Cardiac Cell Lineages Research Articles

Expandable hESC-derived cardiovascular progenitor cells generate functional cardiac lineage cells for microtissue construction

BackgroundCardiovascular progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) are considered valuable cell sources for investigating cardiovascular physiology in vitro. Meeting the diverse needs of this application requires the large-scale production of CPCs in an in vitro environment. This study aimed to use an effective culture system utilizing signaling factors for the large-scale expansion of hESC-derived CPCs with the potential to differentiate into functional cardiac lineage cells.Methods and resultsInitially, CPCs were generated from hESCs using a 4-day differentiation protocol with a combination of four small molecules (CHIR99021, IWP2, SB-431542, and purmorphamine). These CPCs were then expanded and maintained in a medium containing three factors (bFGF, CHIR, and A83-01), resulting in a > 6,000-fold increase after 8 passages. These CPCs were successfully cryopreserved for an extended period in late passages. The expanded CPCs maintained their gene and protein expression signatures as well as their differentiation capacity through eight passages. Additionally, these CPCs could differentiate into four types of cardiac lineage cells: cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts, demonstrating appropriate functionality. Furthermore, the coculture of these CPC-derived cardiovascular lineage cells in rat tail collagen resulted in cardiac microtissue formation, highlighting the potential of this 3D platform for studying cardiovascular physiology in vitro.ConclusionIn conclusion, expandable hESC-derived CPCs demonstrated the ability to self-renewal and differentiation into functional cardiovascular lineage cells consistently across passages, which may apply as potential cell sources for in vitro cardiovascular studies.Graphical

Abstract We091: Maternal hyperglycemia disrupts human cardiac cell lineage differentiation

Maternal diabetes is associated with a 4-fold increased risk of offspring developing congenital heart defects (CHDs). It remains unknown how maternal diabetes interferes with cardiac cell lineage determination and leads to malformations in the heart. In this study, we aim to elucidate the cellular mechanisms by which maternal hyperglycemia causes high risk of CHDs in newborns. Here we leverage an in vitro hyperglycemic model using human induced pluripotent stem cells (iPSCs), which could recapture cardiac differentiation and cell lineage commitment during embryonic heart development. We collected differentiating cells at D5 (cardiac mesoderm), D10 (cardiac progenitors), and D14 (early cardiomyocytes) during cardiac differentiation under normal and hyperglycemic conditions, and performed single-cell transcriptomic analysis. We found that hyperglycemia significantly impedes cardiac differentiation of human iPSCs as robust cardiac differentiation is rarely observed in multiple iPSC lines (n=10) under hyperglycemia. At the cellular level, hyperglycemia interferes with cardiac differentiation of human iPSCs in response to WNT signaling activation, which is manifested by reduced number of cardiac mesoderm (MESP1+ PDGFRA+) cells at D5 of differentiation. In contrast, neural differentiation is enhanced under hyperglycemia, with a high proportion of neural cell lineage (SOX2+ PAX6+). At D10, differentiated neural cell lineage dominates the cell population under hyperglycemia at the expense of cardiac progenitors and early cardiomyocytes. At D14, the prevalence of neural lineage persist whereas early cardiomyocytes only accounts for a small portion of cell population under hyperglycemia. Moreover, we treated D30 iPSC-derived cardiomyocytes (iPSC-CMs) with high glucose concentration (25 mM) for 7 days and found that iPSC-CMs show reduced mitochondria respiration and ATP production, but elevated apoptosis and reactive oxygen species (ROS) generation. Together, our data suggest that maternal hyperglycemia could interrupt human embryonic heart morphogenesis through overriding WNT-medicated cardiac differentiation and promoting neural cell lineage determination by default.

Abstract We041: In vitro characterization of different human-induced pluripotent stem cell-derived cardiac progenitors

Introduction: Myocardial Infarction (MI) is the number one cause of death in the United States. The heart undergoes severe trauma during MI, which results in the death of cardiac cells. Although the cardiac tissue cannot regenerate on its own, regenerative medicine approaches involving cell transplantation have been shown to improve the cardiac function. Among different cell types, human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSCs) have been shown to have the ability to proliferate and differentiate into cardiac lineage cells and therefore, are a promising source for myocardial repair. Here, we characterized two distinct populations of hiPSC-CPCs and assessed their differentiation potential. Methods: We compared hiPSC-CPCs differentiated using the small molecule, isoxazole-9 (ISX-9), ISX9-hiPSC-CPCs with the commercially acquired iCell hiPSC-CPCs (Fujifilm Cellular Dynamics Int.). The expression of different cardiac lineage markers were assessed via immunostaining, qPCR and flow cytometry. Furthermore, we assessed the proliferation of the cells using immunostaining and XTT assays. We also evaluated their in vitro differentiation to cardiomyocytes, via immunostaining and qPCR analysis. Results: Our studies showed similar expression of different cardiac progentior markers (Nkx2.5, GATA4, KFL-4, ISL-1, BRY-T, MESP-1, MYH-6, CTnT) in the two hiPSC-CPC populations. However, while the iCell hiPSC-CPCs predominantly expressed the cell surface marker KDR, the ISX-9-hiPSC-CPCs predominantly expressed SSEA-1. The iCell-hiPSC-CPCs showed higher proliferation rates when compared to the ISX-9-hiPSC-CPCs. However, >95% of both the cell populations expressed the proliferation marker, ki67. Finally, the while both the CPC populations could differentiate into cardiomyocytes (cTNT + ), only a few of these differentiated cardiomyocytes showed spontaneous contractility in vitro. Conclusion: In our study, we have compared and characterized two distinct populations of hiPSC-CPCs to identify a promising candidate for myocardial regeneration therapies.

ScRNA-seq reveals the diversity of the developing cardiac cell lineage and molecular players in heart rhythm regulation

We utilized scRNA-seq to delineate the diversity of cell types in the zebrafish heart. Transcriptome profiling of over 50,000 cells at 48 and 72 hpf defined at least 18 discrete cell lineages of the developing heart. Utilizing well-established gene signatures, we identified a population of cells likely to be the primary pacemaker and characterized the transcriptome profile defining this critical cell type. Two previously uncharacterized genes, atp1b3b and colec10, were found to be enriched in the sinoatrial cardiomyocytes. CRISPR/Cas9-mediated knockout of these two genes significantly reduced heart rate, implicating their role in cardiac development and conduction. Additionally, we describe other cardiac cell lineages, including the endothelial and neural cells, providing their expression profiles as a resource. Our results established a detailed atlas of the developing heart, providing valuable insights into cellular and molecular mechanisms, and pinpointed potential new players in heart rhythm regulation.

Contribution of VEGF-B-Induced Endocardial Endothelial Cell Lineage in Physiological Versus Pathological Cardiac Hypertrophy.

Preclinical studies have shown the therapeutic potential of VEGF-B (vascular endothelial growth factor B) in revascularization of the ischemic myocardium, but the associated cardiac hypertrophy and adverse side effects remain a concern. To understand the importance of endothelial proliferation and migration for the beneficial versus adverse effects of VEGF-B in the heart, we explored the cardiac effects of autocrine versus paracrine VEGF-B expression in transgenic and gene-transduced mice. We used single-cell RNA sequencing to compare cardiac endothelial gene expression in VEGF-B transgenic mouse models. Lineage tracing was used to identify the origin of a VEGF-B-induced novel endothelial cell population and adeno-associated virus-mediated gene delivery to compare the effects of VEGF-B isoforms. Cardiac function was investigated using echocardiography, magnetic resonance imaging, and micro-computed tomography. Unlike in physiological cardiac hypertrophy driven by a cardiomyocyte-specific VEGF-B transgene (myosin heavy chain alpha-VEGF-B), autocrine VEGF-B expression in cardiac endothelium (aP2 [adipocyte protein 2]-VEGF-B) was associated with septal defects and failure to increase perfused subendocardial capillaries postnatally. Paracrine VEGF-B led to robust proliferation and myocardial migration of a novel cardiac endothelial cell lineage (VEGF-B-induced endothelial cells) of endocardial origin, whereas autocrine VEGF-B increased proliferation of VEGF-B-induced endothelial cells but failed to promote their migration and efficient contribution to myocardial capillaries. The surviving aP2-VEGF-B offspring showed an altered ratio of secreted VEGF-B isoforms and developed massive pathological cardiac hypertrophy with a distinct cardiac vessel pattern. In the normal heart, we found a small VEGF-B-induced endothelial cell population that was only minimally expanded during myocardial infarction but not during physiological cardiac hypertrophy associated with mouse pregnancy. Paracrine and autocrine secretions of VEGF-B induce expansion of a specific endocardium-derived endothelial cell population with distinct angiogenic markers. However, autocrine VEGF-B signaling fails to promote VEGF-B-induced endothelial cell migration and contribution to myocardial capillaries, predisposing to septal defects and inducing a mismatch between angiogenesis and myocardial growth, which results in pathological cardiac hypertrophy.

Cardiac Progenitor Cells of the First and Second Heart Fields.

The heart forms from the first and second heart fields, which contribute to distinct regions of the myocardium. This is supported by clonal analyses, which identify corresponding first and second cardiac cell lineages in the heart. Progenitor cells of the second heart field and its sub-domains are controlled by a gene regulatory network and signaling pathways, which determine their behavior. Multipotent cells in this field can also contribute cardiac endothelial and smooth muscle cells. Furthermore, the skeletal muscles of the head and neck are clonally related to myocardial cells that form the arterial and venous poles of the heart. These lineage relationships, together with the genes that regulate the heart fields, have major implications for congenital heart disease.

Human Stem Cell-Derived Cardiomyocytes Integrate Into the Heart of Monkeys With Right Ventricular Pressure Overload.

Cardiac ventricular pressure overload affects patients with congenital heart defects and can cause cardiac insufficiency. Grafts of stem cell-derived cardiomyocytes are proposed as a complementary treatment to surgical repair of the cardiac defect, aiming to support ventricular function. Here, we report successful engraftment of human induced pluripotent stem cell-derived cardiac lineage cells into the heart of immunosuppressed rhesus macaques with a novel surgical model of right ventricular pressure overload. The human troponin+ grafts were detected in low-dose (2 × 106 cells/kg) and high-dose (10 × 106 cells/kg) treatment groups up to 12 weeks post-injection. Transplanted cells integrated and progressively matched the organization of the surrounding host myocardium. Ventricular tachycardia occurred in five out of 16 animals receiving cells, with episodes of incessant tachycardia observed in two animals; ventricular tachycardia events resolved within 19 days. Our results demonstrate that grafted cardiomyocytes mature and integrate into the myocardium of nonhuman primates modeling right ventricular pressure overload.

Single-cell transcriptomics reveals neural stem cell trans-differentiation and cell subpopulations in whole heart decellularized extracellular matrix.

The whole heart decellularized extracellular matrix (ECM) has become a promising scaffold material for cardiac tissue engineering. Our previous research has shown that the whole heart acellular matrix possesses the memory function regulating neural stem cells (NSCs) trans-differentiating to cardiac lineage cells. However, the cell subpopulations and phenotypes in the trans-differentiation of NSCs have not been clearly identified. Here, we performed single-cell RNA sequencing and identified 2,765 cells in the recellularized heart with NSCs revealing the cellular diversity of cardiac and neural lineage, confirming NSCs were capable of trans-differentiating into the cardiac lineage while maintaining the original ability to differentiate into the neural lineage. Notably, the trans-differentiated heart-like cells have dual signatures of neuroectoderm and cardiac mesoderm. This study unveils an in-depth mechanism underlying the trans-differentiation of NSCs and provides a new opportunity and theoretical basis for cardiac regeneration.

The mesencephalic astrocyte-derived neurotrophic factor (MANF) as a novel regulator of post-infarction cardiac sympathetic hyperinnervation

Abstract Background Excessive outgrowth of sympathetic nerve fibers (sympathetic hyperinnervation) around the infarcted myocardium is associated with ventricular arrhythmia and an increased risk of sudden cardiac death. Post-infarction, epicardial cells exit quiescence through epithelial-to-mesenchymal transition (EMT), resulting in the formation of epicardium-derived cells (EPDCs). EPDCs support cardiac repair through differentiation into various cardiac cell lineages, as well as secretion of paracrine factors that stimulate processes such as angiogenesis. Recently, we demonstrated that human EPDCs reinforce neurite outgrowth in vitro. Whether EPDCs regulate cardiac innervation through paracrine signaling and contribute to post-infarction sympathetic hyperinnervation is unknown. Purpose To identify epicardial paracrine factors post-infarction that are involved in nerve growth. Methods Single-cell RNA-sequencing (scRNA-seq) datasets capturing epicardial cells of the healthy and infarcted adult mouse heart were collected from published research articles and were integrated into one object using Seurat. A list of gene products of known involvement in biological processes related to nerve growth (675 unique entries in total) was compiled. Based on this list, differential gene expression analysis on data from the integrated scRNA-seq object was performed to uncover highly-expressed candidates in the epicardial cell population. Epicardial expression of a candidate paracrine factor was validated by immunohistochemistry in the infarcted mouse heart as well as in an adult human in vitro model of EPDC EMT by immunocytochemistry and transcriptomic analysis. Results In our integrated scRNA-seq object, we identified high expression of Manf in epicardial cells post-infarction. Consistent with this finding, immunohistochemistry showed that Manf was up-regulated in the infarcted area, borderzone area, and epicardial region situated close to the infarct, an area where also hyperinnervation is observed. In vitro, MANF transcript expression was significantly down-regulated (p<0.01) in human EPDCs that were stimulated with TGFβ3 to induce EMT compared to EPDCs in which EMT was inhibited. In line with this, immunocytochemistry staining of MANF indicated that MANF expression was down-regulated in human EPDCs stimulated with TGFβ3. Conclusion(s) This study is the first to show that Manf is highly expressed in the epicardial region close to the infarct. In vitro, MANF is down-regulated in human adult EPDCs post-EMT. Future experiments will be directed toward determining the effect of MANF knockdown in human EPDC on neurite outgrowth in co-cultured human sympathetic neurons.MANF expression in human EPDCs after EMTManf expression after infarction

Use of Stem Cells to Treat Left Ventricular Hypertrophy

Left Ventricular Hypertrophy is a multifactorial condition involving genetic and signaling pathways. Current recommendations for treating hypertensive LVH include ACE inhibitors, RAS and CCBs. Despite numerous drug therapies for ventricular hypertrophy, patients often deteriorate and experience a decline in ventricular function ultimately leading to heart failure. For this reason, alternative therapies must be investigated. Stem cells possess the potential to divide at a high capacity, self-renew and differentiate into numerous cardiac cell lineages. Furthermore, stem cells are connected to ventricular remodeling and are vital in maintaining the homeostasis of the heart. Further examination of the role of stem cells in LVH could result in the development of targeted therapeutic interventions for prevention or reversal of the phenomenon.

Xenograft of human pluripotent stem cell-derived cardiac lineage cells on zebrafish embryo heart

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) are a promising cell source for regenerative medicine and drug discovery. However, the use of animal models for studying human cardiomyocytes derived from hiPSCs in vivo is limited and challenging. Given the shared properties between humans and zebrafish, their ethical advantages over mammalian models, and their immature immune system that is rejection-free against xenografted human cells, zebrafish provide a suitable alternative model for xenograft studies. We microinjected fluorescence-labeled cardiac lineage cells derived from hiPSCs, specifically mesoderm or cardiac mesoderm cells, into the yolk and the area proximal to the outflow tract of the linear heart at 24 hours post-fertilization (hpf). The cells injected into the yolk survived and did not migrate to other tissues. In contrast, the cells injected contiguous with the outflow tract of the linear heart migrated into the pericardial cavity and heart. After 1 day post injection (1 dpi, 22–24 hpi), the injected cells migrated into the pericardial cavity and heart. Importantly, we observed heartbeat-like movements of some injected cells in the zebrafish heart after 1 dpi. These results suggested successful xenografting of hiPSC-derived cardiac lineage cells into the zebrafish embryo heart. Thus, we developed a valuable tool using zebrafish embryos as a model organism for investigating the molecular and cellular mechanisms involved in the grafting process. This is essential in developing cell transplantation-based cardiac therapeutics as well as for drug testing, notably contributing to advancements in the field of cardio-medicine.

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.

Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.

NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.

Polycomb repressive complex 2 controls cardiac cell fate decision via interacting with RNA: Promiscuously or well-ordered.

The epigenetic landscape determines cell fate during heart development. Polycomb repressive complex 2 (PRC2) mediates histone methyltransferase activity during cardiac cell differentiation. The PRC2 complex contains the proteins embryonic ectoderm development (EED), suppressor of zeste (SUZ12), the chromatin assembly factor 1 (CAF1) histone-binding proteins RBBP4 and RBBP7, and the histone methyltransferase called enhancer of zeste (EZH2 or EZH1), which incorporates the Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain. Cardiac PRC2-deficient mice display lethal congenital heart malformations. The dynamic process of cardiac cell fate decisions is controlled by PRC2 and the PRC2-mediated epigenetic landscape. Although specific individual long noncoding RNAs (lncRNAs) including Braveheart were widely reported to regulate the recruitments of PRC2 to their specific targets, a promiscuous RNA binding profile by PRC2 was also identified to play an essential role in cardiac cell fate decision. In this review, we focus on RNA-mediated PRC2 recruitment machinery in the process of cardiac cell fate decisions. The roles of individual lncRNAs which recruit PRC2, as well as promiscuous RNA binding by PRC2 in heart development are summarized. Since the binding priority of RNAs with different primary and secondary structures differs in its affinity to PRC2, the competitive relationship between individual lncRNAs binding and promiscuous RNA binding by PRC2 may be important for understanding the machinery by which biding of individual lncRNA and promiscuous RNA by PRC2 coordinately control the well-ordered dynamic cardiac cell lineage differentiation process.

Overexpression of PYGO1 promotes early cardiac lineage development in human umbilical cord mesenchymal stromal/stem cells by activating the Wnt/β-catenin pathway.

Cardiovascular disease still has the highest mortality. Gene-modified mesenchymal stromal/stem cells could be a promising therapy. Pygo plays an important role in embryonic development and regulates life activities with a variety of regulatory mechanisms. Therefore, this study aimed to investigate whether the overexpression of the PYGO1 gene can promote the differentiation of human umbilical cord-derived mesenchymal stromal/stem cells (HUC-MSCs) into early cardiac lineage cells and to preliminary explore the relevant mechanisms. In this study, HUC-MSCs were isolated by the explant method and were identified by flow cytometry and differentiation assay, followed by transfected with lentivirus carrying the PYGO1 plasmid. In PYGO1 group (cells were incubated with lentiviral-PYGO1), the mRNA expressions of cardiac differentiation-specific markers (MESP1, NKX2.5, GATA4, MEF2C, ISL1, TBX5, TNNT2, ACTC1, and MYH6 genes) and the protein expressions of NKX2.5 and cTnT were significantly up-regulated compared with the NC group (cells were incubated with lentiviral-empty vector). In addition, the proportion of NKX2.5, GATA4, and cTnT immunofluorescence-positive cells increased with the inducement time. Overexpression of PYGO1 statistically significantly increased the relative luciferase expression level of Topflash plasmid, the protein expression level of β-catenin and the mRNA expression level of CYCLIND1. Compared with the control group, decreased protein levels of NKX2.5 and cTnT were detected in PYGO1 group after application of XAV-939, the specific inhibitor of the canonical Wnt/β-catenin pathway. Our study suggests that overexpression of PYGO1 significantly promotes the differentiation of HUC-MSCs into early cardiac lineage cells, which is regulated by the canonical Wnt/β-catenin signaling.

SRF-derived miR210 and miR30c both repress beating cardiomyocyte formation in the differentiation system of embryoid body

Serum response factor (SRF) cooperates with various co-factors to manage the specification of diverse cell lineages during heart development. Many microRNAs mediate the function of SRF in this process. However, how are miR210 and miR30c involved in the decision of cardiac cell fates remains to be explored. In this study, we found that SRF directly controlled the cardiac expression of miR210. Both miR210 and miR30c blocked the formation of beating cardiomyocyte during embryoid body (EB) differentiation, a cellular model widely used for studying cardiogenesis. Both of anticipated microRNA targets and differentially expressed genes in day8 EBs were systematically determined and enriched with gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and Reactome. Functional enrichments of prediction microRNA targets and down-regulated genes in day8 EBs of miR210 suggested the importance of PI3K-Akt signal and ETS2 in miR210 inhibition of cardiomyocyte differentiation. Similar analyses revealed that miR30c repressed both developmental progress and the adrenergic signaling in cardiomyocytes during the differentiation of EBs. Taken together, SRF directs the expression of miR210 and miR30c, and they repress cardiac development via inhibiting the differentiation of cardiac muscle cell lineage as well as the cell proliferation. Through the regulation of specific microRNAs, the complication of SRF's function in heart development is emphasized.

Application of Three-Dimensional Culture Method in the Cardiac Conduction System Research.

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.

The Vascular Niche for Adult Cardiac Progenitor Cells.

Research on cardiac progenitor cell populations has generated expectations about their potential for cardiac regeneration capacity after acute myocardial infarction and during physiological aging; however, the endogenous capacity of the adult mammalian heart is limited. The modest efficacy of exogenous cell-based treatments can guide the development of new approaches that, alone or in combination, can be applied to boost clinical efficacy. The identification and manipulation of the adult stem cell environment, termed niche, will be critical for providing new evidence on adult stem cell populations and improving stem-cell-based therapies. Here, we review and discuss the state of our understanding of the interaction of adult cardiac progenitor cells with other cardiac cell populations, with a focus on the description of the B-CPC progenitor population (Bmi1+ cardiac progenitor cell), which is a strong candidate progenitor for all main cardiac cell lineages, both in the steady state and after cardiac damage. The set of all interactions should be able to define the vascular cardiac stem cell niche, which is associated with low oxidative stress domains in vasculature, and whose manipulation would offer new hope in the cardiac regeneration field.

CCBE1 in Cardiac Development and Disease.

The collagen- and calcium-binding EGF-like domains 1 (CCBE1) is a secreted protein extensively described as indispensable for lymphangiogenesis during development enhancing VEGF-C signaling. In human patients, mutations in CCBE1 have been found to cause Hennekam syndrome, an inherited disease characterized by malformation of the lymphatic system that presents a wide variety of symptoms such as primary lymphedema, lymphangiectasia, and heart defects. Importantly, over the last decade, an essential role for CCBE1 during heart development is being uncovered. In mice, Ccbe1 expression was initially detected in distinct cardiac progenitors such as first and second heart field, and the proepicardium. More recently, Ccbe1 expression was identified in the epicardium and sinus venosus (SV) myocardium at E11.5–E13.5, the stage when SV endocardium–derived (VEGF-C dependent) coronary vessels start to form. Concordantly, CCBE1 is required for the correct formation of the coronary vessels and the coronary artery stem in the mouse. Additionally, Ccbe1 was found to be enriched in mouse embryonic stem cells (ESC) and revealed as a new essential gene for the differentiation of ESC-derived early cardiac precursor cell lineages. Here, we bring an up-to-date review on the role of CCBE1 in cardiac development, function, and human disease implications. Finally, we envisage the potential of this molecule’s functions from a regenerative medicine perspective, particularly novel therapeutic strategies for heart disease.

From engineered heart tissue to cardiac organoid.

The advent of human pluripotent stem cells (hPSCs) presented a new paradigm to employ hPSC-derived cardiomyocytes (hPSC-CMs) in drug screening and disease modeling. However, hPSC-CMs differentiated in conventional two-dimensional systems are structurally and functionally immature. Moreover, these differentiation systems generate predominantly one type of cell. Since the heart includes not only CMs but other cell types, such monolayer cultures have limitations in simulating the native heart. Accordingly, three-dimensional (3D) cardiac tissues have been developed as a better platform by including various cardiac cell types and extracellular matrices. Two advances were made for 3D cardiac tissue generation. One type is engineered heart tissues (EHTs), which are constructed by 3D cell culture of cardiac cells using an engineering technology. This system provides a convenient real-time analysis of cardiac function, as well as a precise control of the input/output flow and mechanical/electrical stimulation. The other type is cardiac organoids, which are formed through self-organization of differentiating cardiac lineage cells from hPSCs. While mature cardiac organoids are more desirable, at present only primitive forms of organoids are available. In this review, we discuss various models of hEHTs and cardiac organoids emulating the human heart, focusing on their unique features, utility, and limitations.