Cardiomyocyte Differentiation Research Articles

Polycaprolactone-co-polylactic acid nanofiber scaffold in combination with 5-azacytidine and transforming growth factor-β to induce cardiomyocyte differentiation of adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (Ad-MSCs) are promising candidates for cardiac repair/regeneration. The application of copolymer nanoscaffolds has received great attention in tissue engineering to support differentiation and functional tissue organization toward effective tissue regeneration. The objective of the current study was to develop functional and bioactive scaffolds by combining polycaprolactone (PCL) and polylactic acid (PLA) for cardiomyocyte differentiation of human Ad-MSC (hAd-MSCs) in the absence or presence of 5-azacytidine and transforming growth factor-β (TGF-β). To that end, the human MSCs were extracted from human adipose tissue (AD). The cardiomyocyte differentiation potency of hAd-MSCs was evaluated on the novel synthetic PCL/PLA nanofiber scaffolds prepared in the absence and presence of 5-azacytidine and TGF-β supplements. A PCL/PLA nanofibrous scaffold was fabricated using the electrospinning method and its nanotopography and porous structure werecharacterized using scanning electron microscopy. In addition, the attachment of hAd-MSCs on the PCL/PLA scaffolds was semiquantitatively investigated. Compared with other treatments, the PCL/PLA nanofibrous scaffold supplemented with both 5-azacytidine and TGF-β was observed to differentiate hAd-MSCs into cardiomyocytes at Day 21 as evidenced by real-time PCR for cardiac-specific genes including cardiac troponin I (cTnI), GATA4, MYH7, and NKX2.5. In addition, flow cytometric analysis of cTnI-positive cells demonstrated that the cardiomyocytedifferentiation of hAd-MSCs was more efficient on the PCL/PLA nanofibrous scaffold supplemented with both 5-azacytidine and TGF-β than it was in the other treatment groups. Generally speaking, the results show that PCL/PLA nanofibrous scaffolds may be applied as a platform for efficient differentiation of hAd-MSCs into functional cardiomyocytes.

Fibroblast-derived exosomal miRNA-133 promotes cardiomyocyte-like differentiation

ObjectiveTo investigate the role of exosomal miRNA-133 secreted by cardiac fibroblasts (CFs) in promoting cardiomyocyte differentiation. MethodsNeonatal rat CFs were cultured in vitro, and the cultured CFs were divided into three groups as follows: induction, miRNA-133 high expression, and miRNA-133 inhibition. miRNA-133 was transfected into CFs with lentivirus as a vector. CFs were transfected with the miRNA-133 inhibitor, and the markers of cardiomyocyte were detected through immunofluorescence staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR) at 3, 8, and 14 days, respectively. The expression levels of cardiac troponin T (cTnT) and cardiac actin (α-actin) were determined, and qRT-PCR was used to detect the expression of miRNA-133 in the fibroblast exosomes. ResultsCFs subjected to immunofluorescence staining expressed vimentin and discoid domain receptor 2. The exosomes secreted by CFs were observed as small vesicles of 30–100 nm via transmission electron microscopy, and Western blotting was used to detect exosome-specific protein CD63 and CD9 expression. The expression levels of cTnT, α-actin, and exosomal miRNA-133 secreted into the supernatant of the miRNA-133 high-expression group increased gradually at different time points and reached the highest level at 14 days. The expression levels of cTnT, α-actin, and exosome miRNA-133 in the miRNA-133 inhibition group were the lowest. ConclusionThe exosomal miRNA-133, which is derived from CFs, can promote the differentiation of fibroblasts into cardiomyocyte-like cells.

Energy Metabolism on Mitochondrial Maturation and Its Effects on Cardiomyocyte Cell Fate.

Alterations in energy metabolism play a major role in the lineage of cardiomyocytes, such as the dramatic changes that occur in the transition from neonate to newborn. As cardiomyocytes mature, they shift from a primarily glycolytic state to a mitochondrial oxidative metabolic state. Metabolic intermediates and metabolites may have epigenetic and transcriptional roles in controlling cell fate by increasing mitochondrial biogenesis. In the maturing cardiomyocyte, such as in the postnatal heart, fatty acid oxidation increases in conjunction with increased mitochondrial biogenesis driven by the transcriptional coregulator PGC1-α. PGC1-α is necessary for mitochondrial biogenesis in the heart at birth, with deficiencies leading to postnatal cardiomyopathy. While stem cell therapy as a treatment for heart failure requires further investigation, studies suggest that adult stem cells may secrete cardioprotective factors which may regulate cardiomyocyte differentiation and survival. This review will discuss how metabolism influences mitochondrial biogenesis and how mitochondrial biogenesis influences cell fate, particularly in the context of the developing cardiomyocyte. The implications of energy metabolism on stem cell differentiation into cardiomyocytes and how this may be utilized as a therapy against heart failure and cardiovascular disease will also be discussed.

CIEGAN: A Deep Learning Tool for Cell Image Enhancement.

Long-term live-cell imaging technology has emerged in the study of cell culture and development, and it is expected to elucidate the differentiation or reprogramming morphology of cells and the dynamic process of interaction between cells. There are some advantages to this technique: it is noninvasive, high-throughput, low-cost, and it can help researchers explore phenomena that are otherwise difficult to observe. Many challenges arise in the real-time process, for example, low-quality micrographs are often obtained due to unavoidable human factors or technical factors in the long-term experimental period. Moreover, some core dynamics in the developmental process are rare and fleeting in imaging observation and difficult to recapture again. Therefore, this study proposes a deep learning method for microscope cell image enhancement to reconstruct sharp images. We combine generative adversarial nets and various loss functions to make blurry images sharp again, which is much more convenient for researchers to carry out further analysis. This technology can not only make up the blurry images of critical moments of the development process through image enhancement but also allows long-term live-cell imaging to find a balance between imaging speed and image quality. Furthermore, the scalability of this technology makes the methods perform well in fluorescence image enhancement. Finally, the method is tested in long-term live-cell imaging of human-induced pluripotent stem cell-derived cardiomyocyte differentiation experiments, and it can greatly improve the image space resolution ratio.

Association between donor's age and expression of cardiomyocyte marker quantity on adipocyte-derived mesenchymal stem cell.

Aim Adipocyte-derived mesenchymal stem cells (AMSCs) have recently been studied as a potential new approach for regeneration treatment of heart failure and ischemic myocardium. However, donor's age is thought to affect the ability of AMSC cells to differentiate into cardiomyocytes. This study aimed to analyse the association between donor's age with the expression of cardiomyocyte marker cardiac isoform of Troponin-T (cTnT) quantity on AMSCs. Methods Subcutaneous adipose tissue was taken from donors of 3 different age groups (30-40, 41-50, and >50 years old), and then processed into AMSC cells by insert culture. AMSC cell phenotype was identified by assessing the quantity of mesenchymal markers expression (CD34, CD45, CD90, CD105) using indirect immunofluorescence technique. Afterwards, cTnT expression, a marker for cardiomyocyte differentiation from AMSC cells, was evaluated under the fluorescent microscope. The comparison was done between the 3 different age groups. Results The comparative test and post hoc analysis showed that there were differences in the expression of CD34, CD45, CD90, CD105, and cTnT between the three age groups (all p<0.05). Furthermore, there were strong negative correlations between age and CD34, CD90, CD105 and cTnT (r= -0.844, -0.914, -0.899, and -0.738, respectively), while the correlation was positive between age and CD45 (r=0.899). Conclusion Expression quantity of mesenchymal markers and cardiomyocyte markers from AMSC cells were affected by the donor's age. The higher the donor age group, the lower the expression quantity of mesenchymal markers (except CD45) and cTnT.

Atrial-specific hiPSC-derived cardiomyocytes in drug discovery and disease modeling.

The discovery and application of human-induced pluripotent stem cells (hiPSCs) have been instrumental in the investigation of the pathophysiology of cardiovascular diseases. Patient-specific hiPSCs can now be generated, genome-edited, and subsequently differentiated into various cell types and used for regenerative medicine, disease modeling, drug testing, toxicity screening, and 3D tissue generation. Modulation of the retinoic acid signaling pathway has been shown to direct cardiomyocyte differentiation towards an atrial lineage. A variety of studies have successfully differentiated patient-specific atrial cardiac myocytes (hiPSC-aCM) and atrial engineered heart tissue (aEHT) that express atrial specific genes (e.g., sarcolipin and ANP) and exhibit atrial electrophysiological and contractility profiles. Identification of protocols to differentiate atrial cells from patients with atrial fibrillation and other inherited diseases or creating disease models using genetic mutation studies has shed light on the mechanisms of atrial-specific diseases and identified the efficacy of atrial-selective pharmacological compounds. hiPSC-aCMs and aEHTs can be used in drug discovery and drug screening studies to investigate the efficacy of atrial selective drugs on atrial fibrillation models. Furthermore, hiPSC-aCMs can be effective tools in studying the mechanism, pathophysiology and treatment options of atrial fibrillation and its genetic underpinnings. The main limitation of using hiPSC-CMs is their immature phenotype compared to adult CMs. A wide range of approaches and protocols are used by various laboratories to optimize and enhance CM maturation, including electrical stimulation, culture time, biophysical cues and changes in metabolic factors.

Lack of effect of plasma of myocardial preconditioned, ischemic and ischemic-reperfused rats of myocardium on differentiation of mesenchymal stem cells into cardiomyocytes

Introduction: Numerous studies have shown that enriched plasma protects myocardial cells against ischemia. The aim of this study was to evaluate the effect of rat enriched plasma by preconditioning, ischemia, and reperfusion on the differentiation of bone marrow mesenchymal stem cells (BMMSC) into cardiomyocytes. Methods: In this experimental and laboratory study, BMMSCs were extracted from the femur bone of male Wistar rats weighting 250-300 g. BMMSCs of the third passage were divided into six groups according to the type of plasma received: 1) control, 2) azacytidine, 3) azacytidine and non-ischemic plasma recipient, 4) azacytidine and preconditioned plasma recipient , 5) azacytidine and ischemic plasma recipient 6) azacytidine and plasma reperfusion recipient groups. In all groups except group 1, to initiate cardiomyocyte differentiation, BMMSCs were exposed to azacytidine (10 μM) for 24 hours. Plasma (25 μg / ml) was added to the culture medium each time the culture medium was changed. After 21 days, the expression level of myocardial βMHC, desmin, and troponin I genes was assessed using qRT-PCR. Results: The expression of myocardial βMHC, desmin, and troponin I genes was significantly increased in all groups received azacytidine (groups 2 to 6) (P&lt;0.01). There was no significant difference in the expression of myocardial βMHC, desmin, and troponin I genes, the doubling time, and migration between the plasma receiving groups (groups 3 to 6) compared to the group 2 (P&gt;0.05). Conclusion: The findings of present study showed that the plasma collected during ischemia, ischemia- reperfusion, and ischemia preconditioning had no effect on the differentiation of BMMSCs into cardiomyocytes in the presence of azacytidine.

Differentiation of induced pluripotent stem cells to Cardiomyocytes on Cellulose Nanofibril substrate

In vitro differentiation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) holds great potential in regenerative medicine. Currently the differentiation of iPSCs to cardiomyocytes is mostly done on vitronectin substrate, which is very expensive and difficult to produce on a large scale. For potential future clinical application, an appropriate natural and non-xenogenic substrate for the differentiation of iPS cells is very important. In this study a natural, innocuous, tissue compatible and biodegradable polymer Cellulose Nanofibril (CNF) was used as a fine coat substrate for differentiating iPSCs to cardiomyocytes. We checked the expression of NKX2–5, an early cardiac mesoderm marker and TNNT2, a mature cardiomyocyte marker to confirm the process of becoming iPSCs to cardiomyocytes on both vitronectin and CNF substrates. Our preliminary results suggest that the stage specific markers were expressed on the differentiated cardiomyocytes from both the substrates and were analogous. Thus, we established a natural biomaterial, CNF, that may be used as a potential substrate in tissue engineering applications for the process by which iPSCs become cardiomyocytes.

Circular RNA circ-RCCD promotes cardiomyocyte differentiation in mouse embryo development via recruiting YY1 to the promoter of MyD88.

Congenital heart disease (CHD) is the most common birth defect, affecting approximately 1% of live births. Genetic and environmental factors are leading factors to CHD, but the mechanism of CHD pathogenesis remains unclear. Circular RNAs (circRNAs) are kinds of endogenous non‐coding RNAs (ncRNAs) involved in a variety of physiological and pathological processes, especially in heart diseases. In this study, three significant differently expressed circRNA between maternal embryonic day (E) E13 and E17 was found by microarray assay. Among them, the content of circ‐RCCD increases with the development of heart and was enriched in primary cardiomyocytes of different species, which arouses our attention. Functional experiments revealed that inhibition of circ‐RCCD dramatically suppressed the formation of beating cell clusters, the fluorescence intensity of cardiac differentiation marker MF20, and the expression of the myocardial‐specific markers CTnT, Mef2c, and GATA4. Next, we found that circ‐RCCD was involved in cardiomyocyte differentiation through negative regulation of MyD88 expression. Further experiments proved that circ‐RCCD inhibited MyD88 levels by recruiting YY1 to the promoter of MyD88; circ‐RCCD inhibited nuclear translocation of YY1. These results reported that circ‐RCCD promoted cardiomyocyte differentiation by recruiting YY1 to the promoter of MyD88. And, this study provided a potential role and molecular mechanism of circ‐RCCD as a target for the treatment of CHD.

Cardiomyocytes derived from human-induced pluripotent stem cell culture in rod-shaped micropattern

Cardiomyocytes derived from human-induced pluripotent stem cells (iPS-CMs) have numerous advantages for drug screening and disease modeling but present with structural and functional immaturity features. Experiments in cardiomyocytes isolated from adult human or animal hearts are conditioned by their typical rod-shaped morphology. Here, we aimed at improving iPS-CMs architectural maturity by using a rod-shaped cell micropatterned substrate made of repeated rectangles of 120 μm-length and 30 μm-width and surrounded with a chemical cell-repellent. Cardiomyocyte differentiation was performed using a chemically defined method. Thirty-five days later the generated iPS-CMs were cultured on unpatterned vs. rod-shaped micropatterned substrates for 10 additional days. We performed molecular investigations by qPCR and immunofluorescence, and electrophysiological characterization by patch-clamp technique. The culture of micropatterned substrates resulted in the generation of rod-shaped iPS-CMs with a 4:1 cell ratio, with a more organized troponin T staining pattern exhibiting sarcomere-type striations and higher sarcomere organization score (0.25 ± 0.07 vs. 0.13 ± 0.004, P < 0.0001). The rod-shaped iPS-CMs also showed significant improvements in electrophysiological parameters with a decrease of the spontaneous beating rate and a more hyperpolarized membrane diastolic potential (−68.11 ± 6.25 vs. −59.17 ± 4.94 mV, P < 0.001) as compared to unpatterned iPS-CMs. Rod-shaped iPS-CMs displayed a more predominant ventricular-like type after analyzing action potential recordings (91% ± 1), with an increase in the percentage of late ventricular-like APs compared to unpatterned (64% vs. 29%), suggesting that rod-shaped morphology stimulates ventricular maturation. Notably, the maximum upstroke velocity (dV/dtmax) (98.09 ± 32.63 vs. 59.71 ± 50.43 V/ms, P < 0.1) and the sodium current density (INa) was increased (−89.30 ± 25.09 vs. −64.92 ± 21.63 pA/pF, P < 0.001) in rod-shaped iPS-CMs compared to unpatterned CMs. Importantly, we found no significant change in the RNA expression of the main ion channels or in major sarcomeric components. We are now assessing the Ca2 + signaling properties by confocal analysis. We developed a novel method to generate rod-shaped iPS-CMs, leading to improved functional characteristics. Preliminary results suggest that the architectural re-arrangement is the primary driver of the improved functionality observed in micropatterned iPS-CMs.

CASK is a key organizer of the structural and electrical polarity of the cardiomyocyte

Although the organization of the cardiomyocyte membrane into functional microdomains directly influences the anisotropic propagation of the action potential, the mechanisms underlying the rod-like structural organization of cardiomyocytes during postnatal development are poorly understood. We have previously shown in the adult cardiomyocyte that CASK regulates sodium channel expression at the lateral membrane in interaction with focal adhesion complexes. In addition, preliminary results have shown that the expression level of CASK evolves during postnatal development and is altered during post-infarction myocardial remodeling in rat. To unravel the implication of CASK in postnatal cardiomyocyte differentiation and in the acquisition of structural and electrical function. CASK viral constructs were designed to inhibit the expression of CASK in neonatal and adult cardiac myocytes, in vitro (Ad-shCASK) and in vivo (AAV-shCASK). Proteomic analysis shows that CASK invalidation differentially regulates the expression of different protein families between neonatal and adult myocytes, suggesting a different role for CASK during postnatal development. Immunostaining experiments on isolated neonatal cardiomyocytes showed that CASK is required for the correct organization of the cardiomyocyte cytoskeleton, and impact both mechanical and electrical junction formation. Neonatal cardiomyocytes invalidated for CASK display abnormal contractility, focal adhesion fragility, and anoikis. Echocardiography phenotyping of CASK-invalidated rats at the neonatal and adult stages reveals eccentric ventricular remodeling and decreased systolic performance. However, in neonatal CASK-invalidated rats, the contractile reserve is preserved and the cardiac output is increased. In addition, mild electrical remodeling is observed since only neonatal CASK-invalidated rats display QT prolongation and slowed heart rhythm. This study demonstrates that CASK confers the ability to orchestrate both cardiomyocyte molecular organization and tissue cohesion. CASK appears to be a key modulator in molecular arrangement of electrical and structural polarity of cardiomyocytes during postnatal development.

A combined human gastruloid model of cardiogenesis and neurogenesis

SummaryMulti-lineage development from gastruloids is enabling unprecedented opportunities to model and study human embryonic processes and is expected to accelerate ex vivo strategies in organ development. Reproducing human cardiogenesis with neurogenesis in a multi-lineage context remains challenging, requiring spatiotemporal input of paracrine and mechanical cues. Here we extend elongating multi-lineage organized (EMLO) gastruloids to include cardiogenesis (EMLOC) and describe interconnected neuro-cardiac lineages in a single gastruloid model. Contractile EMLOCs recapitulate numerous interlinked developmental features including heart tube formation and specialization, cardiomyocyte differentiation and remodeling phases, epicardium, ventricular wall morphogenesis, chamber-like structures and formation of a putative outflow tract. The EMLOC cardiac region, which originates anterior to gut tube primordium, is progressively populated by neurons in a spatial pattern mirroring the known distribution of neurons in the innervated human heart. This human EMLOC model represents a multi-lineage advancement for the study of coincident neurogenesis and cardiogenesis.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Calreticulin and the Heart.

Calreticulin is an endoplasmic Ca2+ binding protein and molecular chaperone. As a cardiac embryonic gene, calreticulin is essential for heart development. The protein supports Ca2+-dependent signaling events that are critical to cardiomyocyte differentiation and cardiogenesis. The increased expression of calreticulin and endoplasmic reticulum/sarcoplasmic reticulum Ca2+ capacity produces cardiomyocytes with enhanced efficiency, and detrimental mechanical stretching of cardiac fibroblasts, leading to cardiac pathology. Deletion of the calreticulin gene in adult cardiomyocytes results in left ventricle dilation, an impaired electrocardiogram, and heart failure. These observations indicate that a well-adjusted endoplasmic reticulum and calreticulin-dependent Ca2+ pool in cardiomyocytes are critical for the maintenance of proper cardiac function.

Dynamic Involvement of Telocytes in Modulating Multiple Signaling Pathways in Cardiac Cytoarchitecture.

Cardiac interstitium is a complex and dynamic environment, vital for normal cardiac structure and function. Telocytes are active cellular players in regulating main events that feature myocardial homeostasis and orchestrating its involvement in heart pathology. Despite the great amount of data suggesting (microscopically, proteomically, genetically, etc.) the implications of telocytes in the different physiological and reparatory/regenerative processes of the heart, understanding their involvement in realizing the heart’s mature cytoarchitecture is still at its dawn. Our scrutiny of the recent literature gave clearer insights into the implications of telocytes in the WNT signaling pathway, but also TGFB and PI3K/AKT pathways that, inter alia, conduct cardiomyocytes differentiation, maturation and final integration into heart adult architecture. These data also strengthen evidence for telocytes as promising candidates for cellular therapies in various heart pathologies.

Sbk2, a Newly Discovered Atrium-Enriched Regulator of Sarcomere Integrity.

Heart development relies on tight spatiotemporal control of cardiac gene expression. Genes involved in this intricate process have been identified using animals and pluripotent stem cell-based models of cardio(myo)genesis. Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal line of conditionally immortalized neonatal rat atrial myocytes (NRAMs), which allows toggling between proliferative and differentiated (ie, excitable and contractile) phenotypes in a synchronized and homogenous manner. In this study, the unique properties of conditionally immortalized NRAMs (iAMs) were exploited to identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation. Transcriptome analysis of iAM-1 cells at different stages during one cycle of differentiation and subsequent dedifferentiation identified ≈13 000 transcripts, of which the dynamic changes in expression upon cardiomyogenic differentiation mostly opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many with known (lineage-specific) functions in cardiac muscle formation. Filtering for cardiac-enriched low-abundance transcripts, identified multiple genes with an uncharacterized role during cardio(myo)genesis including Sbk2 (SH3 domain binding kinase family member 2). Sbk2 encodes an evolutionarily conserved putative serine/threonine protein kinase, whose expression is strongly up- and downregulated during iAM-1 cell differentiation and dedifferentiation, respectively. In neonatal and adult rats, the protein is muscle-specific, highly atrium-enriched, and localized around the A-band of cardiac sarcomeres. Knockdown of Sbk2 expression caused loss of sarcomeric organization in NRAMs, iAMs and their human counterparts, consistent with a decrease in sarcomeric gene expression as evinced by transcriptome and proteome analyses. Interestingly, co-immunoprecipitation using Sbk2 as bait identified possible interaction partners with diverse cellular functions (translation, intracellular trafficking, cytoskeletal organization, chromatin modification, sarcomere formation). iAM-1 cells are a relevant and suitable model to identify (lowly expressed) genes with a hitherto unidentified role in cardiomyocyte differentiation as exemplified by Sbk2: a regulator of atrial sarcomerogenesis.

Inducible apelin receptor knockdown reduces differentiation efficiency and contractility of hESC-derived cardiomyocytes.

The apelin receptor, a G protein-coupled receptor, has emerged as a key regulator of cardiovascular development, physiology, and disease. However, there is a lack of suitable human in vitro models to investigate the apelinergic system in cardiovascular cell types. For the first time we have used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and a novel inducible knockdown system to examine the role of the apelin receptor in both cardiomyocyte development and to determine the consequences of loss of apelin receptor function as a model of disease. Expression of the apelin receptor and its ligands in hESCs and hESC-CMs was determined. hESCs carrying a tetracycline-inducible short hairpin RNA targeting the apelin receptor were generated using the sOPTiKD system. Phenotypic assays characterized the consequences of either apelin receptor knockdown before hESC-CM differentiation (early knockdown) or in 3D engineered heart tissues as a disease model (late knockdown). hESC-CMs expressed the apelin signalling system at a similar level to the adult heart. Early apelin receptor knockdown decreased cardiomyocyte differentiation efficiency and prolonged voltage sensing, associated with asynchronous contraction. Late apelin receptor knockdown had detrimental consequences on 3D engineered heart tissue contractile properties, decreasing contractility and increasing stiffness. We have successfully knocked down the apelin receptor, using an inducible system, to demonstrate a key role in hESC-CM differentiation. Knockdown in 3D engineered heart tissues recapitulated the phenotype of apelin receptor down-regulation in a failing heart, providing a potential platform for modelling heart failure and testing novel therapeutic strategies.

Genetic Basis of Left Ventricular Noncompaction.

Left ventricular noncompaction (LVNC) is the third most common pediatric cardiomyopathy characterized by a thinned myocardium and prominent trabeculations. Next-generation genetic testing has led to a rapid increase in the number of genes reported to be associated with LVNC, but we still have little understanding of its pathogenesis. We sought to grade the strength of the gene-disease relationship for all genes reported to be associated with LVNC and identify molecular pathways that could be implicated. Following a systematic PubMed review, all genes identified with LVNC were graded using a validated, semi-quantitative system based on all published genetic and experimental evidence created by the Clinical Genome Resource (ClinGen). Genetic pathway analysis identified molecular processes and pathways associated with LVNC. We identified 189 genes associated with LVNC: 11 (6%) were classified as definitive, 21 (11%) were classified as moderate, and 140 (74%) were classified as limited, but 17 (9%) were classified as no evidence. Of the 32 genes classified as definitive or moderate, the most common gene functions were sarcomere function (n=11; 34%), transcriptional/translational regulator (n=6; 19%), mitochondrial function (n=3; 9%), and cytoskeletal protein (n=3; 9%). Furthermore, 18 (56%) genes were implicated in noncardiac syndromic presentations. Lastly, 3 genetic pathways (cardiomyocyte differentiation via BMP receptors, factors promoting cardiogenesis in vertebrates, and Notch signaling) were found to be unique to LVNC and not overlap with pathways identified in dilated cardiomyopathy and hypertrophic cardiomyopathy. LVNC is a genetically heterogeneous cardiomyopathy. Distinct from dilated or hypertrophic cardiomyopathies, LVNC appears to arise from abnormal developmental processes.

Dynamic Transcriptome Profiling Reveals LncRNA-Centred Regulatory Networks in the Modulation of Pluripotency.

Long noncoding RNAs (lncRNAs) have emerged as vital regulators of gene expression during embryonic stem cell (ESC) self-renewal and differentiation. Here, we systemically analyzed the differentially regulated lncRNAs during ESC-derived cardiomyocyte (CM) differentiation. We established a perspicuous profile of lncRNA expression at four critical developmental stages and found that the differentially expressed lncRNAs were grouped into six distinct clusters. The cluster with specific expression in ESC enriches the largest number of lncRNAs. Investigation of lncRNA-protein interaction network revealed that they are not only controlled by classic key transcription factors, but also modulated by epigenetic and epitranscriptomic factors including N6-methyladenosine (m6A) effector machineries. A detailed inspection revealed that 28 out of 385 lncRNAs were modified by methylation as well as directly recruited by the nuclear m6A reader protein Ythdc1. Unlike other 27 non-coding transcripts, the ESC-specific lncRNA Gm2379, located in both nucleus and cytoplasm, becomes dramatically upregulated in response to the depletion of m6A or Ythdc1. Consistent with the role of m6A in cell fate regulation, depletion of Gm2379 results in dysregulated expressions of pluripotent genes and crucial genes required for the formation of three germ layers. Collectively, our study provides a foundation for understanding the dynamic regulation of lncRNA transcriptomes during ESC differentiation and identifies the interplay between epitranscriptomic modification and key lncRNAs in the regulation of cell fate decision.

Stage-specific regulation of signalling pathways to differentiate pluripotent stem cells to cardiomyocytes with ventricular lineage.

BackgroundPluripotent stem cells (PSCs) can be an ideal source of differentiation of cardiomyocytes in vitro and during transplantation to induce cardiac regeneration. However, differentiation of PSCs into a heterogeneous population is associated with an increased incidence of arrhythmia following transplantation. We aimed to design a protocol to drive PSCs to a ventricular lineage by regulating Wnt and retinoic acid (RA) signalling pathways.MethodsMouse embryonic stem cells were cultured either in monolayers or three-dimensional hanging drop method to form embryonic bodies (EBs) and exposed to different treatments acting on Wnt and retinoic acid signalling. Samples were collected at different time points to analyse cardiomyocyte-specific markers by RT-PCR, flow cytometry and immunofluorescence.ResultsTreatment of monolayer and EBs with Wnt and RA signalling pathways and ascorbic acid, as a cardiac programming enhancer, resulted in the formation of an immature non-contractile cardiac population that expressed many of the putative markers of cardiac differentiation. The population exhibited upregulation of ventricular specific markers while suppressing the expression of pro-atrial and pro-sinoatrial markers. Differentiation of EBs resulted in early foetal like non-contractile ventricular cardiomyocytes with an inherent propensity to contract when stimulated.ConclusionOur results provide the first evidence of in vitro differentiation that mimics the embryonic morphogenesis towards ventricular specific cardiomyocytes through regulation of Wnt and RA signalling pathways.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-022-02845-9.