Fibroblasts can be reprogrammed into cardiovascular progenitor cells (CPCs) using transgenic approaches, although the underlying mechanism remains unclear. We determined whether activation of endogenous genes such as Gata4, Nkx2.5, and Tbx5 can rapidly establish autoregulatory loops and initiate CPC generation in adult extracardiac fibroblasts using a CRISPR activation system. The induced fibroblasts (>80%) showed phenotypic changes as indicated by an Nkx2.5 cardiac enhancer reporter. The progenitor characteristics were confirmed by colony formation and expression of cardiovascular genes. Cardiac sphere induction segregated the early and late reprogrammed cells that can generate functional cardiomyocytes and vascular cells in vitro. Therefore, they were termed CRISPR-induced CPCs (ciCPCs). Transcriptomic analysis showed that cell cycle and heart development pathways were important to accelerate CPC formation during the early reprogramming stage. The CRISPR system opened the silenced chromatin locus, thereby allowing transcriptional factors to access their own promoters and eventually forming a positive feedback loop. The regenerative potential of ciCPCs was assessed after implantation in mouse myocardial infarction models. The engrafted ciCPCs differentiated into cardiovascular cells in vivo but also significantly improved contractile function and scar formation. In conclusion, multiplex gene activation was sufficient to drive CPC reprogramming, providing a new cell source for regenerative therapeutics.

Continuous loss of cardiomyocytes (CMs) is one of the fundamental characteristics of many heart diseases, which eventually can lead to heart failure. Due to the limited proliferation ability of human adult CMs, treatment efficacy has been limited in terms of fully repairing damaged hearts. It has been shown that cell lineage conversion can be achieved by using cell reprogramming approaches, including human induced pluripotent stem cells (hiPSCs), providing a promising therapeutic for regenerative heart medicine. Recent studies using advanced cellular reprogramming-based techniques have also contributed some new strategies for regenerative heart repair. In this review, hiPSC-derived cell therapeutic methods are introduced, and the clinical setting challenges (maturation, engraftment, immune response, scalability, and tumorigenicity), with potential solutions, are discussed. Inspired by the iPSC reprogramming, the approaches of direct cell lineage conversion are merging, such as induced cardiomyocyte-like cells (iCMs) and induced cardiac progenitor cells (iCPCs) derived from fibroblasts, without induction of pluripotency. The studies of cellular and molecular pathways also reveal that epigenetic resetting is the essential mechanism of reprogramming and lineage conversion. Therefore, CRISPR techniques that can be repurposed for genomic or epigenetic editing become attractive approaches for cellular reprogramming. In addition, viral and non-viral delivery strategies that are utilized to achieve CM reprogramming will be introduced, and the therapeutic effects of iCMs or iCPCs on myocardial infarction will be compared. After the improvement of reprogramming efficiency by developing new techniques, reprogrammed iCPCs or iCMs will provide an alternative to hiPSC-based approaches for regenerative heart therapies, heart disease modeling, and new drug screening.

Cardiovascular diseases resulting from myocardial infarction (MI) remain a leading cause of death worldwide, imposing a substantial burden on global health systems. Current MI treatments, primarily pharmacological and surgical, do not regenerate lost myocardium, leaving patients at high risk for heart failure. Engineered heart tissue (EHT) offers a promising solution for MI and related cardiac conditions by replenishing myocardial loss. However, challenges like immune rejection, inadequate vascularization, limited mechanical strength, and incomplete tissue maturation hinder clinical application. The discovery of human-induced pluripotent stem cells (hiPSCs) has transformed the EHT field, enabling new bioengineering innovations. This review explores recent advancements and future directions in hiPSC-derived EHTs, focusing on innovative materials and fabrication methods like bioprinting and decellularization, and assessing their therapeutic potential through preclinical and clinical studies. Achieving functional integration of EHTs in the heart remains challenging due to the need for synchronized contraction, sufficient vascularization, and mechanical compatibility. Solutions such as genome editing, personalized medicine, and AI technologies offer promising strategies to address these translational barriers. Beyond MI, EHTs also show potential in treating ischemic cardiomyopathy, heart valve engineering, and drug screening, underscoring their promise in cardiovascular regenerative medicine.

Myocardial infarction-induced cardiovascular diseases remain a leading cause of mortality worldwide. Excessive post-infarct fibrosis contributes to adverse cardiac remodeling and the progression to heart failure. In vivo reprogramming strategies offer a promising avenue for heart regeneration by directly converting resident fibroblasts into cardiomyocytes through enforced expression of cardiogenic genes. This approach circumvents the need for invasive biopsies, cell expansion, induction of pluripotency, or autologous transplantation. Despite these advantages, key challenges persist, including low reprogramming efficiency and limited cellular targeting specificity. A critical factor for effective anti-fibrotic therapy is the precise and efficient delivery of reprogramming effectors specifically to fibrotic fibroblasts, while minimizing off-target effects on non-fibroblast cardiac cells and fibroblasts in non-cardiac tissues. In this review, we discuss the cellular and molecular mechanisms underlying in vivo cardiac reprogramming, with a focus on fibroblast heterogeneity, key transcriptional drivers, and relevant intercellular interactions. We also examine current advances in fibroblast-specific delivery systems employing both viral and non-viral vectors for the administration of lineage-reprogramming factors such as cDNA overexpressions or microRNAs. Finally, we underscore innovative strategies that hold promise for enhancing the precision and efficacy of cellular reprogramming, ultimately fostering translational development and paving the way for rigorous preclinical assessment.

Despite advances in prenatal screening and a notable decrease in mortality rates, congenital heart disease (CHD) remains the most prevalent congenital disorder in newborns globally. Current therapeutic surgical approaches face challenges due to the significant rise in complications and disabilities. Emerging cardiac regenerative therapies offer promising adjuncts for CHD treatment. One novel avenue involves investigating methods to stimulate cardiomyocyte proliferation. However, the mechanism of altered cardiomyocyte proliferation in CHD is not fully understood, and there are few feasible approaches to stimulate cardiomyocyte cell cycling for optimal healing in CHD patients. In this review, we explore recent progress in understanding genetic and epigenetic mechanisms underlying defective cardiomyocyte proliferation in CHD from development through birth. Targeting cell cycle pathways shows promise for enhancing cardiomyocyte cytokinesis, division, and regeneration to repair heart defects. Advancements in human disease modeling techniques, clustered regularly interspaced short palindromic repeats ‐based genome and epigenome editing, and next‐generation sequencing technologies will expedite the exploration of abnormal machinery governing cardiomyocyte differentiation, proliferation, and maturation across diverse genetic backgrounds of CHD. Ongoing studies on screening drugs that regulate cell cycling are poised to translate this nascent technology of enhancing cardiomyocyte proliferation into a new therapeutic paradigm for CHD surgical interventions.

Limited adult cardiac cell proliferation after cardiovascular disease, such as heart failure, hampers regeneration, resulting in a major loss of cardiomyocytes (CMs) at the site of injury. Recent studies in cellular reprogramming approaches have provided the opportunity to improve upon previous techniques used to regenerate damaged heart. Using these approaches, new CMs can be regenerated from differentiation of iPSCs (similar to embryonic stem cells), the direct reprogramming of fibroblasts [induced cardiomyocytes (iCMs)], or induced cardiac progenitors. Although these CMs have been shown to functionally repair infarcted heart, advancements in technology are still in the early stages of development in research laboratories. In this review, reprogramming-based approaches for generating CMs are briefly introduced and reviewed, and the challenges (including low efficiency, functional maturity, and safety issues) that hinder further translation of these approaches into a clinical setting are discussed. The creative and combined optimal methods to address these challenges are also summarized, with optimism that further investigation into tissue engineering, cardiac development signaling, and epigenetic mechanisms will help to establish methods that improve cell-reprogramming approaches for heart regeneration.