Understanding the origin and fate of cardiac progenitor cells is of great significance for unraveling the developmental properties of main cardiac cell lineages and the pathogenesis of various cardiac diseases. The heart contains a extraordinarily complex lineage of cells, primarily cardiomyocytes and non-cardiomyocytes. Nuclear bomb test-derived ¹⁴C and genetic cell-lineage tracing analysis revealed that cardiomyocytes account for 25–35% of all cells in the heart, with the remainder being non-cardiomyocytes including endothelial cells (ECs), mesenchymal cells, hematopoietic stem cells, and fibroblasts (1–3). To further reveal the characteristics of cardiac cell lineages, cardiac cardiomyocyte counts were measured in 29 subjects ranging in age from 1 month to 73 years. The number of cardiomyocytes remains constant throughout human life, and the cardiomyocyte turnover rate is less than 1% per year in adult cardiomyocytes (1). In recent years, the continuous development and innovation of genetic lineage tracing technology have promoted the study of the developmental characteristics and function of cardiac cell lineages. The genetic lineage tracing tools based on cyclization recombinase (Cre) and site-specific locus of x-over, P1 (loxP) homologous recombination elucidate the characteristics of the developmental origin and fate of cells in different organs (4). Cre-loxP provides a new research tool for further revealing the regeneration, homeostasis and pathogenesis of cardiac tissue (5). The regulatory mechanisms of cell origin and fate elucidated the fate and fate plasticity of various cell progeny in humans.

With the advancement of a prospective study, the accuracy of Cre-loxP-mediated genetic lineage-tracing systems has been constantly iterated and updated. Many new genetic lineage tracking systems for different cell lineages have been developed, such as epicardial progenitors, epicardial cells, and cycling cardiomyocytes. Zhou Bin et al. (6) applied a genetic lineage tracing system to effectively monitor the development of endocardial cell progeny over a prolonged period, denying the contribution of cardiac fibroblasts (CFs) to angiogenesis. Mapping all proliferating cells and their progeny in the heart by Ki67 single-cell mRNA sequencing and genetic lineage tracing found no evidence of a quiescent cardiac stem cell population. Dual recombinases dramatically improved the precision of genetic lineage tracing and reduced pitfalls in lineage-tracing studies due to Cre expression in pre-existing non-targeted cells. The lineage tracing system based on dual-recombinase-induced cross-reporter genes is called dual-recombinase-activated lineage tracing with an interleaved reporter (DeaLT-IR) (7). This site-specific recombination at the DNA level is permanent and irreversible and is an indispensable aid in studying cardiac structural development and congenital heart disease.

CVDs remains the leading cause of death in Western countries, including myocardial infarction, myocardial hypertrophy, hypertension, and congenital diseases, which seriously threaten human life and health (8). In recent years, the American Heart Association (AHA) and the Centers for Disease Control and Prevention (CDC) have reported that mortality from CVDs has declined. Still, it remains a heavy health and economic burden for the United States and the world (9). Studies have shown that most cardiomyocytes exit the cell cycle shortly after birth and eventually become terminally differentiated cells (10, 11). The heart has minimal regenerative capacity compared to organs, such as the liver, skin, and skeletal muscles (12). Even if the heart is injured, it is difficult to re-enter the proliferative cycle. Therefore, any loss of cardiomyocytes is irreversible and may cause or exacerbate heart failure. In 2011, genetic fate mapping revealed that most cardiomyocytes in regenerating tissues were derived from pre-existing cardiomyocytes, that is, by direct division of cardiomyocytes (13). Genetic lineagetracing studies of epicardial cells have revealed several previously unexplored cardiomyocyte progenitor populations, with significant implications for cardiomyocyte origin in the interventricular septum and atrioventricular wall. Therefore, achieving selective and more precise control is a core criterion for Cre recombinase-based cell fate lineage tracing. Following cardiac injury, genetic fate mapping provides a potential therapeutic strategy and theoretical framework for vascular and myocardial tissue regeneration and repair.

In the 1960s, as the use of lineage tracing in cell development increased, the technology was constantly updated and reinvented. In the process of egg cell division, cell lineage effectively infers that the differentiation fate of specific cells in the early embryo is uncertain due to several factors (14). Using genetic lineage tracing to effectively reveal the growth pattern of high clonal potential cancer stem cells and the fate determination and proliferative potential of individual tumor cells (15). It is also an essential tool for studying the fate properties of adult mammalian tissue stem cells. Throughout the lineage tracing system, the fate of a single cell is indiscriminately passed on to offspring, resulting in a set of stable and permanently marked clones (16). It is worth noting that genetic markers will not affect the normal life activities and properties of neighboring cells. At the same time, recent advances in single-cell sequencing (17–19) to elucidate cell type composition present enormous opportunities and challenges. To enable this marker to identify and classify cells as accurately as possible, single-cell transcriptomic-based lineage tracing improves the resolution and reliability of progeny differentiation trajectories. Lineage tracing provides a powerful means of understanding the relationship between tissue regeneration, homeostasis, and disease, especially when it is cross-talked with signals that regulate cell fate decisions.

In addition to the traditional Cre-loxP system, Dre expression from the En1 (Dre)-rox recombinase system is also an essential technical means to precisely track the cell fate of some progenitor and stem cells (20, 21). However, Cre recombinase may be confounded in non-target cells and thus affect the accuracy of cell fate tracking. The Cre-loxP and Dre-rox recombinase systems carry different reporter genes, such as the ZsGreen and tdTomato reporter genes, which can eliminate the interference of non-target cells by precisely identifying specific and staggered sites (7). This was further demonstrated by the tracer study of cardiomyocytes, which were labeled and induced by crossing IR and constitutive Tnni3-Dre and αMHC-MerCreMer mice. As a result, the Tnni3-labeled cardiomyocytes were all tdTomato⁺. Thus, expression of the Dre-rox mediated recombination system inhibited tamoxifen (TAM)-induced Cre-loxP recombination in cardiomyocytes. This system of dual recombinases precisely acting on cross-reporter genes addresses untargeted and unintentional lineage tracing (22). By the same principle, using a dual histone-mediated genetic system revealed the inability of peritoneal or pleural macrophages to invade deep organs for organ regeneration during lung and liver injury (23).

Cre recombinase is a 38 kDa protein encoded using P1 bacteriophage, which is highly conserved and catalytically active and can efficiently achieve site-specific DNA fragmentation and recombination both in vivo and in vitro (24–26). Similarly, the locus of crossing over in phage P1 (27, 28) is also derived from the P1 phage, composed of two 13 bp inverted repeats and an 8 bp sequence in the middle. It is a specific site for Cre recognition and recombination. According to the site orientation of the loxP sequence, Cre recombinase can efficiently excise site-specific DNA, sequence inversion, the exchange of double strands or chromosomal translocation (29, 30). The primary condition for the Cre-loxp system to recombine is that the flanking sequence of a DNA fragment to be deleted or recombined must contain 13 bp of floxed DNA sequence. Second, cells and animals carrying loxP DNA must drive the cell-specific expression of Cre recombinase, including DAG, ATCB, c-Kit, Ki67, and αMHC. Since the birth of Cre-loxP technology in 1981, as of 2015, more than 3,000 research reports have been recorded in the PubMed database (31). Consequently, the potential for the application of Cre technology is unparalleled. Cre-loxP system initially focuses on achieving the expression or deletion of target cell genes and constructing conditional gene expression and knockout mice (32, 33). The Cre recombinase-constructed mice are crossed with flox mice, and Cre accurately recognizes the specific site of the target gene to obtain transgenic or knockout mice. Notably, conditional gene expression mice showed a lox-stop-lox (LSL) sequence upstream of the target gene. When the specific Cre recombinase excises the LSL sequence, it further activates the expression of the downstream gene of interest (GOI) (34).

With the development and application of Cre-loxP technology, gene regulatory functions and disease prevention of cell populations have been widely monitored in various tissues and environments. TAM binds to the estrogen receptor (ER) to activate the expression of Cre recombinase, collectively referred to as the inducible Cre-loxP system, also known as the CreER system. After TAM was injected into mice, the complex formed by CreER and heat shock protein 90 (HSP90) (35, 36) dissociated and nuclearly translocated, in turn activating the Cre-loxP system (Figure 1). In addition, several indicators were selected for specific single-cell lineage tracing, such as LacZ, GFP, red fluorescent protein (RFP), tdTomato, and Cherry. Activation of specific ERs using TAM and doxycycline (DOX) forms an inducible Cre-loxP system that can be used to study gene function at specific stages of an organism’s developmental process, mainly in the CreER and CreTet systems (4, 37, 38). The inducible Cre-loxP system improves its precision and spatiotemporal specificity, revealing gene function and cell lineage tracing studies at specific stages.

The Cre-loxP system (39) controls the activation or repression of tissue-specific gene function, and expression in progenitor cells generates progeny cells that are labeled by reporter genes, and this genetic marker is heritable and permanent (40, 41). To comprehensively understand the characteristics and limitations of tissue development at various levels, stages, time, and space, new genetic lineage tools based on Cre-loxP have emerged, including the DeaLT-IR system (7) and the proliferation tracer (ProTracer) system (42) (Figure 2). In recent years, the Cre-loxP system has been widely used in cardiac structural development, especially in cardiac cardiomyocyte proliferation, angiogenesis, epicardial formation, and cardiac fibroblast fate determination. Therefore, the evolving Cre-loxP is an effective research tool for studying CVDs and pathogenesis.

The epicardium is a layer of mesothelial tissue covering the surface of the heart, and epicardium-derived progenitor cells with EMT properties (78–80). Epicardial-derived progenitor cells (EDPCs) are an essential source of vascular smooth muscle cells (SMCs), pericytes (PCs), and CFs (Figure 3). In addition, transcription factors released by paracrine in the epicardium further trigger cardiomyocyte proliferation and cardiac regeneration. Therefore, using a genetic lineage tracing system to explore the differentiation fate of epicardial cells has prospective implications for cardiac development and regenerative repair. Numerous studies have demonstrated that epicardial cells provide nutritional and structural support for cardiac development and adult cardiac repair (81, 82). Transplantation of epicardial cells significantly drives the proliferative and regenerative capacity of cardiomyocytes, improving cardiac contractile function and therapeutic efficacy.

Moreover, cardiogenic follistatin-like 1 (Fstl1) in the heart stimulates cell cycle entry and cardiomyocyte division, which to some extent reverses cardiac remodeling after myocardial injury (83). Therefore, the precise delineation of cardiac progenitor cell fate determination and structural hierarchy will be an essential link in the study of cardiac development and disease treatment. In previous reports, cardiac progenitor cells expressing pluripotent Nkx2-5 and Isl1 transcription factors were reportedly actively involved in forming myocardial tissue, ECs, SMCs, and epicardium (84, 85). Since Wilms tumor 1(Wt1) and Tbx18 are strongly expressed as markers of the epicardium, it has been demonstrated that Nkx2-5 and Isl1 cardiac progenitors are involved in forming the epicardium during cardiac development. The Cre-loxP system was used to precisely track epicardium-specifically expressed transcription factors or markers, such as Wt1, Tbx18, Scx and Sema3D, TCF21, and GATA4/5. Genetic fate mapping was used to study the progeny development of epicardial progenitor cells, highlighting the remarkable contribution of cardiac precursors to the various cell lineages of the heart.

When myocardial infarction occurs, normal coronary blood is blocked, and myocardial cells die in large numbers because they cannot get oxygen. In the process of heart repair, exploring the regeneration mechanism of coronary vessels is an important research field in the treatment of CVDs. Coronary vessels primarily develop from sinus endocardial cells (ECs), ventricular ECs, and epicardial cells (110, 111). In the early embryonic stage, endocardial cells form coronary ECs, and epicardial cells form vascular SMCs, both significantly contributing to angiogenesis (112–114). However, whether cardiac injury in adulthood leads to coronary angiogenesis remains a mystery. Ubil et al. (115) suggest that CFs take on ECs properties through mesenchymal endothelial cell transformation after cardiac injury, contributing to angiogenesis. However, using genetic lineage-tracing analysis, CFs were not further differentiated into ECs involved in angiogenesis (111). In 2017, Zhou Bin et al. generated a new genetic lineage-tracing system, using Npr3 as an EC marker, to extensively study the fate and origin of cardiac ECs throughout the developmental stages. Cardiac cushion MCs form as the early embryonic endocardium undergoes an endothelial-to-mesenchymal transition.

However, the specific differentiation mechanism of the endocardium is unclear. Based on the genetic lineage tracing performed by Cre recombinase, a dual-recombinase cross-targeting method was developed to improve the specificity of cell targeting (116). Fibroblasts, coronary parietal cells and adipocytes (ACs) in the heart are all derived from endocardial-derived cushion MCs, contributing to understanding the origin of cardiac development. ECs ultimately contribute to the coronary vessels of most of the heart. Lineage tracing and single-cell RNA sequencing in embryonic and adult mouse hearts uncover differential changes and plasticity in the transcriptional heterogeneity of coronary ECs (117). VEGF-B potently activates the ECs cycle via CreERT recombinase-mediated genetic cell lineage tracing (118). Endocardial-derived blood vessels develop toward the myocardium, promote the formation of coronary arteries, and improve the structure and remodeling of cardiac function. The widespread application of gene lineage tracing technology provides a strong guarantee of vascular reconstruction after myocardial infarction.

Genetic lineage tracing sheds light on the mysteries of developmental biology and facilitates deciphering the origin and fate of single-cell progeny. Widespread lineage-tracing systems have significantly advanced research in cardiac development, homeostasis, and tissue regeneration. To meet the requirements of heart development and disease prevention, Cre-loxP-based lineage-tracing technologies, such as dual recombinases, ProTracer, and MADM systems, are constantly innovating, improving the accuracy and stability of lineage-tracing. Tracking the development of epicardial and ECs reveals the components of each structural level of the heart, ruling out previous misconceptions. For example, epicardial cells substantially contribute to ventricular myocytes, whereas fibroblasts have little involvement in forming vascular ECs. The loss of cardiomyocytes after myocardial infarction is irreversible, exacerbating pathological remodeling and fibrosis of the heart. Supplementation of pre-existing cardiomyocytes is an effective means of cardiac regeneration. The lineage-tracing system uncovers the scientific question of the origin of cardiomyocytes and provides a reliable theoretical basis for the proliferation of cardiomyocytes. Moreover, it is controversial whether c-kit-expressing cardiac progenitors are the primary source of new cardiomyocytes. Lineage tracing has shown that c-kit cells proliferate cardiac ECs, but not cardiomyocytes (119, 120). TAM and DOX are generally used as time-controlled switches for inducible Cre-loxP initiation. The efficiency of site-specific recombination still needs to be improved, especially for gene deletion. These emerging tracers dissect cell origin and fate more precisely, offering new therapeutic strategies for congenital diseases and cardiac regeneration.

The extensive application value of the Cre-loxp system is compelling, but has some obvious constraints. The specific expression of Cre recombinase in targeted cells guarantees the accuracy of lineage tracing. Unfortunately, Cre recombinase expression in non-target cells may complicate lineage tracing, confusing tagged progeny cells. After the generation of mouse alleles and transgenic lines, the involvement of foreign genes may lead to tortuous structures of related genes, which may affect the functional expression of normal genes. Therefore, the accuracy of cell-lineage tracing is significantly compromised. Developing more recombination sites and tracers is an effective strategy for circumventing untargeted DNA site-specific recombination. Moreover, to enhance the recombination efficiency of inducible Cre-loxP, a self-cleavage-inducible CreER (sCreER) (121) came into being. Compared with traditional Cre-loxP, sCreER induces gene recombination more efficiently in ECs or fibroblasts. The iteration and innovation of genetic lineage tracing will provide further contributions and technical support for CVDs.

TW is mainly responsible for drafting the manuscript and organizing the graphics. XC, KaW, JJ, XY, and SW assisted in drafting the manuscript and design of ideas. CL and KuW reviewed and revised the final manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grant from the National Natural Science Foundation of China (81870236, 82070313, and 81770275), Taishan Scholar Program of Shandong Province, Major Research Program of the National Natural Science Foundation of China (No. 91849209), and Qingdao Scientific Program (No. 18–6-1-63-nsh).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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