Human Heart Development Research Articles

High resolution single cell analysis of the developing human heart

Abstract The human heart is composed of various cell types with distinct embryonic origins and functions. Having an understanding of the components of the developing heart at a molecular level should provide better insights into its function in health and disease. Current single cell studies of human fetal hearts are limited by low cell numbers and poor cell type resolution. Here, we aimed to increase the number of cells studied while adding a spatial resolution. We analysed human hearts aged between post conception weeks of 8 and 11, where apex and base in each were processed, providing spatial resolution on cells constituting the myocardium and the great vessels. Our analysis suggests 6 main cell types in the developing hearts with a total of 28 subtypes and provides a differential expression signature for similar cell types with different origins and anatomical locations, including the smooth muscle cells and endothelial cells of the myocardium and the great vessels. Through immunohistochemistry, we validated our findings on these subtypes, as well as on the cells that were less clearly described before, such sinoatrial node cells or resident macrophages. Cell to cell interaction analysis inferred distinct molecular crosstalk across subtypes, including the cells of the great vessels or the vessels of the myocardium. Our findings suggest a list of transcription factors that might be involved in governing the identity of these cells. Collectively, our analysis provides a better understanding of human heart development, and will inform studies into disease modelling and disease progression. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Human-gained heart enhancers are associated with species-specific cardiac attributes.

The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers.

Uncovering the origins and lineage markers of human heart fields.

In this issue of Cell Stem Cell, Yang etal. devise a protocol for induction and differentiation of human heart-field precursors, drawing on inspiration from invivo development. Intricate computational analyses uncovered conserved factors governing heart-field segregation, which facilitate enhanced study of human heart development and disease in the dish.

Propeller: testing for differences in cell type proportions in single cell data.

MotivationSingle cell RNA-Sequencing (scRNA-seq) has rapidly gained popularity over the last few years for profiling the transcriptomes of thousands to millions of single cells. This technology is now being used to analyse experiments with complex designs including biological replication. One question that can be asked from single cell experiments, which has been difficult to directly address with bulk RNA-seq data, is whether the cell type proportions are different between two or more experimental conditions. As well as gene expression changes, the relative depletion or enrichment of a particular cell type can be the functional consequence of disease or treatment. However, cell type proportion estimates from scRNA-seq data are variable and statistical methods that can correctly account for different sources of variability are needed to confidently identify statistically significant shifts in cell type composition between experimental conditions.ResultsWe have developed propeller, a robust and flexible method that leverages biological replication to find statistically significant differences in cell type proportions between groups. Using simulated cell type proportions data, we show that propeller performs well under a variety of scenarios. We applied propeller to test for significant changes in cell type proportions related to human heart development, ageing and COVID-19 disease severity.Availability and implementationThe propeller method is publicly available in the open source speckle R package (https://github.com/phipsonlab/speckle). All the analysis code for the article is available at the associated analysis website: https://phipsonlab.github.io/propeller-paper-analysis/. The speckle package, analysis scripts and datasets have been deposited at https://doi.org/10.5281/zenodo.7009042.Supplementary information Supplementary data are available at Bioinformatics online.

De novo spatiotemporal modelling of cell-type signatures in the developmental human heart using graph convolutional neural networks.

With the emergence of high throughput single cell techniques, the understanding of the molecular and cellular diversity of mammalian organs have rapidly increased. In order to understand the spatial organization of this diversity, single cell data is often integrated with spatial data to create probabilistic cell maps. However, targeted cell typing approaches relying on existing single cell data achieve incomplete and biased maps that could mask the true diversity present in a tissue slide. Here we applied a de novo technique to spatially resolve and characterize cellular diversity of in situ sequencing data during human heart development. We obtained and made accessible well defined spatial cell-type maps of fetal hearts from 4.5 to 9 post conception weeks, not biased by probabilistic cell typing approaches. With our analysis, we could characterize previously unreported molecular diversity within cardiomyocytes and epicardial cells and identified their characteristic expression signatures, comparing them with specific subpopulations found in single cell RNA sequencing datasets. We further characterized the differentiation trajectories of epicardial cells, identifying a clear spatial component on it. All in all, our study provides a novel technique for conducting de novo spatial-temporal analyses in developmental tissue samples and a useful resource for online exploration of cell-type differentiation during heart development at sub-cellular image resolution.

Abstract P3101: Chromatin Remodeling BAF (SWI/SNF) Complex Controls Both Cardiogenesis And Neurogenesis From Human Pluripotent Stem Cells

Background: Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. Results: Here, we report ARID1A, a DNA-binding subunit of the BAF(SWI/SNF) epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Loss-of-ARID1A (ARID1A –/– ) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A –/– hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF. Conclusions: We uncover the key and opposite roles by the BAF(SWI/SNF) complex to govern early cardiac and neural development. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A-REST/NRSF interaction.

Abstract EC102: RBPMS, An RNA-binding Protein That Mediates Cardiomyocyte Binucleation And Cardiovascular Development

Noncompaction cardiomyopathy is a common congenital cardiac disorder associated with abnormal ventricular cardiomyocyte trabeculation and impaired pump function. The genetic basis and underlying mechanisms of this disorder remain elusive. We show that genetic deletion of RNA binding protein with multiple splicing (Rbpms), an uncharacterized RNA binding and splicing factor, causes perinatal lethality in mice due to congenital cardiovascular defects, including noncompaction cardiomyopathy, ventricular septal defects, and patent ductus arteriosus. Loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development, thus disturbing embryonic heart growth. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis, and Rbpms expression is decreased in human patients with congenital cardiomyopathy, implicating an essential role of Rbpms in human heart development. Paired-end RNA sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates isoform switching of the heart-specific LIM domain protein Pdlim5. Loss of Rbpms leads to abnormal accumulation of Pdlim5-short isoforms, disrupting cardiomyocyte cytokinesis. Our findings connect premature cardiomyocyte binucleation to noncompaction cardiomyopathy and highlight the role of Rbpms in this process.

Abstract P2064: High Resolution Single Cell Analysis Of The Developing Human Heart

The human heart is composed of various cell types with distinct embryonic origins and functions. Having an understanding of the components of the developing heart at a molecular level should provide better insights into its function in health and disease. Current single cell studies of human fetal hearts are limited by low cell numbers and poor cell type resolution. Here, we aimed to increase the number of cells studied while adding a spatial resolution. We analysed human hearts aged between post conception weeks of 8 and 11, where apex and base in each were processed, providing spatial resolution on cells constituting the myocardium and the great vessels. Our analysis suggests 6 main cell types in the developing hearts with a total of 28 subtypes and provides a differential expression signature for similar cell types with different origins and anatomical locations, including the smooth muscle cells and endothelial cells of the myocardium and the great vessels. Through immunohistochemistry, we validated our findings on these subtypes, as well as on the cells that were less clearly described before, such sinoatrial node cells or resident macrophages. Cell to cell interaction analysis inferred distinct molecular crosstalk across subtypes, including the cells of the great vessels or the vessels of the myocardium. Our findings suggest a list of transcription factors that might be involved in governing the identity of these cells. Collectively, our analysis provides a better understanding of human heart development, and will inform studies into disease modelling and disease progression.

Single-cell atlas of multilineage cardiac organoids derived from human induced pluripotent stem cells

Abstract Human induced pluripotent stem cell (hiPSC)-derived cardiac organoids can be used to model human heart development and cardiovascular disease, and provide therapeutic cells to repair the heart. We used single-cell transcriptome analysis to dissect the development of 3D mini-cardiac organoids (MCOs) consisting of hiPSC-derived cardiomyocytes, and endothelial and smooth muscle cells. We found that the 3D matrix-rich microenvironment significantly promoted the maturation of cardiomyocytes, and mixing endothelial and smooth muscle cells with cardiomyocytes led to the formation of cardiac fibroblast highly expressing DLK1. Modulation of DLK1 signaling affected immunomodulatory gene expression in 2D cultured cardiomyocytes. Transplantation of multilineage MCO into a rat model of myocardial infarction significantly improved cardiac function and reduced fibrosis in the infarcted area. Our single-cell analysis of MCO provided rich information about cell state and fate dynamics in the 3D multilineage microenvironment and brought new insight into the molecular mechanism that promotes cardiomyocyte maturation and heart repair.

Modeling Human Heart Development and Congenital Defects Using Organoids: How Close Are We?

The emergence of human-induced Pluripotent Stem Cells (hiPSCs) has dramatically improved our understanding of human developmental processes under normal and diseased conditions. The hiPSCs have been differentiated into various tissue-specific cells in vitro, and the advancement in three-dimensional (3D) culture has provided a possibility to generate those cells in an in vivo-like environment. Tissues with 3D structures can be generated using different approaches such as self-assembled organoids and tissue-engineering methods, such as bioprinting. We are interested in studying the self-assembled organoids differentiated from hiPSCs, as they have the potential to recapitulate the in vivo developmental process and be used to model human development and congenital defects. Organoids of tissues such as those of the intestine and brain were developed many years ago, but heart organoids were not reported until recently. In this review, we will compare the heart organoids with the in vivo hearts to understand the anatomical structures we still lack in the organoids. Specifically, we will compare the development of main heart structures, focusing on their marker genes and regulatory signaling pathways.

PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

Hemodynamic and Structural Comparison of Human Fetal Heart Development Between Normally Growing and Hypoplastic Left Heart Syndrome-Diagnosed Hearts.

Congenital heart defects (CHDs) affect a wide range of societies with an incidence rate of 1.0–1.2%. These defects initiate at the early developmental stage and result in critical health disorders. Although genetic factors play a role in the formation of CHDs, the occurrence of cases in families with no history of CHDs suggests that mechanobiological forces may also play a role in the initiation and progression of CHDs. Hypoplastic left heart syndrome (HLHS) is a critical CHD, which is responsible for 25–40% of all prenatal cardiac deaths. The comparison of healthy and HLHS hearts helps in understanding the main hemodynamic differences related to HLHS. Echocardiography is the most common imaging modality utilized for fetal cardiac assessment. In this study, we utilized echocardiographic images to compare healthy and HLHS human fetal hearts for determining the differences in terms of heart chamber dimensions, valvular flow rates, and hemodynamics. The cross-sectional areas of chamber dimensions are determined from 2D b-mode ultrasound images. Valvular flow rates are measured via Doppler echocardiography, and hemodynamic quantifications are performed with the use of computational fluid dynamics (CFD) simulations. The obtained results indicate that cross-sectional areas of the left and right sides of the heart are similar for healthy fetuses during gestational development. The left side of HLHS heart is underdeveloped, and as a result, the hemodynamic parameters such as flow velocity, pressure, and wall shear stress (WSS) are significantly altered compared to those of healthy hearts.

Advances in Manufacturing Cardiomyocytes from Human Pluripotent Stem Cells.

The emergence of human pluripotent stem cell (hPSC) technology over the past two decades has provided a source of normal and diseased human cells for a wide variety of in vitro and in vivo applications. Notably, hPSC-derived cardiomyocytes (hPSC-CMs) are widely used to model human heart development and disease and are in clinical trials for treating heart disease. The success of hPSC-CMs in these applications requires robust, scalable approaches to manufacture large numbers of safe and potent cells. Although significant advances have been made over the past decade in improving the purity and yield of hPSC-CMs and scaling the differentiation process from 2D to 3D, efforts to induce maturation phenotypes during manufacturing have been slow. Process monitoring and closed-loop manufacturing strategies are just being developed. We discuss recent advances in hPSC-CM manufacturing, including differentiation process development and scaling and downstream processes as well as separation and stabilization.

Restoring Ravaged Heart: Molecular Mechanisms and Clinical Application of miRNA in Heart Regeneration.

Human heart development is a complex and tightly regulated process, conserving proliferation, and multipotency of embryonic cardiovascular progenitors. At terminal stage, progenitor cell type gets suppressed for terminal differentiation and maturation. In the human heart, most cardiomyocytes are terminally differentiated and so have limited proliferation capacity. MicroRNAs (miRNAs) are non-coding single-stranded RNA that regulate gene expression and mRNA silencing at the post-transcriptional level. These miRNAs play a crucial role in numerous biological events, including cardiac development, and cardiomyocyte proliferation. Several cardiac cells specific miRNAs have been discovered. Inhibition or overexpression of these miRNAs could induce cardiac regeneration, cardiac stem cell proliferation and cardiomyocyte proliferation. Clinical application of miRNAs extends to heart failure, wherein the cell cycle arrest of terminally differentiated cardiac cells inhibits the heart regeneration. The regenerative capacity of the myocardium can be enhanced by cardiomyocyte specific miRNAs controlling the cell cycle. In this review, we focus on cardiac-specific miRNAs involved in cardiac regeneration and cardiomyocyte proliferation, and their potential as a new clinical therapy for heart regeneration.

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.

Abstract 9102: Cytosolic and Nuclear LncRNA HBL1 Crosstalk to Orchestrate Human Cardiogenesis via Interactions With MicroRNA-1 and PRC2 Epigenetic Complex

Introduction: Compared to rodents, human embryos exhibit unique dynamics of cardiogenesis, suggesting the existence of novel human-specific genetic programs underlying early-stage human heart development. Due to lack of in vivo model, human-specific mechanisms underlying cardiogenesis remain elusive. Hypothesis: Human Heart Brake LncRNA 1 (HBL1) functions as a new layer of regulator controlling cardiogenic gene transcription via coordinating with microRNA-1 and PRC2 epigenetic complex. Methods: Human pluripotent stem cells (hPSCs) were utilized to model human cardiogenesis. CRISPR/Cas-9 was utilized to knockout HBL1 and coding genes, and disrupted microRNA-1 binding sites on HBL1. HBL1 interacting proteins were pulled down by Co-IP-MS. EMSA and RNA-IP tested HBL1 interactions with PRC2 subunits. ChIP-seq uncovered the impact of HBL1 on genome-wide PRC2 occupancy. Luciferase assay confirmed JARID2 is a target of microRNA-1. Results: HBL1 expresses in cytoplasm and nucleus of hPSCs. Gain-of-HBL1 represses, whereas loss-of-HBL1 increases cardiomyocyte differentiation from hPSCs. Cytosolic HBL1 modulates cardiac development from hPSCs by counteracting microRNA-1. In hPSCs, disruption of microRNA-1 binding sites on HBL1 phenocopies HBL1 deficiency. Nuclear HBL1 interacts with two PRC2 subunits, JARID2 and EED. Loss-of-JARID2 or EED phenocopies HBL1 deficiency to enhance cardiac differentiation from hPSCs. HBL1 deficiency disrupts genome-wide PRC2 occupancy and H3K27me3 deposition on essential cardiogenic genes, and consequently enhances their transcription in hPSCs. Both HBL1 and JARID2 are required for PRC2 deposition to determine the epigenetic state of critical cardiogenic genes in hPSCs. Furthermore, the cytosolic and nuclear fractions of HBL1 could crosstalk via a conserved “microRNA-1-JARID2” axis to modulate transcription of cardiogenic genes. Conclusions: Our findings delineate the indispensable role of human-specific HBL1 in early human cardiogenesis, and expand the mechanistic scope of lncRNA(s) that cytosolic and nuclear portions of HBL1 could coordinate to precisely control human cardiogenesis.

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

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. 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. 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. Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs. The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

In vitro models of the human heart.

Cardiac congenital disabilities are the most common organ malformations, but we still do not understand how they arise in the human embryo. Moreover, although cardiovascular disease is the most common cause of death globally, the development of new therapies is lagging compared with other fields. One major bottleneck hindering progress is the lack of self-organizing human cardiac models that recapitulate key aspects of human heart development, physiology and disease. Current in vitro cardiac three-dimensional systems are either engineered constructs or spherical aggregates of cardiomyocytes and other cell types. Although tissue engineering enables the modeling of some electro-mechanical properties, it falls short of mimicking heart development, morphogenetic defects and many clinically relevant aspects of cardiomyopathies. Here, we review different approaches and recent efforts to overcome these challenges in the field using a new generation of self-organizing embryonic and cardiac organoids.

Myosin light chain 2 marks differentiating ventricular cardiomyocytes derived from human embryonic stem cells.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great value for studies of human cardiac development, drug discovery, disease modeling, and cell therapy. However, the mixed cardiomyocyte subtypes (ventricular-, atrial-, and nodal-like myocytes) and the maturation heterogeneity of hPSC-CMs restrain their application in vitro and in vivo. Myosin light chain 2 (MYL2, encoding the ventricular/cardiac muscle isoform MLC2v protein) is regarded as a ventricular-specific marker of cardiac myocardium; however, its restricted localization to ventricles during human heart development has been questioned. Consequently, it is currently unclear whether MYL2 definitively marks ventricular hESC-CMs. Here, by using a MYL2-Venus hESC reporter line, we characterized a time-dependent increase of the MYL2-Venus positive (MLC2v-Venus+) hESC-CMs during differentiation. We also compared the molecular, cellular, and functional properties between the MLC2v-Venus+ and MYL2-Venus negative (MLC2v-Venus-) hESC-CMs. At early differentiation stages of hESC-CMs, we reported that both MLC2v-Venus- and MLC2v-Venus+ CMs displayed ventricular-like traits but the ventricular-like cells from MLC2v-Venus+ hESC-CMs displayed more developed action potential (AP) properties than that from MLC2v-Venus- hESC-CMs. Meanwhile, about a half MLC2v-Venus- hESC-CM population displayed atrial-like AP properties, and a half showed ventricular-like AP properties, whereas only ~ 20% of the MLC2v-Venus- hESC-CMs expressed the atrial marker nuclear receptor subfamily 2 group F member 2 (NR2F2, also named as COUPTFII). At late time points, almost all MLC2v-Venus+ hESC-CMs exhibited ventricular-like AP properties. Further analysis demonstrates that the MLC2v-Venus+ hESC-CMs had enhanced Ca2+ transients upon increase of the MLC2v level during cultivation. Concomitantly, the MLC2v-Venus+ hESC-CMs showed more defined sarcomeric structures and better mitochondrial function than those in the MLC2v-Venus- hESC-CMs. Moreover, the MLC2v-Venus+ hESC-CMs were more sensitive to hypoxic stimulus than the MLC2v-Venus- hESC-CMs. These results provide new insights into the development of human ventricular myocytes and reveal a direct correlation between the expression profile of MLC2v and ventricular hESC-CM development. Our findings that MLC2v is predominantly a ventricular marker in developmentally immature hESC-CMs have implications for human development, drug screening, and disease modeling, and this marker should prove useful in overcoming issues associated with hESC-CM heterogeneity.