Embryonic Stem Cell Cardiac Differentiation Research Articles

Dioxin and AHR impairs mesoderm gene expression and cardiac differentiation in human embryonic stem cells

Dioxin and dioxin-related polychlorinated biphenyls are potent toxicants with association with developmental heart defects and congenital heart diseases. However, the underlying mechanism of their developmental toxicity is not fully understood. Further, different animals show distinct susceptibility and phenotypes after exposure, suggesting possible species-specific effects. Using a human embryonic stem cell (ESC) cardiomyocyte differentiation model, we examined the impact, susceptible window, and dosage of 2,3,7,8‑tetrachlorodibenzo‑p‑dioxin (TCDD) on human cardiac development. We showed that treatment of human ESCs with TCDD at the ESC stage inhibits cardiomyocyte differentiation, and the effect is largely mediated by the aryl hydrocarbon receptor (AHR). We further identified genes that are differentially expressed after TCDD treatment by RNA-sequencing, and genomic regions that are occupied by AHR by chromatin immunoprecipitation and high-throughput sequencing. Our results support the model that TCDD impairs human ESC cardiac differentiation by promoting AHR binding and repression of key mesoderm genes. More importantly, our study demonstrates the toxicity of dioxin in human embryonic development and uncovered a novel mechanism by which dioxin and AHR regulates lineage commitment. It also illustrates the power of ESC-based models in the systematic study of developmental toxicology.

Small RNA-directed epigenetic programming of embryonic stem cell cardiac differentiation

Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine.

Abstract 139: Role of Histone Lysine Demethylase PHF8 in Epigenetic Regulation of Embryonic Stem Cell Cardiac Differentiation

Epigenetic control mechanisms play a key role in the regulation of lineage commitment of stem/progenitor cells, while the epigenetic regulators involved in the determination of cardiomyogenic lineage are incompletely defined. Using in vitro cardiac differentiation system of mESCs with Brachyury and Nkx2.5 selection, we analyzed expression profiles of epigenetic regulators at critical stages of cardiomyogenesis by RT 2 profiler PCR arrays. To identify the potential epigenetic regulators in the cardiac lineage decision, we compared their expression levels in Brachyury + mesodermal cells with Brachyury - cells, and in Nkx2.5 + cardiac progenitor cells with the Nkx2.5 - cells, respectively. To further understand the role of these epigenetic regulators in cardiac lineage commitment, we knockout these genes and investigate their function. For example, deletion of the H3K9me2 demethylase PHF8 in mESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis, without significant differences between differentiating wide-type (ph8 +/Y ) and ph8 -/Y ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. These results reveal the new epigenetic control mechanism in mesodemal and cardiac differentiation and establish a link between the apoptosis and cell lineage decision as well as cardiogenesis.

An Optimized and Simplified System of Mouse Embryonic Stem Cell Cardiac Differentiation for the Assessment of Differentiation Modifiers

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation.

Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

Identification of factors that direct embryonic stem (ES) cell (ESC) differentiation into functional cardiomyocytes is essential for successful use of ESC-based therapy for cardiac repair. Neuregulin-1 (NRG1) and microRNA play important roles in the cardiac differentiation of ESCs. Understanding how NRG1 regulates microRNA will provide new mechanistic insights into the role of NRG1 on ESCs. It may also lead to the discovery of novel microRNAs that are important for ESC cardiac differentiation. The objective of this study was to assess the microRNA expression profile during NRG1-induced ESC cardiac differentiation. Murine ESCs were incubated with a recombinant NRG1β or an inhibitor of ErbB2 or ErbB4 during hanging drop-induced cardiac differentiation. The expression of cardiac-specific markers and microRNAs was analyzed by RT-PCR and microRNA array, respectively. We found that the expression of NRG1 and the ErbB receptors was increased during hanging drop-induced cardiac differentiation of ESCs. NRG1 stimulation during a specific developmental window enhanced, while inhibition of the ErbB2 or ErbB4 receptor inhibited, cardiac differentiation of ESCs. NRG1 increased the expression of mmu-miR-296-3p and mmu-miR-200c*, and decreased mmu-miR-465b-5p. Inhibition of mmu-miR-296-3p or mmu-miR-200c* decreased, while inhibition of mmu-miR-465-5p increased, the differentiation of ESCs into the cardiac lineage. This is the first report demonstrating that microRNAs are differentially regulated by NRG1-ErbB signaling during cardiac differentiation of ESCs. This study has also identified new microRNAs that are important for ESC cardiac differentiation.

Glycolytic network restructuring integral to the energetics of embryonic stem cell cardiac differentiation

Decoding of the bioenergetic signature underlying embryonic stem cell cardiac differentiation has revealed a mandatory transformation of the metabolic infrastructure with prominent mitochondrial network expansion and a distinctive switch from glycolysis to oxidative phosphorylation. Here, we demonstrate that despite reduction in total glycolytic capacity, stem cell cardiogenesis engages a significant transcriptome, proteome, as well as enzymatic and topological rearrangement in the proximal, medial, and distal modules of the glycolytic pathway. Glycolytic restructuring was manifested by a shift in hexokinase (Hk) isoforms from Hk-2 to cardiac Hk-1, with intracellular and intermyofibrillar localization mapping mitochondrial network arrangement. Moreover, upregulation of cardiac-specific enolase 3, phosphofructokinase, and phosphoglucomutase and a marked increase in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) phosphotransfer activity, along with apparent post-translational modifications of GAPDH and phosphoglycerate kinase, were all distinctive for derived cardiomyocytes compared to the embryonic stem cell source. Lactate dehydrogenase (LDH) isoforms evolved towards LDH-2 and LDH-3, containing higher proportions of heart-specific subunits, and pyruvate dehydrogenase isoforms rearranged between E1α and E1β, transitions favorable for substrate oxidation in mitochondria. Concomitantly, transcript levels of fetal pyruvate kinase isoform M2, aldolase 3, and transketolase, which shunt the glycolytic with pentose phosphate pathways, were reduced. Collectively, changes in glycolytic pathway modules indicate active redeployment, which would facilitate connectivity of the expanding mitochondrial network with ATP utilization sites. Thus, the delineated developmental dynamics of the glycolytic phosphotransfer network is integral to the remodeling of cellular energetic infrastructure underlying stem cell cardiogenesis.

Abstract 5442: Oxidative Stress Regulates Cardiac Stem Cell Differentiation in a MicroRNA-Mediated Mechanism

Oxidative stress, specifically hydrogen peroxide (H 2 O 2 ) is significantly increased following myocardial infarction and its effect on adult cardiac stem cell (CSC) differentiation is largely unknown. To promote endogenous and implanted CSC differentiation into functional cardiac cells in ischemic tissue, mechanisms of deleterious redox signaling on cardiomyogenesis must be determined. Our data demonstrate that H 2 O 2 time- and dose-dependently inhibits Nkx2.5 expression in CSCs exposed to differentiating medium. Since overexpression of microRNA (miR)-1 increases embryonic stem cell cardiac differentiation, we hypothesized that H 2 O 2 regulates CSC differentiation in a miRNA-mediated mechanism. CSCs exposed to H 2 O 2 (1–100 μ M) in differentiating media demonstrated time-dependent, decreased expression of miR1 compared to cells exposed to differentiating media alone (1 μ M: 37±7.6% decrease from Day 5 control; 100 μ M: 47±11% decrease from Day 8 control). MiR133a, essential for normal cardiac development, was not time-dependently regulated by H 2 O 2 , suggesting it is not redox sensitive. Since cardiac differentiation in Drosophila is regulated by miR-1 binding to the Notch ligand Delta and Notch activation attenuates embryonic stem cell cardiogenesis, we next examined the effect of miR1 on the Notch pathway in CSCs. Delta1 expression increased 24±5% above the time-matched control exposed to differentiating media alone after 8 days exposure to H 2 O 2 (0.1 μ M). Since miR1 exhibited the inverse response to H 2 O 2 , the data support Delta1 as a target for miR1-mediated silencing in CSCs. In summary, our data suggest a mechanism for redox-mediated attenuation of CSC differentiation. H 2 O 2 decreases miR1 expression, which prevents normal Delta1 silencing by miR-1 that occurs during differentiation. This failure of Delta1 degradation increases Notch activation, which subsequently suppresses cardiogenic differentiation. This study underscores the importance of H 2 O 2 scavenging following cardiac injury to promote CSC differentiation, and suggests that targeting anti-cardiogenic miRNA regulation may be a promising strategy for successful CSC-based therapy.

Developmental Restructuring of the Creatine Kinase System Integrates Mitochondrial Energetics with Stem Cell Cardiogenesis

Differentiation of pluripotent low-energy requiring stem cells into the high-energy expenditure cardiac lineage requires coordination of genomic programming and energetic system maturation. Here, in a murine embryonic stem cell cardiac differentiation model, emergence of electrical and beating activity in cardiomyocytes developing within embryoid bodies was coupled with the establishment of the mitochondrial network and expansion of the creatine kinase (CK) phosphotransfer system. Stem cell cardiogenesis was characterized by increased total CK activity, an isoform shift manifested by amplified muscle CK-M mRNA levels and protein content, and the appearance of cardiac-specific CK-MB dimers. Treatment of differentiating stem cells with BMP2, a cardiogenic growth factor, promoted CK activity. CK-M clustered around developing myofibrils, sarcolemma, and the perinuclear compartment, whereas CK-B was tightly associated with myofibrillar alpha-actinin, forming wire-like structures extending from the nuclear compartment to the sarcolemma. Developmentally enhanced phosphotransfer enzyme-anchoring protein FHL2 coalesced the myofibrillar CK metabolic signaling circuit, providing an energetic continuum between mitochondria and the nascent contractile machinery. Thus, the evolving CK-catalyzed phosphotransfer network integrates mitochondrial energetics with cardiogenic programming, securing the emergence of energy-consuming cardiac functions in differentiating embryonic stem cells.

Cardioinductive Network Guiding Stem Cell Differentiation Revealed by Proteomic Cartography of Tumor Necrosis Factor α-Primed Endodermal Secretome

In the developing embryo, instructive guidance from the ventral endoderm secures cardiac program induction within the anterolateral mesoderm. Endoderm-guided cardiogenesis, however, has yet to be resolved at the proteome level. Here, through cardiopoietic priming of the endoderm with the reprogramming cytokine tumor necrosis factor alpha (TNFalpha), candidate effectors of embryonic stem cell cardiac differentiation were delineated by comparative proteomics. Differential two-dimensional gel electrophoretic mapping revealed that more than 75% of protein species increased >1.5-fold in the TNFalpha-primed versus unprimed endodermal secretome. Protein spot identification by linear ion trap quadrupole (LTQ) tandem mass spectrometry (MS/MS) and validation by shotgun LTQ-Fourier transform MS/MS following multidimensional chromatography mapped 99 unique proteins from 153 spot assignments. A definitive set of 48 secretome proteins was deduced by iterative bioinformatic screening using algorithms for detection of canonical and noncanonical indices of secretion. Protein-protein interaction analysis, in conjunction with respective expression level changes, revealed a nonstochastic TNFalpha-centric secretome network with a scale-free hierarchical architecture. Cardiovascular development was the primary developmental function of the resolved TNFalpha-anchored network. Functional cooperativity of the derived cardioinductive network was validated through direct application of the TNFalpha-primed secretome on embryonic stem cells, potentiating cardiac commitment and sarcomerogenesis. Conversely, inhibition of primary network hubs negated the procardiogenic effects of TNFalpha priming. Thus, proteomic cartography establishes a systems biology framework for the endodermal secretome network guiding stem cell cardiopoiesis.