Placental growth factor exerts a dual function for cardiomyogenesis and vasculogenesis during heart development.

Cardiogenic growth factors play important roles in heart development. Placental growth factor (PLGF) has previously been reported to have angiogenic effects; however, its potential role in cardiogenesis has not yet been determined. We analyze single-cell RNA-sequencing data derived from human and primate embryonic hearts and find PLGF shows a biphasic expression pattern, as it is expressed specifically on ISL1+ second heart field progenitors at an earlier stage and on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) at later stages. Using chemically modified mRNAs (modRNAs), we generate a panel of cardiogenic growth factors and test their effects on enhancing cardiomyocyte (CM) and EC induction during different stages of human embryonic stem cell (hESC) differentiations. We discover that only the application of PLGF modRNA at early time points of hESC-CM differentiation can increase both CM and EC production. Conversely, genetic deletion of PLGF reduces generation of CMs, SMCs and ECs in vitro. We also confirm in vivo beneficial effects of PLGF modRNA for development of human heart progenitor-derived cardiac muscle grafts on murine kidney capsules. Further, we identify the previously unrecognized PLGF-related transcriptional networks driven by EOMES and SOX17. These results shed light on the dual cardiomyogenic and vasculogenic effects of PLGF during heart development.

Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease.

The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration.

Recent Advances in Generation of In Vitro Cardiac Organoids.

Cardiac organoids are in vitro self-organizing and three-dimensional structures composed of multiple cardiac cells (i.e., cardiomyocytes, endothelial cells, cardiac fibroblasts, etc.) with or without biological scaffolds. Since cardiac organoids recapitulate structural and functional characteristics of the native heart to a higher degree compared to the conventional two-dimensional culture systems, their applications, in combination with pluripotent stem cell technologies, are being widely expanded for the investigation of cardiogenesis, cardiac disease modeling, drug screening and development, and regenerative medicine. In this mini-review, recent advances in cardiac organoid technologies are summarized in chronological order, with a focus on the methodological points for each organoid formation. Further, the current limitations and the future perspectives in these promising systems are also discussed.

STRA6 is essential for induction of vascular smooth muscle lineages in human embryonic cardiac outflow tract development.

AIMS: Retinoic acid (RA) signalling is essential for heart development, and dysregulation of the RA signalling can cause several types of cardiac outflow tract (OFT) defects, the most frequent congenital heart disease (CHD) in humans. Matthew-Wood syndrome is caused by inactivating mutations of a transmembrane protein gene STRA6 that transports vitamin A (retinol) from extracellular into intracellular spaces. This syndrome shows a broad spectrum of malformations including CHD, although murine Stra6-null neonates did not exhibit overt heart defects. Thus, the detailed mechanisms by which STRA6 mutations could lead to cardiac malformations in humans remain unclear. Here, we investigated the role of STRA6 in the context of human cardiogenesis and CHD. METHODS AND RESULTS: To gain molecular signatures in species-specific cardiac development, we first compared single-cell RNA sequencing (RNA-seq) datasets, uniquely obtained from human and murine embryonic hearts. We found that while STRA6 mRNA was much less frequently expressed in murine embryonic heart cells derived from the Mesp1+ lineage tracing mice (Mesp1Cre/+; Rosa26tdTomato), it was expressed predominantly in the OFT region-specific heart progenitors in human developing hearts. Next, we revealed that STRA6-knockout human embryonic stem cells (hESCs) could differentiate into cardiomyocytes similarly to wild-type hESCs, but could not differentiate properly into mesodermal nor neural crest cell-derived smooth muscle cells (SMCs) in vitro. This is supported by the population RNA-seq data showing down-regulation of the SMC-related genes in the STRA6-knockout hESC-derived cells. Further, through machinery assays, we identified the previously unrecognized interaction between RA nuclear receptors RARα/RXRα and TBX1, an OFT-specific cardiogenic transcription factor, which would likely act downstream to STRA6-mediated RA signalling in human cardiogenesis. CONCLUSION: Our study highlights the critical role of human-specific STRA6 progenitors for proper induction of vascular SMCs that is essential for normal OFT formation. Thus, these results shed light on novel and human-specific CHD mechanisms, driven by STRA6 mutations.

Progress in Bioengineering Strategies for Heart Regenerative Medicine.

The human heart has the least regenerative capabilities among tissues and organs, and heart disease continues to be a leading cause of mortality in the industrialized world with insufficient therapeutic options and poor prognosis. Therefore, developing new therapeutic strategies for heart regeneration is a major goal in modern cardiac biology and medicine. Recent advances in stem cell biology and biotechnologies such as human pluripotent stem cells (hPSCs) and cardiac tissue engineering hold great promise for opening novel paths to heart regeneration and repair for heart disease, although these areas are still in their infancy. In this review, we summarize and discuss the recent progress in cardiac tissue engineering strategies, highlighting stem cell engineering and cardiomyocyte maturation, development of novel functional biomaterials and biofabrication tools, and their therapeutic applications involving drug discovery, disease modeling, and regenerative medicine for heart disease.

Uncovering the molecular identity of cardiosphere-derived cells (CDCs) by single-cell RNA sequencing.

Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders.

Sequencing of a Chinese tetralogy of Fallot cohort reveals clustering mutations in myogenic heart progenitors.

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole-genome sequencing analysis on 146 nonsyndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this data set with data from a European cohort identified both overlapping and potentially novel gene loci and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene coexpression modules along the cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation.

BACKGROUND: The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. METHODS: A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. RESULTS: Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. CONCLUSION: Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.

Isolation of human ESC-derived cardiac derivatives and embryonic heart cells for population and single-cell RNA-seq analysis.

The combination of population and single-cell RNA sequencing analysis using human embryonic stem cell (hESC) differentiation and developmental tissues is a powerful approach to elucidate an organ-specific cellular and molecular atlas in human embryogenesis. This protocol describes (1) cardiac-directed differentiation and isolation of hESC-derived cardiac derivatives with fluorescence-activated cell sorting, (2) isolation of human embryonic heart-derived single cardiac cells, and (3) construction of cDNA libraries with Smart-seq2. These allow for the preparation of human developmental samples for comprehensive transcriptional analysis. For complete details on the use and execution of this protocol, please refer to Sahara et al. (2019).

Genome-wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors.

Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA-Seq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis.

Cardiac progenitors and paracrine mediators in cardiogenesis and heart regeneration.

The mammalian hearts have the least regenerative capabilities among tissues and organs. As such, heart regeneration has been and continues to be the ultimate goal in the treatment against acquired and congenital heart diseases. Uncovering such a long-awaited therapy is still extremely challenging in the current settings. On the other hand, this desperate need for effective heart regeneration has developed various forms of modern biotechnologies in recent years. These involve the transplantation of pluripotent stem cell-derived cardiac progenitors or cardiomyocytes generated in vitro and novel biochemical molecules along with tissue engineering platforms. Such newly generated technologies and approaches have been shown to effectively proliferate cardiomyocytes and promote heart repair in the diseased settings, albeit mainly preclinically. These novel tools and medicines give somehow credence to breaking down the barriers associated with re-building heart muscle. However, in order to maximize efficacy and achieve better clinical outcomes through these cell-based and/or cell-free therapies, it is crucial to understand more deeply the developmental cellular hierarchies/paths and molecular mechanisms in normal or pathological cardiogenesis. Indeed, the morphogenetic process of mammalian cardiac development is highly complex and spatiotemporally regulated by various types of cardiac progenitors and their paracrine mediators. Here we discuss the most recent knowledge and findings in cardiac progenitor cell biology and the major cardiogenic paracrine mediators in the settings of cardiogenesis, congenital heart disease, and heart regeneration.

In search of the next super models.

The advent of pluripotent stem cell biology and facile genetic manipulation via CRISPR technology has ushered in a new era of human disease models for drug discovery and development. While these precision "super models" hold great promise for tailoring personalized therapy, their full potential and in vivo validation have remained elusive.

Cell-mediated delivery of VEGF modified mRNA enhances blood vessel regeneration and ameliorates murine critical limb ischemia.

Synthetic chemically modified mRNAs (modRNA) encoding vascular endothelial growth factor (VEGF) represents an alternative to gene therapy for the treatment of ischemic cardiovascular injuries. However, novel delivery approaches of modRNA are needed to improve therapeutic efficacy in the diseased setting. We hypothesized that cell-mediated modRNA delivery may enhance the in vivo expression kinetics of VEGF protein thus promoting more potent angiogenic effects. Here, we employed skin fibroblasts as a "proof of concept" to probe the therapeutic potential of a cell-mediated mRNA delivery system in a murine model of critical limb ischemia (CLI). We show that fibroblasts pre-treated with VEGF modRNA have the potential to fully salvage ischemic limbs. Using detailed molecular analysis we reveal that a fibroblast-VEGF modRNA combinatorial treatment significantly reduced tissue necrosis and dramatically improved vascular densities in CLI-injured limbs when compared to control and vehicle groups. Furthermore, fibroblast-delivered VEGF modRNA treatment increased the presence of Pax7+ satellite cells, indicating a possible correlation between VEGF and satellite cell activity. Our study is the first to demonstrate that a cell-mediated modRNA therapy could be an alternative advanced strategy for cardiovascular diseases.

Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract.

The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5+ population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1+TNNT2+ intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations.

Lnc'ed in to Cardiogenesis.

Despite the continuous discovery of long noncoding RNAs (lncRNAs) with critical developmental roles, our knowledge of lncRNAs that control cardiac lineage commitment is still limited. In this issue, Guo et al. (2018) report a novel lncRNA-mediated multiprotein complex assembly that directly regulates the key transcriptional programs of murine cardiogenesis.

Cardiac Progenitor Cells in Basic Biology and Regenerative Medicine.

Major cardiovascular events including myocardial infarction (MI) continue to dominate morbidity rates in the developed world. Although multiple device therapies and various pharmacological agents have been shown to improve patient care and reduce mortality rates, clinicians and researchers alike still lack a true panacea to regenerate damaged cardiac tissue. Over the previous two to three decades, cardiovascular stem cell therapies have held great promise. Several stem cell-based approaches have now been shown to improve ventricular function and are documented in preclinical animal models as well as phase I and phase II clinical trials. More recently, the cardiac progenitor cell has begun to gain momentum as an ideal candidate for stem cell therapy in heart disease. Here, we will highlight the most recent advances in cardiac stem/progenitor cell biology in regard to both the basics and applied settings.