FIG 2
Induction of Purkinje fiber phenotype in micromass cultures of embryonic myocytes. Confocal images of myocyte-micromass aggregates incubated for 48 hr in media containing no added ET (a, c, e) or 10 8 M ET (b, d, f ) and immunolabeled for cMyBP-C (a, b), Cx42 (c, d), and sMHC (e, f ). Inset shows the sarcomeric distribution of sMHC labeling adjacent the asterisk in a ET-treated aggregate.
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A regular heart beat is dependent on a specialized network of pacemaking and conductive cells. There has been a longstanding controversy regarding the developmental origin of these cardiac tissues which also manifest neural-like properties. Recently, we have shown conclusively that during chicken embryogenesis, impulse-conducting Purkinje cells are...
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... myocytes from embryonic day 3-5 (E3-5) chicken ventricles were isolated and maintained as either substrate- adherent ( Fig. 1 g and i) or suspended-aggregate micromass cultures (Fig. 2 a-f ). Under these conditions, myocytes estab- lished spontaneous, rhythmic contractions within 18-20 hr. The cultures were then incubated with a recombinant ET peptide. On the first day of culture, beating myocytes were Conversion of isolated E3 myocytes from cMyBP-C positive (red signal) to sMHC-positive (green signal) after exposure to ...
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... proteins, cMyBP-C was down-regulated significantly ( Fig. 1 h and i). The ET-dependent induction of Cx42 was then examined in the micromass cultures. Because of the three-dimensional arrangement of cells in aggregates, micromass cultures were found to be a more appropriate system for studying the formation of the intermyocyte gap junctions (Fig. 2). Up-regulation of sMHC ( Fig. 2 a and b) and down-regulation of cMyBP-C ( Fig. 2 e and f ) were both induced in myocytes exposed to ET. These changes in the aggregates occurred over a similar time course to that seen in substrate-adherent cultures. Cx42 was significantly up- regulated by ET ( Fig. 2 c and d), resembling what is seen ...
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... significantly ( Fig. 1 h and i). The ET-dependent induction of Cx42 was then examined in the micromass cultures. Because of the three-dimensional arrangement of cells in aggregates, micromass cultures were found to be a more appropriate system for studying the formation of the intermyocyte gap junctions (Fig. 2). Up-regulation of sMHC ( Fig. 2 a and b) and down-regulation of cMyBP-C ( Fig. 2 e and f ) were both induced in myocytes exposed to ET. These changes in the aggregates occurred over a similar time course to that seen in substrate-adherent cultures. Cx42 was significantly up- regulated by ET ( Fig. 2 c and d), resembling what is seen during Purkinje fiber differentiation in ...
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... induction of Cx42 was then examined in the micromass cultures. Because of the three-dimensional arrangement of cells in aggregates, micromass cultures were found to be a more appropriate system for studying the formation of the intermyocyte gap junctions (Fig. 2). Up-regulation of sMHC ( Fig. 2 a and b) and down-regulation of cMyBP-C ( Fig. 2 e and f ) were both induced in myocytes exposed to ET. These changes in the aggregates occurred over a similar time course to that seen in substrate-adherent cultures. Cx42 was significantly up- regulated by ET ( Fig. 2 c and d), resembling what is seen during Purkinje fiber differentiation in vivo (3). sMHC-positive cells increased in ...
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... formation of the intermyocyte gap junctions (Fig. 2). Up-regulation of sMHC ( Fig. 2 a and b) and down-regulation of cMyBP-C ( Fig. 2 e and f ) were both induced in myocytes exposed to ET. These changes in the aggregates occurred over a similar time course to that seen in substrate-adherent cultures. Cx42 was significantly up- regulated by ET ( Fig. 2 c and d), resembling what is seen during Purkinje fiber differentiation in vivo (3). sMHC-positive cells increased in response to ET in a dose-dependent manner over a range from 10 11 to 10 7 M ET (Fig. 3a). In contrast to the robust effects seen with ET, no detectable induction of Purkinje fiber phenotype was obtained in adherent cultures ...
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... of Purkinje fiber phenotype appeared to be specific to ET. Indeed, the response to ET was neutralized by addition of either BQ123 or BQ788, selective antagonists of the ET-receptor subtypes (16), ET-A and ET-B respectively (Fig. 3c). On rare occasions, some cells in control cultures not exposed to ET were found to be sMHC-positive (e.g., Fig. 2e). The appearance of sMHC-positive cells in control cultures was not completely suppressed by addition of the receptor antag- onists. These observations led us to speculate that the ET- dependent increase of cells exhibiting a Purkinje-like pheno- type may have resulted from activated proliferation of cells already committed to a ...
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In an earlier study, we suggested that adaptive gap junctions (GJs) might be a basis of cardiac memory, a phenomenon which refers to persistent electrophysiological response of the heart to external pacing. Later, it was also shown that the proposed mechanism of adaptation of GJs is consistent with known electrophysiology of GJs. In the present art...
Citations
... 3,4 Recent studies uncovered critical roles of paracrine/autocrine factors such as neuregulin-1 (NRG-1), endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) as well as various transcriptional factor networks in the VCS formation. [5][6][7][8][9] While the ability of NRG-1 to induce VCS specific reporter gene expression was restricted to E8.5 to E10.5 stages of mouse heart development, 7 ANP treatment was effective beyond E11.5 stage in increasing the expression of VCS markers such as connexin 40 (Cx40) and hyperpolarizationactivated cyclic nucleotide-gated channel-4 (HCN4). 8 Additionally, genetic ablation of the high-affinity receptor for ANP (natriuretic peptide receptor-A/NPRA) revealed reductions in the expression of Cx40 and HCN4, and a hypoplastic Purkinje fiber phenotype. ...
... Homozygous deletions of genes coding for carnitine Exogenous ANP treatment increases VCS marker protein levels and VCS cell proliferation in E11. 5 ...
It has been shown that atrial natriuretic peptide (ANP) and its high affinity receptor (NPRA) are involved in the formation of ventricular conduction system (VCS). Inherited genetic variants in fatty acid oxidation (FAO) genes are known to cause conduction abnormalities in newborn children. Although the effect of ANP on energy metabolism in noncardiac cell types is well documented, the role of lipid metabolism in VCS cell differentiation via ANP/NPRA signaling is not known. In this study, histological sections and primary cultures obtained from E11.5 mouse ventricles were analyzed to determine the role of metabolic adaptations in VCS cell fate determination and maturation. Exogenous treatment of E11.5 ventricular cells with ANP revealed a significant increase in lipid droplet accumulation, FAO and higher expression of VCS marker Cx40. Using specific inhibitors, we further identified PPARγ and FAO as critical downstream regulators of ANP-mediated regulation of metabolism and VCS formation.
... The origin of the cell, cellular differentiation, and the maturation of the various components of the CCS, in particular the ventricular CCS, are incompletely understood. In murine hearts, it has been accepted that the Purkinje fibre (PKJ) network arises from developing trabecular cardiomyocytes through endocardially-derived inductive signals [5][6][7][8] . Recently, it has also been suggested that a polyclonal PKJ network forms by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors 9 . ...
The heterogeneity of functional cardiomyocytes arises during heart development, which is essential to the complex and highly coordinated cardiac physiological function. Yet the biological and physiological identities and the origin of the specialized cardiomyocyte populations have not been fully comprehended. Here we report a previously unrecognised population of cardiomyocytes expressing Dbhgene encoding dopamine beta-hydroxylase in murine heart. We determined how these myocytes are distributed across the heart by utilising advanced single-cell and spatial transcriptomic analyses, genetic fate mapping and molecular imaging with computational reconstruction. We demonstrated that they form the key functional components of the cardiac conduction system by using optogenetic electrophysiology and conditional cardiomyocyte Dbh gene deletion models. We revealed their close relationship with sympathetic innervation during cardiac conduction system formation. Our study thus provides new insights into the development and heterogeneity of the mammalian cardiac conduction system by revealing a new cardiomyocyte population with potential catecholaminergic endocrine function.
... The origin of the cell, cellular differentiation, and the maturation of the various components of the CCS, in particular the ventricular CCS, are incompletely understood. In general, it has been widely accepted that the Purkinje fiber (PKJ) network arises from developing trabecular cardiomyocytes through endothelial-derived inductive signals 5,6 . Recently, it has als o been suggested that a polyclonal PKJ network forms by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors 7 . ...
... The ssDNA images were captured with Ti-7 Nikon Eclipse microscope using FITC channel. After imaging, tissue permeabilization was performed with 0.1% pepsin (Sigma, P7000) in 0.01 M HCl buffer (pH = 2) at 37 incubator for optimal time, which is 6 min for E12.5, E14.5 and P3 heart sections, and 18 min for adult heart sections. After washing with 0.1× SSC+RI, Reverse transcription was performed with SuperScript II (Invitrogen, 18064014, 10 U/μL reverse transcriptase, Stereo-TSO and 1× First-Strand buffer) at 42 incubator for overnight. ...
Cardiac conduction system (CCS) morphogenesis is essential for correct heart function yet is incompletely understood. Here we established the transcriptional landscape of cell types populating the developing heart by integrating single-cell RNA sequencing and spatial enhanced resolution omics-sequencing (Stereo-seq). Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. This led to the identification of a previously unrecognized cardiomyocyte population expressing dopamine beta-hydroxylase ( Dbh ⁺ -CMs), which is closely associated with the CCS in transcriptomic analyses. To confirm this finding, genetic fate mapping by using Dbh Cre /Rosa26-tdTomato reporter mouse line was performed with Stereo-seq, RNAscope, and immunohistology. We revealed that Dbh ⁺ -derived CMs first emerged in the sinus venosus at E12.5, then populated the atrial and ventricular CCS components at E14.5, with increasing abundance towards perinatal stages. Further tracing by using Dbh CFP reporter and Dbh CreERT /Rosa26-tdTomato inducible reporter, we confirmed that Dbh ⁺ -CMs are mostly abundant in the AVN and ventricular CCS and this persists in the adult heart. By using Dbh Cre /Rosa26-tdTomato/Cx40-eGFP compound reporter line, we validated a clear co-localization of tdTomato and eGFP signals in both left and right ventricular Purkinje fibre networks. Finally, electrophysiological optogenetic study using cell-type specific Channelrhodopsin2 (ChR2) expression further elucidated that Dbh ⁺ -derived CMs form a functional part of the ventricular CCS and display similar photostimulation-induced electrophysiological characteristics to Cx40 CreERT /ChR2-tdTomato CCS components. Thus, by utilizing advanced transcriptomic, mouse genetic, and optogenetic functional analyses, our study provides new insights into mammalian CCS development and heterogeneity by revealing novel Dbh ⁺ -CMs.
Highlights
Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS.
Established the transcriptional landscape of cell types populating the developing murine heart.
Revealed previously unreported catecholaminergic cardiomyocyte populations contributing to the developing and mature murine cardiac conduction system.
... The gap junction protein, connexin 40 (Cx40) expression is uniform in the trabecular myocardium at E11 stage and it becomes restricted to the VCS and atria by E16 through neonatal stages [8,9]. There is evidence that factors secreted from the coronary endothelium, endocardium, and trabecular myocardium (e.g., endothelin-1, neuregulin-1 and atrial natriuretic peptide) provide instructive cues for the VCS cell fate [10][11][12][13]. Although two-dimensional (2-D) cultures of embryonic ventricular cells and embryonic stem cells have been used to study cell proliferation and differentiation patterns [14][15][16][17][18], mechanisms regulating VCS cell formation are not very well characterized. ...
Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells.
... Kahr et al. additionally showed that the Purkinje cells did not exhibit significantly increased cell cycle activity in response to ischemia, but that preexisting cardiomyocytes may be recruited to become new Purkinje cells during the process of natural heart regeneration. RNA sequencing of the neonatal mouse Purkinje cells after MI further revealed strong upregulation of genes involved in inflammation, chemotaxis, and angiogenesis, including Endothelin1, which is known to govern the transdifferentiation of cardiomyocytes into Purkinje cells during embryonic cardiac development (42). These data suggest that, during natural heart regeneration after MI in neonatal mammals, preexisting cardiomyocytes are not only the source of new cardiomyocytes to maintain normal myocardial architecture and ventricular function (20), but potentially also the source of new conducting cells in the His-Purkinje system to maintain normal conduction dynamics as well. ...
Newborn mammals, including piglets, exhibit natural heart regeneration after myocardial infarction (MI) on postnatal day 1 (P1), but this ability is lost by postnatal day 7 (P7). The electrophysiologic properties of this naturally regenerated myocardium have not been examined. We hypothesized that epicardial conduction is preserved after P1 MI in piglets. Yorkshire-Landrace piglets underwent left anterior descending coronary artery ligation at age P1 (n = 6) or P7 (n = 7), After 7 weeks, cardiac magnetic resonance imaging was performed with late gadolinium enhancement for analysis of fibrosis. Epicardial conduction mapping was performed using custom 3D-printed high-resolution mapping arrays. Age- and weight-matched healthy pigs served as controls (n = 6). At the study endpoint, left ventricular (LV) ejection fraction was similar for controls and P1 pigs (46.4 ± 3.0% vs. 40.3 ± 4.9%, p = 0.132), but significantly depressed for P7 pigs (30.2 ± 6.6%, p < 0.001 vs. control). The percentage of LV myocardial volume consisting of fibrotic scar was 1.0 ± 0.4% in controls, 9.9 ± 4.4% in P1 pigs (p = 0.002 vs. control), and 17.3 ± 4.6% in P7 pigs (p < 0.001 vs. control, p = 0.007 vs. P1). Isochrone activation maps and apex activation time were similar between controls and P1 pigs (9.4 ± 1.6 vs. 7.8 ± 0.9 ms, p = 0.649), but significantly prolonged in P7 pigs (21.3 ± 5.1 ms, p < 0.001 vs. control, p < 0.001 vs. P1). Conduction velocity was similar between controls and P1 pigs (1.0 ± 0.2 vs. 1.1 ± 0.4 mm/ms, p = 0.852), but slower in P7 pigs (0.7 ± 0.2 mm/ms, p = 0.129 vs. control, p = 0.052 vs. P1). Overall, our data suggest that epicardial conduction dynamics are conserved in the setting of natural heart regeneration in piglets after P1 MI.
... Most PF markers analyzed to date are expressed in surrounding contractile tissue prior to their restriction to conductive cells including Cx40, Irx3, Etv1, and Sema3a which are expressed in ventricular trabeculae before being progressively restricted to the mature VCS [13,[52][53][54]. Consistent with this model, the chick VCS has been shown to develop by a process of induction and recruitment (ingrowth model) of localized cardiomyocytes through endothelial derived signals, i.e., endothelin, explaining in part why the distal avian VCS, unlike in the mouse, develops next to coronary arteries [55][56][57]. In contrast to chick, specification of the murine cardiac conduction occurs in the absence of endothelin signaling [58,59]. ...
The rapid propagation of electrical activity through the ventricular conduction system (VCS) controls spatiotemporal contraction of the ventricles. Cardiac conduction defects or arrhythmias in humans are often associated with mutations in key cardiac transcription factors that have been shown to play important roles in VCS morphogenesis in mice. Understanding of the mechanisms of VCS development is thus crucial to decipher the etiology of conduction disturbances in adults. During embryogenesis, the VCS, consisting of the His bundle, bundle branches, and the distal Purkinje network, originates from two independent progenitor populations in the primary ring and the ventricular trabeculae. Differentiation into fast-conducting cardiomyocytes occurs progressively as ventricles develop to form a unique electrical pathway at late fetal stages. The objectives of this review are to highlight the structure–function relationship between VCS morphogenesis and conduction defects and to discuss recent data on the origin and development of the VCS with a focus on the distal Purkinje fiber network.
... Concomitant with these events, the "primitive" myocardium is also re-structured, leading to the formation of the major components of the ventricular conduction system, i.e., the atrioventricular node, the bundle of His, the left and right bundle branches and the peripheral Purkinje fiber network [351][352][353][354][355][356][357][358][359][360][361][362][363] (Figure 3B). Several evidence in chicken demonstrate that certain components of the cardiac conduction system were derived from "working" myocardium by endothelin signaling [364][365][366][367][368][369][370][371]. Curiously, such mechanism seems not be involved in mice, where cardiac conduction system and working myocardium display a progressive mechanism of differentiation as revealed by systematic retrospective clonal analyses [372][373][374][375][376][377]. ...
Cardiac development is a complex developmental process that is initiated soon after gastrulation, as two sets of precardiac mesodermal precursors are symmetrically located and subsequently fused at the embryonic midline forming the cardiac straight tube. Thereafter, the cardiac straight tube invariably bends to the right, configuring the first sign of morphological left–right asymmetry and soon thereafter the atrial and ventricular chambers are formed, expanded and progressively septated. As a consequence of all these morphogenetic processes, the fetal heart acquired a four-chambered structure having distinct inlet and outlet connections and a specialized conduction system capable of directing the electrical impulse within the fully formed heart. Over the last decades, our understanding of the morphogenetic, cellular, and molecular pathways involved in cardiac development has exponentially grown. Multiples aspects of the initial discoveries during heart formation has served as guiding tools to understand the etiology of cardiac congenital anomalies and adult cardiac pathology, as well as to enlighten novels approaches to heal the damaged heart. In this review we provide an overview of the complex cellular and molecular pathways driving heart morphogenesis and how those discoveries have provided new roads into the genetic, clinical and therapeutic management of the diseased hearts.
... As in mammals, EDN and EDNR genes have also been predicted or identified in birds and other non-mammalian vertebrate species (Watanabe et al., 1989;Braasch and Schartl, 2014). In birds, there are lines of evidence to suggest that EDN-EDNR signaling plays critical roles in many physiological/pathological processes, such as cardiovascular development (Gourdie et al., 1998;Groenendijk et al., 2008;Kanzawa et al., 2002), angiogenesis (Cruz et al., 2001), pulmonary arterial hypertension (Gomez et al., 2008;Gomez et al., 2007), morphogenesis of the face and heart (Kempf et al., 1998;Nataf et al., 1998), trunk NCCs' proliferation, migration and their final differentiation into the enteric nervous system (Nagy and Goldstein, 2006), or melanocytes (Harris et al., 2008;Lahav et al., 1996;Pla and Larue, 2003), which are likely controlled by EDN3-EDNRB2 signaling essential for pigmentation and plumage patterning in quails (Lecoin et al., 1998;Miwa et al., 2007;Miwa et al., 2006), ducks Wu et al., 2017), and chickens (Dorshorst et al., 2011;Han et al., 2014;Kinoshita et al., 2014;Shinomiya et al., 2012). In zebrafish and Xenopus, EDN-EDNR signaling has also been reported to play important roles in pigmentation and cranial NCC migration and development (Kawasaki-Nishihara et al., 2011;Nair et al., 2007;Spiewak et al., 2018). ...
Endothelins (EDNs) and their receptors (EDNRs) are reported to be involved in the regulation of many physiological/pathological processes, such as cardiovascular development and functions, pulmonary hypertension, neural crest cell proliferation, differentiation and migration, pigmentation, and plumage in chickens. However, the functionality, signaling, and tissue expression of avian EDN-EDNRs have not been fully characterized, thus impeding our comprehensive understanding of their roles in this model vertebrate species. Here, we reported the cDNAs of three EDN genes (EDN1, EDN2, EDN3) and examined the functionality and expression of the three EDNs and their receptors (EDNRA, EDNRB and EDNRB2) in chickens. The results showed that: 1) chicken (c-) EDN1, EDN2, and EDN3 cDNAs were predicted to encode bioactive EDN peptides of 21 amino acids, which show remarkable degree of amino acid sequence identities (91-95%) to their respective mammalian orthologs; 2) chicken (c-) EDNRA expressed in HEK293 cells could be preferentially activated by chicken EDN1 and EDN2, monitored by the three cell-based luciferase reporter assays, indicating that cEDNRA is a functional receptor common for both cEDN1 and cEDN2. In contrast, both cEDNRB and cEDNRB2 could be activated by all three EDN peptides with similar potencies, indicating that both receptors can function as common receptors for the three EDNs and share functional similarity. Moreover, activation of three EDNRs could stimulate intracellular calcium, MAPK/ERK, and cAMP/PKA signaling pathways. 3) qPCR assay revealed that cEDNs and cEDNRs are widely, but differentially, expressed in adult chicken tissues. Taken together, our data establishes a clear molecular basis to uncover the physiological/pathological roles of EDN-EDNR system in birds and helps to reveal the conserved actions of EDN-EDNR signaling across vertebrates.
... Extensive research from multiple groups have clearly shown Notch to regulate not only trabecular formation but also Purkinje cell development via downstream regulators including Neuregulin 1 and its receptor ErbB, Ephrin B2, and BMP10 [34][35][36][37]. Loss of function mutant mice for either RBPJk, a downstream transcriptional repressor in canonical Notch signaling, and to a lesser extent Notch1, results in poorly formed trabeculae and less-structured myocardium [34]. ...
The cardiac conduction system is a network of distinct cell types necessary for the coordinated contraction of the cardiac chambers. The distal portion, known as the ventricular conduction system, allows for the rapid transmission of impulses from the atrio-ventricular node to the ventricular myocardium and plays a central role in cardiac function as well as disease when perturbed. Notably, its patterning during embryogenesis is intimately linked to that of ventricular wall formation, including trabeculation and compaction. Here, we review our current understanding of the underlying mechanisms responsible for the development and maturation of these interdependent processes.
... Myocardium also releases TGF-β, BMP, Wnt, and Notch signals regulating the EMT of cells in the endocardium during valve development [31]. Conduction cells differentiate from a subset of contractile cardiomyocytes in response to paracrine signals including endothelin-1 [34]. Epicardial retinoic acid (RA) activates FGF signalling important for proliferation in compact myocardium and for inducing downstream Wnt signalling promoting EMT for growth of the coronary vasculature. ...
Heart development in mammals is followed by a postnatal decline in cell proliferation and cell renewal from stem cell populations. A better understanding of the developmental changes in cardiac microenvironments occurring during heart maturation will be informative regarding the loss of adult regenerative potential. We reevaluate the adult heart’s mitotic potential and the reported adult cardiac stem cell populations, as these are two topics of ongoing debate. The heart’s early capacity for cell proliferation driven by progenitors and reciprocal signalling is demonstrated throughout development. The mature heart architecture and environment may be more restrictive on niches that can host progenitor cells. The engraftment issues observed in cardiac stem cell therapy trials using exogenous stem cells may indicate a lack of supporting stem cell niches, while tissue injury adds to a hostile microenvironment for transplanted cells. Engraftment may be improved by preconditioning the cultured stem cells and modulating the microenvironment to host these cells. These prospective areas of further research would benefit from a better understanding of cardiac progenitor interactions with their microenvironment throughout development and may lead to enhanced cardiac niche support for stem cell therapy engraftment.
