic mechanisms underlying cardiac reprogramming, with a focus on key early-stage molecular events, including epigenetic barriers and regulatory mechanisms that facilitate reprogramming. Despite substantial progress, human cardiac fibroblast reprogramming and iCM maturation remain areas for further exploration. We also discuss the combinatorial roles of reprogramming factors in governing transcriptional and epigenetic changes. This review consolidates current knowledge and proposes future directions for promoting the translational potential of cardiac reprogramming techniques.

Direct conversion is an innovative new technology that involves the conversion of somatic cells to target cells without passing through a pluripotent state. Forced expression alone or in combination with transcription factors (TFs), which are critical for the generation of target cells, is important for successful direct conversion. However, most somatic cells are unable to directly convert into target cells even with forced expression. We herein demonstrated that epithelial-mesenchymal transition (EMT) is advantageous for the direct conversion of somatic cells. We previously reported that mouse keratinocytes converted into neural crest cells (NCCs) following the forced expression of the NCC specifier Sox10 in combination with expression of the TFs Snail1, Slug, Twist1, and Tcfap2a (4 TFs). 4 TFs induced EMT in keratinocytes; therefore, EMT was considered to be advantageous for direct conversion. The direct conversion of mouse mammary gland epithelial cells (NMuMG cells) into NCCs was not observed with the forced expression of Sox10, but was detected with the expression of Sox10 following the induction of EMT by 4 TFs. Furthermore, TGF-β1-induced EMT and Sox10 expression directly converted NMuMG cells into NCCs. These results suggest that the induction of EMT in somatic cells is advantageous for direct conversion.

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by Yamanaka factors, including pioneer transcription factors (TFs), has greatly reshaped our traditional understanding of cell plasticity and demonstrated the remarkable potential of pioneer TFs. In addition to iPSC reprogramming, pioneer TFs are pivotal in direct reprogramming or transdifferentiation where somatic cells are converted into different cell types without passing through a pluripotent state. Pioneer TFs initiate a reprogramming process through chromatin opening, thereby establishing competence for new gene regulatory programs. The action of pioneer TFs is both influenced by and exerts influence on epigenetic regulation. Despite significant advances, many direct reprogramming processes remain inefficient, which limits their reliability for clinical applications. In this review, we discuss the molecular mechanisms underlying pioneer TF-driven reprogramming, with a focus on their interactions with epigenetic modifiers, including Polycomb repressive complexes (PRCs), nucleosome remodeling and deacetylase (NuRD) complexes, and the DNA methylation machinery. A deeper understanding of the dynamic interplay between pioneer TFs and epigenetic modifiers will be essential for advancing reprogramming technologies and unlocking their full clinical potential.

Direct reprogramming has garnered considerable attention due to its capacity to directly convert differentiated cells into desired cells. Fibroblasts are frequently employed in reprogramming studies due to their abundance and accessibility. However, they are also the key drivers in the progression of fibrosis, a pathological condition characterized by excessive extracellular matrix deposition and tissue scarring. Furthermore, the initial stage of reprogramming typically involves deactivating fibrotic pathways. Hence, direct reprogramming offers a valuable method to regenerate target cells for tissue repair while simultaneously reducing fibrotic tendencies. Understanding the link between reprogramming and fibrosis could help develop effective strategies to treat damaged tissue with a potential risk of fibrosis. This review summarizes the advances in direct reprogramming and reveals their anti-fibrosis effects in various organs such as the heart, liver, and skin. Furthermore, we dissect the mechanisms of reprogramming influenced by fibrotic molecules including TGF-β signaling, mechanical signaling, inflammation signaling, epigenetic modifiers, and metabolic regulators. Innovative methods for fibroblast reprogramming like small molecules, CRISPRa, modified mRNA, and the challenges of cellular heterogeneity and senescence faced by in vivo direct reprogramming, are also discussed.

Heart disease is the leading cause of mortality in developed countries, and novel regenerative procedures are warranted. Direct cardiac conversion (DCC) of adult fibroblasts can create induced cardiomyocytes (iCMs) for gene and cell-based heart therapy, and in addition to holding great promise, still lacks effectiveness as metabolic and age-associated barriers remain elusive. Here, by employing MGT (Mef2c, Gata4, Tbx5) transduction of mouse embryonic fibroblasts (MEFs) and adult (dermal and cardiac) fibroblasts from animals of different ages, we provide evidence that the direct reprogramming of fibroblasts into iCMs decreases with age. Analyses of histone posttranslational modifications and ChIP-qPCR revealed age-dependent alterations in the epigenetic landscape of DCC. Moreover, DCC is accompanied by profound mitochondrial metabolic adaptations, including a lower abundance of anabolic metabolites, network remodeling, and reliance on mitochondrial respiration. In vitro metabolic modulation and dietary manipulation in vivo improve DCC efficiency and are accompanied by significant alterations in histone marks and mitochondrial homeostasis. Importantly, adult-derived iCMs exhibit increased accumulation of oxidative stress in the mitochondria and activation of mitophagy or dietary lipids; they improve DCC and revert mitochondrial oxidative damage. Our study provides evidence that metaboloepigenetics plays a direct role in cell fate transitions driving DCC, highlighting the potential use of metabolic modulation to improve cardiac regenerative strategies.

Sox17-Erg direct reprogramming is a potent tool for the in vitro and in vivo generation of arterial-like induced-endothelial cells from fibroblasts. In this study, we illustrate the pioneering roles of both Sox17 and Erg in the endothelial cell reprogramming process and demonstrate that emergent gene expression only occurs when both factors are co-expressed. Bioinformatic analyses and molecular validation reveal both Bach2 and Etv4 as integral mediators of Sox17-Erg reprogramming with different roles in lung and heart fibroblast reprogramming. The generated organ-specific induced endothelial cells express molecular signatures similar to vasculature found in the starting cell's organ of origin and the starting chromatin architecture plays a role in the acquisition of this organ-specific identity. Overall, the Sox17-Erg reprogramming mechanism provides foundational knowledge for the future recapitulation of vascular heterogeneity through direct reprogramming.

Direct cardiac reprogramming or transdifferentiation is a relatively new and promising area in regenerative therapy, cardiovascular disease modeling, and drug discovery. Effective reprogramming of fibroblasts is limited by their plasticity, that is, their ability to reprogram, and depends on solving several levels of tasks: inducing cardiomyocyte-like cells and obtaining functionally and metabolically mature cardiomyocytes. Currently, in addition to the use of more classical approaches such as overexpression of exogenous transcription factors, activation of endogenous cardiac transcription factors via controlled nucleases, such as CRISPR, represents another interesting way to obtain cardiomyocytes. Therefore, special attention is given to the potential of synthetic biology, in particular the CRISPR system, for the targeted conversion of only certain subpopulations of fibroblasts into cardiomyocytes. However, obtaining functionally and metabolically mature cardiomyocytes remains a challenge despite the range of recently developed approaches. In this review, we summarized current knowledge on the function and diversity of human cardiac fibroblasts and alternative cell sources for in vitro human cardiomyocyte models. We examined in detail the transcription factors that initiate cardiomyogenic reprogramming and their interactions. Additionally, we critically analyzed the strategies used for the metabolic and physiological maturation of induced cardiomyocytes.

Cellular identity requires the concerted action of multiple transcription factors (TFs) bound together to enhancers of cell-type-specific genes. Despite TFs recognizing specific DNA motifs within accessible chromatin, this information is insufficient to explain how TFs select enhancers1. Here we compared four different TF combinations that induce different cell states, analysing TF genome occupancy, chromatin accessibility, nucleosome positioning and 3D genome organization at the nucleosome resolution. We show that motif recognition on mononucleosomes can decipher only the individual binding of TFs. When bound together, TFs act cooperatively or competitively to target nucleosome arrays with defined 3D organization, displaying motifs in particular patterns. In one combination, motif directionality funnels TF combinatorial binding along chromatin loops, before infiltrating laterally to adjacent enhancers. In other combinations, TFs assemble on motif-dense and highly interconnected loop junctions, and subsequently translocate to nearby lineage-specific sites. We propose a guided-search model in which motif grammar on nucleosome fibres acts as signpost elements, directing TF combinatorial binding to enhancers.

Sall4 as a pivotal transcription factor has been extensively studied across diverse biological processes, including stem cell biology, embryonic development, hematopoiesis, tissue stem/progenitor maintenance, and the progression of various cancers. Recent research highlights Sall4's emerging roles in modulating cardiac progenitors and cellular reprogramming, linking its functions to early heart development and regenerative medicine. These findings provide new insights into the critical functions of Sall4 in cardiobiology. This review explores Sall4's complex molecular mechanisms and their implications for advancing cardiac regenerative medicine.

Cardiovascular diseases as myocardial infarction (MI) represent a major cause for morbidity and mortality worldwide. Even though, patients who survive MI are susceptible to high risk of heart failure. This is mainly attributed to the major loss of cardiomyocytes and limited regenerative potential of myocardium. Despite the availability of various cardiovascular drugs, they fail to address the main cause of MI. The optimum therapeutic goal should therefore focus on enhancing cardiac regeneration through cellular and cell-free therapeutic approaches. This review focused on different mechanisms of cardiac regeneration that can be achieved via non-cellular therapeutic modalities. Passive and active targeting of the infarcted myocardium using various nanoparticles that can be loaded with growth factors, drugs or affordable natural products can reduce negative ventricular remodeling, infarct size and the apoptotic rate of cardiomyocytes. In addition, injectable biomaterials-based nanocomposite can be used as a scaffold to support infarcted heart and recruit cells. Innovative affordable and less invasive cell-free approaches can be implemented to enhance cardiac regeneration post MI.

Dosage-sensitive transcription factors (TFs) underlie altered gene regulation in human developmental disorders, and cell-type specific gene regulation is linked to the reorganization of 3D chromatin during cellular differentiation. Here, we show dose-dependent regulation of chromatin organization by the congenital heart disease (CHD)-linked, lineage-restricted TF TBX5 in human cardiomyocyte differentiation. Genome organization, including compartments, topologically associated domains, and chromatin loops, are sensitive to reduced TBX5 dosage in a human model of CHD, with variations in response across individual cells. Regions normally bound by TBX5 are especially sensitive, while co-occupancy with CTCF partially protects TBX5-bound TAD boundaries and loop anchors. These results highlight the importance of lineage-restricted TF dosage in cell-type specific 3D chromatin dynamics, suggesting a new mechanism for TF-dependent disease.

Engineering and reprogramming cells has significant therapeutic potential to treat a wide range of diseases, by replacing missing or defective proteins, to provide transcription factors (TFs) to reprogram cell phenotypes, or to provide enzymes such as RNA-guided Cas9 derivatives for CRISPR-based cell engineering. While viral vector-mediated gene transfer has played an important role in this field, the use of mRNA avoids safety concerns associated with the integration of DNA into the host cell genome, making mRNA particularly attractive for in vivo applications. Widespread application of mRNA for cell engineering is limited by its instability in the biological environment and challenges involved in the delivery of mRNA to its target site. In this review, we examine challenges that must be overcome to develop effective therapeutics.

Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) can be achieved by ectopic expression of cardiac transcription factors (TFs) via viral vectors. However, risks like genomic mutations, viral toxicity, and immune response limited its clinical application. Transactivation of endogenous TFs emerges as an alternative approach that may partially mitigate some of the risks. In this study, we utilized a modified CRISPRa/dCas9 strategy to transactivate endogenous reprogramming factors MEF2C, GATA4, and TBX5 (MGT) to induce iCMs from both mouse and human fibroblasts. We identified single-guide RNAs (sgRNAs) targeting promoters and enhancers of the TFs capable of activating various degrees of endogenous gene expression. CRISPRa-mediated Gata4 activation, combined with exogenous expression of Mef2c and Tbx5, successfully converted fibroblasts into iCMs. Despite extensive sgRNA screening, transactivation of Mef2c and Tbx5 via CRISPRa remained less effective, potentially due to de novo epigenetic barriers. While future work and refined technologies are needed to determine whether cardiac reprogramming could be achieved solely through CRISPRa activation of endogenous factors, our findings provide proof of concept that reliance on exogenous TFs for reprogramming can be reduced through CRISPRa-mediated activation of endogenous factors, particularly Gata4, offering a novel strategy to convert scar-forming fibroblasts into iCMs for regenerative purposes.

The dynamic regulation of chromatin accessibility is one of the prominent characteristics of eukaryotic genome. The inaccessible regions are mainly located in heterochromatin, which is multilevel compressed and access restricted. The remaining accessible loci are generally located in the euchromatin, which have less nucleosome occupancy and higher regulatory activity. The opening of chromatin is the most important prerequisite for DNA transcription, replication, and damage repair, which is regulated by genetic, epigenetic, environmental, and other factors, playing a vital role in multiple biological progresses. Currently, based on the susceptibility difference of occupied or free DNA to enzymatic cleavage, solubility, methylation, and transposition, there are many methods to detect chromatin accessibility both in bulk and single-cell level. Through combining with high-throughput sequencing, the genome-wide chromatin accessibility landscape of many tissues and cells types also have been constructed. The chromatin accessibility feature is distinct in different tissues and biological states. Research on the regulation network of chromatin accessibility is crucial for uncovering the secret of various biological processes. In this review, we comprehensively introduced the major functions and mechanisms of chromatin accessibility variation in different physiological and pathological processes, meanwhile, the targeted therapies based on chromatin dynamics regulation are also summarized.

Heart disease, including myocardial infarction (MI), remains a leading cause of morbidity and mortality worldwide, necessitating the development of more effective regenerative therapies. Direct reprogramming of cardiomyocyte-like cells from resident fibroblasts offers a promising avenue for myocardial regeneration, but its efficiency and consistency in generating functional cardiomyocytes remain limited. Alternatively, reprogramming induced cardiac progenitor cells (iCPCs) could generate essential cardiac lineages, but existing methods often involve complex procedures. These limitations underscore the need for advanced mechanistic insights and refined reprogramming strategies to improve reparative outcomes in the heart. Partial cellular fate transitions, while still a relatively less well-defined area and primarily explored in longevity and neurobiology, hold remarkable promise for cardiac repair. It enables the reprogramming or rejuvenation of resident cardiac cells into a stem or progenitor-like state with enhanced cardiogenic potential, generating the reparative lineages necessary for comprehensive myocardial recovery while reducing safety risks. As an emerging strategy, partial cellular fate transitions play a pivotal role in reversing myocardial infarction damage and offer substantial potential for therapeutic innovation. This review will summarize current advances in these areas, including recent findings involving two transcription factors that critically regulate stemness and cardiogenesis. It will also explore considerations for further refining these approaches to enhance their therapeutic potential and safety.

Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-p-STAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-p-STAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.

Adult mammals are susceptible to substantial cardiomyocyte (CM) loss following various cardiac diseases due to the limited capacity of CM proliferation and regeneration. Recently, direct cardiac reprogramming, converting fibroblasts into induced CMs, has been achieved both in vitro and in vivo through forced expression of transcription factors (TFs). This review encapsulates the advancements made in enhancing reprogramming efficiency and underlying molecular mechanisms. It covers the optimization of TF-based reprogramming cocktails and in vivo delivery platform and recently identified regulators in enhancing reprogramming efficiency. In addition, we discuss recent insights into the molecular mechanisms of direct cardiac reprogramming from single-cell omics analyses. Finally, we briefly touch on remaining challenges and prospective direction of this field.

Fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) and constitutes a central pathophysiological process that underlies tissue dysfunction, across organs, in multiple chronic diseases and during aging. Myocardial fibrosis is a key contributor to dysfunction and failure in numerous diseases of the heart and is a strong predictor of poor clinical outcome and mortality. The excess structural and matricellular ECM proteins deposited by cardiac fibroblasts, is found between cardiomyocytes (interstitial fibrosis), in focal areas where cardiomyocytes have died (replacement fibrosis), and around vessels (perivascular fibrosis). Although myocardial fibrosis has important clinical prognostic value, access to cardiac tissue biopsies for histological evaluation is limited. Despite challenges with sensitivity and specificity, cardiac magnetic resonance imaging (CMR) is the most applicable diagnostic tool in the clinic, and the scientific community is currently actively searching for blood biomarkers reflecting myocardial fibrosis, to complement the imaging techniques. The lack of mechanistic insights into specific pro- and anti-fibrotic molecular pathways has hampered the development of effective treatments to prevent or reverse myocardial fibrosis. Development and implementation of anti-fibrotic therapies is expected to improve patient outcomes and is an urgent medical need. Here, we discuss the importance of the ECM in the heart, the central role of fibrosis in heart disease, and mechanistic pathways likely to impact clinical practice with regards to diagnostics of myocardial fibrosis, risk stratification of patients, and anti-fibrotic therapy.

Heart degenerative diseases pose a significant challenge due to the limited ability of native heart to restore lost cardiomyocytes. Direct cellular reprogramming technology, particularly the use of small molecules, has emerged as a promising solution to prepare functional cardiomyocyte through faster and safer processes without genetic modification. However, current methods of direct reprogramming often exhibit low conversion efficiencies and immature characteristics of the generated cardiomyocytes, limiting their use in regenerative medicine. This study proposes the use of mitochondrial delivery to metabolically reprogram chemically induced cardiomyocyte-like cells (CiCMs), fostering enhanced maturity and functionality. Our findings show that mitochondria sourced from high-energy-demand organs (liver, brain, and heart) can enhance structural maturation and metabolic functions. Notably, heart-derived mitochondria resulted in CiCMs with a higher oxygen consumption rate capacity, enhanced electrical functionality, and higher sensitivity to hypoxic condition. These results are related to metabolic changes caused by increased number and size of mitochondria and activated mitochondrial fusion after mitochondrial treatment. In conclusion, our study suggests that mitochondrial delivery into CiCMs can be an effective strategy to promote cellular maturation, potentially contributing to the advancement of regenerative medicine and disease modeling.

Pathological transdifferentiation, where differentiated cells aberrantly transform into other cell types that exacerbate disease rather than promote healing, represents a novel and significant concept. This perspective discusses its role and potential targeting in cardiovascular diseases and chronic inflammation. Current therapies mainly focus on mitigating early inflammatory response through proinflammatory cytokines and pathways targeting, including corticosteroids, TNF-α inhibitors, IL-1β monoclonal antibodies and blockers, IL-6 blockers, and nonsteroidal anti-inflammatory drugs (NSAIDs), along with modulating innate immune memory (trained immunity). However, these approaches often fail to address long-term tissue damage and functional regeneration. For instance, fibroblasts can transdifferentiate into myofibroblasts in cardiac fibrosis, and endothelial cells may undergo endothelial to mesenchymal transition (EndMT) in vascular remodeling, resulting in fibrosis and impaired tissue function. Targeting pathological transdifferentiation represents a promising therapeutic avenue by focusing on key signaling pathways that drive these aberrant cellular phenotypic and transcriptomic transitions. This approach seeks to inhibit these pathways or modulate cellular plasticity to promote effective tissue regeneration and prevent fibrosis. Such strategies have the potential to address inflammation, cell death, and the resulting tissue damage, providing a more comprehensive and sustainable treatment solution. Future research should focus on understanding the mechanisms behind pathological transdifferentiation, identifying relevant biomarkers and master regulators, and developing novel therapies through preclinical and clinical trials. Integrating these new therapies with existing anti-inflammatory treatments could enhance efficacy and improve patient outcomes. Highlighting pathological transdifferentiation as a therapeutic target could transform treatment paradigms, leading to better management and functional recovery of cardiovascular tissues in diseases and chronic inflammation.

Cardiovascular diseases (CVD) are the leading cause of death worldwide, and advanced age is a main contributor to the prevalence of CVD. Cellular senescence is an irreversible state of cell cycle arrest that occurs in old age or after cells encounter various stresses. Senescent cells not only result in the reduction of cellular function, but also produce senescence-associated secretory phenotype (SASP) to affect surrounding cells and tissue microenvironment. There is increasing evidence that the gradual accumulation of senescent cardiomyocytes is causally involved in the decline of cardiovascular system function. To highlight the role of senescent cardiomyocytes in the pathophysiology of age-related CVD, we first introduced that senescent cardiomyoyctes can be identified by structural changes and several senescence-associated biomarkers. We subsequently provided a comprehensive summary of existing knowledge, outlining the compelling evidence on the relationship between senescent cardiomyocytes and age-related CVD phenotypes. In addition, we discussed that the significant therapeutic potential represented by the prevention of accelerated senescent cardiomyocytes, and the current status of some existing geroprotectors in the prevention and treatment of age-related CVD. Together, the review summarized the role of cardiomyocyte senescence in CVD, and explored the molecular knowledge of senescent cardiomyocytes and their potential clinical significance in developing senescent-based therapies, thereby providing important insights into their biology and potential therapeutic exploration.