The conserved long non-coding RNA CARMA regulates cardiomyocyte differentiation.

AIMS: Production of functional cardiomyocytes from pluripotent stem cells requires tight control of the differentiation process. Long non-coding RNAs (lncRNAs) exert critical regulatory functions in cell specification during development. In this study, we designed an integrated approach to identify lncRNAs implicated in cardiogenesis in differentiating human embryonic stem cells (ESCs). METHODS AND RESULTS: We identified CARMA (CARdiomyocyte Maturation-Associated lncRNA), a conserved lncRNA controlling cardiomyocyte differentiation and maturation in human ESCs. CARMA is located adjacent to MIR-1-1HG, the host gene for two cardiogenic miRNAs: MIR1-1 and MIR-133a2, and transcribed in an antisense orientation. The expression of CARMA and the miRNAs are negatively correlated, and CARMA knockdown increases MIR1-1 and MIR-133a2 expression. In addition, CARMA possesses MIR-133a2 binding sites, suggesting the lncRNA could be also a target of miRNA action. Upon CARMA down-regulation, MIR-133a2 target protein-coding genes are coordinately down-regulated. Among those, we found RBPJ, the gene encoding the effector of the NOTCH pathway. NOTCH has been shown to control a binary cell fate decision between the mesoderm and the neuroectoderm lineages, and NOTCH inhibition leads to enhanced cardiomyocyte differentiation at the expense of neuroectodermal derivatives. Interestingly, two lncRNAs, linc1230 and linc1335, which are known repressors of neuroectodermal specification, were found up-regulated upon Notch1 silencing in ESCs. Forced expression of either linc1230 or linc1335 improved ESC-derived cardiomyocyte production. These two lncRNAs were also found up-regulated following CARMA knockdown in ESCs. CONCLUSIONS: Altogether, these data suggest the existence of a network, implicating three newly identified lncRNAs, the two myomirs MIR1-1 and MIR-133a2 and the NOTCH signalling pathway, for the coordinated regulation of cardiogenic differentiation in ESCs.

Oxygen-rich Environment Ameliorates Cell Therapy Outcomes of Cardiac Progenitor Cells for Myocardial Infarction.

To some extent, cell therapy for myocardial infarction (MI) has supported the idea of cardiac repair; however, further optimizations are inevitable. Combined approaches that comprise suitable cell sources and supporting molecules considerably improved its effect. Here, we devised a strategy of simultaneous transplantation of human cardiac progenitor cells (CPCs) and an optimized oxygen generating microparticles (MPs) embedded in fibrin hydrogel, which was injected into a left anterior descending artery (LAD) ligating-based rat model of acute myocardial infarction (AMI). Functional parameters of the heart, particularly left ventricular systolic function, markedly improved and reached pre-AMI levels. This functional restoration was well correlated with substantially lower fibrotic tissue formation and greater vascular density in the infarct area. Our novel approach promoted CPCs retention and differentiation into cardiovascular lineages. We propose this novel co-transplantation strategy for more efficient cell therapy of AMI which may function by providing an oxygen-rich microenvironment, and thus regulate cell survival and differentiation.

Automated feature detection in dental periapical radiographs by using deep learning.

OBJECTIVE: The aim of this study was to investigate automated feature detection, segmentation, and quantification of common findings in periapical radiographs (PRs) by using deep learning (DL)-based computer vision techniques. STUDY DESIGN: Caries, alveolar bone recession, and interradicular radiolucencies were labeled on 206 digital PRs by 3 specialists (2 oral pathologists and 1 endodontist). The PRs were divided into "Training and Validation" and "Test" data sets consisting of 176 and 30 PRs, respectively. Multiple transformations of image data were used as input to deep neural networks during training. Outcomes of existing and purpose-built DL architectures were compared to identify the most suitable architecture for automated analysis. RESULTS: The U-Net architecture and its variant significantly outperformed Xnet and SegNet in all metrics. The overall best performing architecture on the validation data set was "U-Net+Densenet121" (mean intersection over union [mIoU] = 0.501; Dice coefficient = 0.569). Performance of all architectures degraded on the "Test" data set; "U-Net" delivered the best performance (mIoU = 0.402; Dice coefficient = 0.453). Interradicular radiolucencies were the most difficult to segment. CONCLUSIONS: DL has potential for automated analysis of PRs but warrants further research. Among existing off-the-shelf architectures, U-Net and its variants delivered the best performance. Further performance gains can be obtained via purpose-built architectures and a larger multicentric cohort.

Heart Repair Induced by Cardiac Progenitor Cell Delivery within Polypyrrole-Loaded Cardiogel Post-ischemia.

Myocardial infarction (MI) irreversibly injures the heart tissue. Cardiovascular tissue engineering has been developed as a promising therapeutic approach for post-MI repair. Previously, we discovered the ability of a polypyrrole (PPy)-incorporated cardiogel (CG) for improvement of maturity and functional synchrony of rat neonatal cardiomyocytes. Here, we used the cross-linked form of PPy-incorporated CG (CG-PPy), in order to improve electromechanical properties of scaffold, for application in cardiac progenitor cell (CPC) transplantation on post-MI rat hearts. Improved mechanical property and electrical conductivity (sixfold) were evident in the cross-linked CG-PPy (P1) compared to cross-linked CG (C1) scaffolds. Transplantation of CPC-loaded P1 (P1-CPC) resulted in substantial improvement of cardiac functional properties. Furthermore, lower fibrotic tissue and higher CPC retention were observed. The grafted cells showed cardiomyocyte characteristics when stained with human cardiac troponin T and connexin43 antibodies, while neovessel formation was similarly prominent. These findings highlight the therapeutic promise of the P1 scaffold as a CPC carrier for functional restoration of the heart post-MI.

Vascular endothelial growth factor sustained delivery augmented cell therapy outcomes of cardiac progenitor cells for myocardial infarction.

Cell therapy has become a novel promising approach for improvement of cardiac functional capacity in the instances of ventricular remodeling and fibrosis caused by episodes of coronary artery occlusion and hypoxia. The challenge toward enhancing cell engraftment as well as formation of functional tissue, however, necessitated combinatorial approaches. Here, we complemented human embryonic stem cell-derived cardiac progenitor cell (hESC-CPC) therapy by heparin-conjugated, vascular endothelial growth factor (VEGF)-loaded fibrin hydrogel as VEGF delivery system. Transplantation of these cardiac committed cells along with sustained VEGF release could surpass the cardiac repair effects of each constituent alone in a rat model of acute myocardial infarction. The histological sections of rat hearts revealed improved vascularization as well as inclusion of hESC-CPC-derived cardiomyocytes, endothelial, and smooth muscle cells in host myocardium. Thus, co-transplantation of hESC-CPC and proangiogenic factor by a suitable delivery rate may resolve the shortcomings of conventional cell therapy.

Expansion of Human Pluripotent Stem Cell-derived Early Cardiovascular Progenitor Cells by a Cocktail of Signaling Factors.

Cardiovascular progenitor cells (CPCs) derived from human pluripotent stem cells (hPSCs) are proposed to be invaluable cell sources for experimental and clinical studies. This wide range of applications necessitates large-scale production of CPCs in an in vitro culture system, which enables both expansion and maintenance of these cells. In this study, we aimed to develop a defined and efficient culture medium that uses signaling factors for large-scale expansion of early CPCs, called cardiogenic mesodermal cells (CMCs), which were derived from hPSCs. Chemical screening resulted in a medium that contained a reproducible combination of three factors (A83-01, bFGF, and CHIR99021) that generated 1014 CMCs after 10 passages without the propensity for tumorigenicity. Expanded CMCs retained their gene expression pattern, chromosomal stability, and differentiation tendency through several passages and showed both the safety and possible cardio-protective potentials when transplanted into the infarcted rat myocardium. These CMCs were efficiently cryopreserved for an extended period of time. This culture medium could be used for both adherent and suspension culture conditions, for which the latter is required for large-scale CMC production. Taken together, hPSC-derived CMCs exhibited self-renewal capacity in our simple, reproducible, and defined medium. These cells might ultimately be potential, promising cell sources for cardiovascular studies.

Down-Regulation of a Male-Specific H3K4 Demethylase, KDM5D, Impairs Cardiomyocyte Differentiation.

Despite the small number of Y chromosome genes, their adequate expression is required for regulation of transcription, translation, and protein stability in males, not just for sex determination. In addition to the role in male fertility, the Y chromosome has a significant role in the development and sexual dimorphism of healthy and disease phenotypes. We observed that KDM5D along with its X-counterpart, KDM5C, are up-regulated during the cardiac mesoderm stage of development. Down-regulation of KDM5D using siRNA resulted in accumulation of differentiating cells in the S-phase of the cell cycle and impaired progression to cardiomyocytes as reflected by an altered expression pattern of cardiac progenitor specific markers. Furthermore, while control cells started spontaneous beating at a normal physiological range on day 7 of differentiation induction, no spontaneous beating was observed in KDM5D down-regulated cells. Interestingly, the knockdown of KDM5D had no significant effect on the expression level of its X-counterpart, KDM5C. Thus, we suggest that KDM5D, in cooperation with its X homologue as a dose-sensitive gene, may have an important role in cardiomyocyte differentiation. Our study presents further evidence on the contribution of Y chromosome genes to sex-dependent development outside of sex determination.

Hsa-miR-335 regulates cardiac mesoderm and progenitor cell differentiation.

BACKGROUND: WNT and TGFß signaling pathways play critical regulatory roles in cardiomyocyte fate determination and differentiation. MiRNAs are also known to regulate different biological processes and signaling pathways. Here, we intended to find candidate miRNAs that are involved in cardiac differentiation through regulation of WNT and TGFß signaling pathways. METHODS: Bioinformatics analysis suggested hsa-miR-335-3p and hsa-miR-335-5p as regulators of cardiac differentiation. Then, RT-qPCR, dual luciferase, TOP/FOP flash, and western blot analyses were done to confirm the hypothesis. RESULTS: Human embryonic stem cells (hESCs) were differentiated into beating cardiomyocytes, and these miRNAs showed significant expression during the differentiation process. Gain and loss of function of miR-335-3p and miR-335-5p resulted in BRACHYURY, GATA4, and NKX2-5 (cardiac differentiation markers) expression alteration during the course of hESC cardiac differentiation. The overexpression of miR-335-3p and miR-335-5p also led to upregulation of CNX43 and TNNT2 expression, respectively. Our results suggest that this might be mediated through enhancement of WNT and TGFß signaling pathways. CONCLUSION: Overall, we show that miR-335-3p/5p upregulates cardiac mesoderm (BRACHYURY) and cardiac progenitor cell (GATA4 and NKX2-5) markers, which are potentially mediated through activation of WNT and TGFß signaling pathways. Our findings suggest miR-335-3p/5p to be considered as a regulator of the cardiac differentiation process.

Human cardiomyocytes undergo enhanced maturation in embryonic stem cell-derived organoid transplants.

Human cardiomyocytes (CM) differentiated from pluripotent stem cells (PSCs) are relatively immature when generated in two-dimensional (2D) in vitro cultures, which limits their biomedical applications. Here, we devised a strategy to enhance maturation of human CM in vitro by assembly of three-dimensional (3D) cardiac organoids (CO) containing human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs), endothelial cells (ECs), and mesenchymal stem cells (MSCs). In contrast to corresponding 2D cultures, 3D CO not only developed into structures containing spontaneously beating CM, but also showed enhanced maturity as indicated by increased expressions of sarcomere and ion channel genes and reduced proliferation. Heterotopic implantation of CO into the peritoneal cavity of immunodeficient mice induced neovascularization, and further stimulated upregulation of genes coding for the contractile apparatus, Ca2+ handling and ion channel proteins. In addition, CM in implanted CO were characterized by a more mature ultrastructure compared to CM implanted without CO support. Functional analysis revealed the presence of working cardiomyocytes in both in vivo and ex ovo chorioallantoic membrane implanted CO. Our results demonstrate that cultivation in 3D CO and subsequent heterotopic implantation enhance maturation of CM towards an adult-like phenotype. We reason that CO-derived CM represent an attractive source for drug discovery and other biomedical applications.

Reversible permeabilization of the mitochondrial membrane promotes human cardiomyocyte differentiation from embryonic stem cells.

Cell death and differentiation appear to share similar cellular features. In this study, we aimed to investigate whether differentiation and mitochondrial cell death use a common pathway. We assessed the hallmarks of apoptosis during cardiomyocyte differentiation of human embryonic stem cells and found remarkable changes in P53, reactive oxygen species, apoptotic protease-activating factor 1, poly[ADP-ribose]polymerase 1, cellular adenosine triphosphate, and mitochondrial complex I activity. Furthermore, we observed reversible mitochondrial membrane permeabilization during cardiomyocyte differentiation accompanied by reversible loss of mitochondrial membrane potential, and these changes coincided with the fluctuating patterns of cytosolic cytochrome c accumulation and subsequent caspase-9 and -3/7 activation. Moreover, the use of apoptosis inhibitors (BCL2-associated X protein [BAX] inhibitor and caspase-3/7 inhibitor) during differentiation impaired cardiomyocyte development, resulting in substantial downregulation of T, MESP1, NKX2.5, and α-MHC. Additionally, although the expression of specific differentiation markers (T, MESP1, NKX2.5, MEF2C, GATA4, and SOX17) was enhanced in doxorubicin-induced human embryonic stem cells, the stemness-specific markers (OCT4 and NANOG) showed significant downregulation. With increasing doxorubicin concentration (0.03-0.6 µM; IC50 = 0.5 µM), we observed a marked increase in the expression of mesoderm and endoderm markers. In summary, we suggest that reversible mitochondrial outer membrane permeabilization promotes cardiomyocyte differentiation through an attenuated mitochondria-mediated apoptosis-like pathway.

Exosomes Secreted by Normoxic and Hypoxic Cardiosphere-derived Cells Have Anti-apoptotic Effect.

Cardiosphere-derived cells (CDCs) have emerged as one of the most promising stem cell types for cardiac protection and repair. Exosomes are required for the regenerative effects of human CDCs and mimic the cardioprotective benefits of CDCs such as anti-apoptotic effect in animal myocardial infarction (MI) models. Here we aimed to investigate the anti-apoptotic effect of the hypoxic and normoxic human CDCs-derived exosomes on induced apoptosis in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). In this study, CDCs were cultured under normoxic (18% O2) and hypoxic (1% O2) conditions and CDC-exosomes were isolated from conditioned media by differential ultracentrifugation. Cobalt chloride as hypoxia-mimetic agents at a high concentration was used to induce apoptosis in hESC-CMs. The caspase-3/7 activity was determined in apoptosis-induced hESC-CMs. The results indicated that the caspase-positive hESC-CMs were significantly decreased from 30.63 ± 1.44% (normalized against untreated cardiomyocytes) to 1.65 ± 0.1 and 1.1 ± 1.09 in the presence of normoxic exosomes (N-exo) at concentration of 25 and 50 µg/mL, respectively. Furthermore, hypoxic exosomes (H-exo) at concentration of 25 and 50 µg/mL led to 8.75 and 12.86 % reduction in caspase-positive cells, respectively. The anti-apoptotic activity of N-exo at the concentrations of 25 and 50 µg/mL was significantly higher than H-exo. These results could provide insights into optimal preparation of CDCs which would greatly influence the anti-apoptotic effect of CDC-exosomes. Totally, CDC-secreted exosomes have the potential to increase the survival of cardiomyocytes by inhibiting apoptosis. Therefore, CDC-exosomes can be developed as therapeutic strategy in ischemic cardiac disease.

Effects of hawthorn ( Crataegus pentagyna) leaf extract on electrophysiologic properties of cardiomyocytes derived from human cardiac arrhythmia-specific induced pluripotent stem cells.

Cardiac arrhythmias are major life-threatening conditions. The landmark discovery of induced pluripotent stem cells has provided a promising in vitro system for modeling hereditary cardiac arrhythmias as well as drug development and toxicity testing. Nowadays, nutraceuticals are frequently used as supplements for cardiovascular therapy. Here we studied the cardiac effects of hawthorn ( Crataegus pentagyna) leaf extract using cardiomyocytes (CMs) differentiated from healthy human embryonic stem cells, long QT syndrome type 2 (LQTS2), and catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) patient-specific induced pluripotent stem cells. The hydroalcoholic extract resulted in a dose-dependent negative chronotropic effect in all CM preparations leading to a significant reduction at 1000 µg/ml. This was accompanied by prolongation of field potential durations, although with different magnitudes in CMs from different human embryonic stem cell and iPSC lines. Hawthorn further prolonged field potential durations in LQTS2 CMs but reduced the beating frequencies and occurrence of immature field potentials triggered by ß1-adrenergic stimulation in CPVT1 CMs at 300 and 1000 µg/ml. Furthermore, isoquercetin and vitexin flavonoids significantly slowed down isoproterenol (5 µM)-induced beating frequencies at 3 and 10 µg/ml. Therefore, C. pentagyna leaf extract and its isoquercetin and vitexin flavonoids may be introduced as a novel nutraceutical with antiarrhythmic potential for CPVT1 patients.-Pahlavan, S., Tousi, M. S., Ayyari, M., Alirezalu, A., Ansari, H., Saric, T., Baharvand, H. Effects of hawthorn ( Crataegus pentagyna) leaf extract on electrophysiologic properties of cardiomyocytes derived from human cardiac arrhythmia-specific induced pluripotent stem cells.

Human embryonic stem cell-derived cardiovascular progenitor cells efficiently colonize in bFGF-tethered natural matrix to construct contracting humanized rat hearts.

Bioengineering of whole hearts using human embryonic stem cells (hESCs)-derived cardiovascular progenitor cells (CPCs) and natural matrices is a promising approach to overcome organ donor shortage threatening millions of patients awaiting for heart transplantation. Here, we developed a novel strategy for generation of heart constructs by repopulating engineered decellularized rat hearts using hESCs-derived CPCs. Careful expansion of CPCs in a scalable stirred-suspension bioreactor combined with step-wise seeding (60 million cells in 3 steps of 20 million per 1.5 h) onto decellularized hearts containing immobilized basic fibroblast growth factor (bFGF) resulted in improved retention of CPCs and differentiation to cardiomyocytes, smooth muscle cells and endothelial cells as evaluated by immunohistochemistry and qRT-PCR. We observed spontaneous and synchronous contractions of humanized hearts after 12 days of perfusion as well as advanced alignment of myofilaments. Our study provides a robust platform for generation of artificial human hearts and resolves major bottlenecks hindering further development of this technology.

Y Chromosome Missing Protein, TBL1Y, May Play an Important Role in Cardiac Differentiation.

Despite evidence for sex-specific cardiovascular physiology and pathophysiology, the biological basis for this dimorphism remains to be explored. Apart from hormonal factors, gender-related characteristics may reside in the function of sex chromosomes during cardiac development. In this study, we investigated the differential expression of the male-specific region of the Y chromosome (MSY) genes and their X counterparts during cardiac differentiation of human embryonic stem cells (hESC). We observed alterations in mRNA and protein levels of TBL1Y, PCDH11Y, ZFY, KDM5D, USP9Y, RPS4Y1, DDX3Y, PRY, XKRY, BCORP1, RBMY, HSFY, and UTY, which accompanied changes in intracellular localization. Of them, the abundance of a Y chromosome missing protein, TBL1Y, showed a significant increase during differentiation while the expression level of its X counterpart decreased. Consistently, reducing TBL1Y cellular level using siRNA approach influenced cardiac differentiation by reducing its efficacy as well as increasing the probability of impaired contractions. TBL1Y knockdown may have negatively impacted cardiogenesis by CtBP stabilization. Furthermore, we presented compelling experimental evidence to distinguish TBL1Y from TBL1X, its highly similar X chromosome homologue, and proposed reclassification of TBL1Y as "found missing protein" (PE1). Our results demonstrated that MSY proteins may play an important role in cardiac development.

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-ß, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing > 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.

UNLABELLED: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-ß and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. SIGNIFICANCE: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.

Efficient induction of pluripotency in primordial germ cells by dual inhibition of TGF-ß and ERK signaling pathways.

Primordial germ cells (PGCs) have the ability to be reprogrammed into a pluripotent state and are defined as embryonic germ cells (EGCs) in vitro. EGC formation is more efficient, has a shorter culture period than somatic cell reprogramming, and does not require exogenous genetic manipulation. Therefore, EGCs are a good model to analyze mechanisms by which committed cells acquire a pluripotent state. In the present study we have attempted to elucidate a more defined and robust protocol that promotes EGC generation through the suppression of transforming growth factor-ß (TGF-ß) and extracellular signal-regulated kinase (ERK) signaling pathways by SB431542 (SB) and PD0325901 (PD), respectively. Under this condition the efficiency of transformation of PGCs into EGCs was more than the inhibition of glycogen synthase kinase 3 and ERK signaling pathways. Pluripotency of the resultant-derived EGC lines were further confirmed by gene expression, immunofluorescent staining, directed differentiation ability, teratoma formation, and their contribution to chimeric mice and germ-line transmission. These results showed that PGCs from different embryonic stages (E8.5 and E12.5) could be reprogrammed, maintained, and expanded efficiently under feeder- and serum-free chemically defined conditions by dual inhibition of TGF-ß and ERK signaling pathways, regardless of the animal's genetic background.