Enhanced Cardiomyocyte Differentiation Research Articles

Hydrogel microsphere stem cell encapsulation enhances cardiomyocyte differentiation and functionality in scalable suspension system

A reliable suspension-based platform for scaling engineered cardiac tissue (ECT) production from human induced pluripotent stem cells (hiPSCs) is crucial for regenerative therapies. Here, we compared the production and functionality of ECTs formed using our scaffold-based, engineered tissue microsphere differentiation approach with those formed using the prevalent scaffold-free aggregate platform. We utilized a microfluidic system for the rapid (1 million cells/min), high density (30, 40, 60 million cells/ml) encapsulation of hiPSCs within PEG-fibrinogen hydrogel microspheres. HiPSC-laden microspheres and aggregates underwent suspension-based cardiac differentiation in chemically defined media. In comparison to aggregates, microspheres maintained consistent size and shape initially, over time, and within and between batches. Initial size and shape coefficients of variation for microspheres were eight and three times lower, respectively, compared to aggregates. On day 10, microsphere cardiomyocyte (CM) content was 27 % higher and the number of CMs per initial hiPSC was 250 % higher than in aggregates. Contraction and relaxation velocities of microspheres were four and nine times higher than those of aggregates, respectively. Microsphere contractile functionality also improved with culture time, whereas aggregate functionality remained unchanged. Additionally, microspheres displayed improved β-adrenergic signaling responsiveness and uniform calcium transient propagation. Transcriptomic analysis revealed that while both microspheres and aggregates demonstrated similar gene regulation patterns associated with cardiomyocyte differentiation, heart development, cardiac muscle contraction, and sarcomere organization, the microspheres exhibited more pronounced transcriptional changes over time. Taken together, these results highlight the capability of the microsphere platform for scaling up biomanufacturing of ECTs in a suspension-based culture platform.

The conserved long non-coding RNA CARMA regulates cardiomyocyte differentiation

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). 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. 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.

Stem Cells and Their Cardiac Derivatives for Cardiac Tissue Engineering and Regenerative Medicine.

Significance: Heart failure is among the leading causes of morbidity worldwide with a 5-year mortality rate of ∼50%. Therefore, major efforts are invested to reduce heart damage upon injury or maintain and at best restore heart function. Recent Advances: In clinical trials, acellular constructs succeeded in improving cardiac function by stabilizing the infarcted heart. In addition, strategies utilizing stem-cell-derived cardiomyocytes have been developed to improve heart function postmyocardial infarction in small and large animal models. These strategies range from injection of cell-laden hydrogels to unstructured hydrogel-based and complex biofabricated cardiac patches. Importantly, novel methods have been developed to promote differentiation of stem-cell-derived cardiomyocytes to prevascularized cardiac patches. Critical Issues: Despite substantial progress in vascularization strategies for heart-on-the-chip technologies, little advance has been made in generating vascularized cardiac patches with clinically relevant dimensions. In addition, proper electrical coupling between engineered and host tissue to prevent and/or eliminate arrhythmia remains an unresolved issue. Finally, despite advanced approaches to include hierarchical structures in cardiac tissues, engineered tissues do not generate forces in the range of native adult cardiac tissue. Future Directions: It involves utilizing novel materials and advancing biofabrication strategies to generate prevascularized three-dimensional multicellular constructs of clinical relevant size; inclusion of hierarchical structures, electroconductive materials, and biologically active factors to enhance cardiomyocyte differentiation for optimized force generation and vascularization; optimization of bioreactor strategies for tissue maturation. Antioxid. Redox Signal. 35, 143-162.

Sustained Activation of AMPK Enhances Differentiation of Human iPSC-Derived Cardiomyocytes via Sirtuin Activation

SummaryRecent studies suggest that metabolic regulation may improve differentiation of cardiomyocytes derived from induced pluripotent stem cells (iPSCs). AMP-activated protein kinase (AMPK) is a master regulator of metabolic activities. We investigated whether AMPK participates in iPSC-derived cardiomyocyte differentiation. We observed that AMPK phosphorylation at Thr172 increased at day 9 but then decreased after day 11 of differentiation to cardiomyocytes. Inhibition of AMPK with compound C significantly reduced mRNA and protein expression of cardiac troponins TNNT2 and TNNI3. Moreover, sustained AMPK activation using AICAR from days 9 to 14 of differentiation increased mRNA and protein expression of both TNNT2 and TNNI3. AICAR decreased acetylation of histone 3 at Lys9 and 56 and histone 4 at Lys16 (known target sites for nuclear-localized sirtuins [SIRT1, SIRT6]), suggesting that AMPK activation enhances sirtuin activity. Sustained AMPK activation during days 9–14 of differentiation induces sirtuin-mediated histone deacetylation and may enhance cardiomyocyte differentiation from iPSCs.

Discovery of Natural Compounds Promoting Cardiomyocyte Differentiation.

The commitment of pluripotent stem cells to the cardiac lineage has enormous potential in regenerative medicine interventions for several cardiac diseases. Thus, it is necessary to understand and regulate this differentiation process for potential clinical application. In this study, we developed defined conditions with chemical inducers for effective cardiac lineage commitment and elucidated the mechanism for high-efficiency differentiation. First, we designed a robust reporter-based platform to screen chemical inducers of cardiac differentiation in the mouse P19 teratocarcinoma cell line. Using this system, we identified two natural alkaloids, lupinine and ursinoic acid, which enhanced cardiomyocyte differentiation of P19 cells in terms of beating colony numbers with respect to oxytocin, and confirmed their activity in mouse embryonic stem cells. By analyzing the expression of key markers, we found that this enhancement can be attributed to the early and rapid induction of the Wnt signaling pathway. We also found that these natural compounds could not only supersede the action of the Wnt3a ligand but also had a very quick response time, allowing them to act as efficient cardiac mesoderm inducers that subsequently promoted cardiomyocyte differentiation. Thus, this study offers a way to develop chemical-based differentiation strategy for high-efficiency cardiac lineage commitment, which has an advantage over currently available methods with complex medium composition and parameters. Furthermore, it also provides an opportunity to pinpoint the key molecular mechanisms pivotal to the cardiac differentiation process, which are necessary to design an efficient strategy for cardiomyocyte differentiation.

A Novel Atypical PKC-Iota Inhibitor, Echinochrome A, Enhances Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells.

Echinochrome A (EchA) is a marine bioproduct extracted from sea urchins having antioxidant, antimicrobial, anti-inflammatory, and chelating effects, and is the active component of the clinical drug histochrome. We investigated the potential use of Ech A for inducing cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). We also assessed the effects of Ech A on mitochondrial mass, inner membrane potential (Δψm), reactive oxygen species generation, and levels of Ca2+. To identify the direct target of Ech A, we performed in vitro kinase activity and surface plasmon resonance binding assays. Ech A dose-dependently enhanced cardiomyocyte differentiation with higher beating rates. Ech A (50 μM) increased the mitochondrial mass and membrane potential but did not alter the mitochondrial superoxide and Ca2+ levels. The in vitro kinase activity of the atypical protein kinase C-iota (PKCι) was significantly decreased by 50 μM of Ech A with an IC50 for PKCι activity of 107 μM. Computational protein-ligand docking simulation results suggested the direct binding of Ech A to PKCι, and surface plasmon resonance confirmed the direct binding with a low KD of 6.3 nM. Therefore, Ech A is a potential drug for enhancing cardiomyocyte differentiation from mESCs through direct binding to PKCι and inhibition of its activity.

Aspirin inhibits growth and enhances cardiomyocyte differentiation of bone marrow mesenchymal stem cells

This study aimed to examine the effects of aspirin on the growth and cardiomyocyte differentiation grade of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs were divided into five differentiation groups with different concentrations of aspirin (0 mM, 0.5 mM, 1 mM, 2 mM, or 5 mM), and a undifferentiated control group. Cell growth was measured by cell proliferation, apoptosis assays and DNA cycle analysis. The differentiation grade of BMMSC-derived cardiomyocyte-like cells was examined by measuring the levels of cardiac-specific proteins with cyto-immunofluorescence staining, flow cytometry, and Western blotting. Electrophysiological analyses were performed by patch-clamp experiments and calcium transients were measured by a laser scanning confocal microscope. Cell proliferation decreased as the concentration of aspirin increased. Cell apoptosis increased with increasing aspirin concentration. DNA replication was inhibited in the high dose-aspirin group compared to the low dose- or non-aspirin groups. The number of α-myosin heavy chain (α-MHC) and cardiac troponin I (cTnI) positive cells, cardiac troponin T (cTnT) and connexin 43 (Cx43) positive rates, expression levels of Cx43, Nkx2.5, GATA4 and β1 adrenoceptor increased with increasing aspirin concentration. No sarcomeric cross-striations, spontaneous or induced beating activity or action potentials was observed in each group. Calcium transients were measured in small number cells in 2 mM aspirin group, but the features are atypical. Consequently, aspirin inhibits proliferation and survival of BMMSCs and enhances cardiomyocyte differentiation of BMMSCs.

Nanopillar Surface Topology Promotes Cardiomyocyte Differentiation through Cofilin-Mediated Cytoskeleton Rearrangement.

Nanoscaled surface patterning is an emerging potential method of directing the fate of stem cells. We adopted nanoscaled pillar gradient patterned cell culture plates with three diameter gradients [280-360 (GP 280/360), 200-280 (GP 200/280), and 120-200 nm (GP 120/200)] and investigated their cell fate-modifying effect on multipotent fetal liver kinase 1-positive mesodermal precursor cells (Flk1+ MPCs) derived from embryonic stem cells. We observed increased cell proliferation and colony formation of the Flk1+ MPCs on the nanopattern plates. Interestingly, the 200-280 nm-sized (GP 200/280) pillar surface dramatically increased cardiomyocyte differentiation and expression of the early cardiac marker gene Mesp1. The gradient nanopattern surface-induced cardiomyocytes had cardiac sarcomeres with mature cardiac gene expression. We observed Vinculin and p-Cofilin-mediated cytoskeleton reorganization during this process. In summary, the gradient nanopattern surface with 200-280 nm-sized pillars enhanced cardiomyocyte differentiation in Flk1+ MPCs.

Ascorbic acid-mediated enhanced cardiomyocyte differentiation of mouse ES-cells involves interplay of DNA methylation and multiple-signals

Embryonic stem cells (ES-cells) provide a good model system to study lineage-specific differentiation. Though, the differentiation of ES-cells to cardiomyocytes is documented, a clear understanding of the molecular mechanism of differentiation and improved functional-differentiation efficiency are yet to be achieved. In this regard, ascorbic acid (Aa) is shown to be one of the effective cardiac inducers in ES-cells. But, its mechanism is poorly understood. We therefore, investigated the mechanism of Aa-mediated cardiomyocyte differentiation of ES-cells. Here, we describe the potential involvement of epigenetic (DNA methylation) as well as integrin- and Erk- signaling systems during cardiomyocyte differentiation. Transgenic GS-2 ES-cells and wild-type D3 ES-cells were differentiated to cardiomyocytes, in the presence or absence of Aa and with or without inhibitors of Erk-, collagen- and integrin- pathways. At specific time points, differentiated states of ES-cells were scored by gene expression analyses and the proportion of functional cTnI+ cardiomyocytes. DNA methylation changes of Isl-1, BMP-2, GATA-4 and α-MHC in cardiogenic cells, following stimulation with Aa, were analyzed by using methylation specific PCR (MSP). We observed that Aa, when applied in initial phase of ES-cell differentiation, consistently enhanced cardiac differentiation (99%) over that observed during spontaneous differentiation (70%). This was associated with enhanced expressions of cardiogenesis-associated genes. A two-fold increase in cTnI+ cells was observed, with appropriate myofibril arrangement. The observed effect of Aa was due to enhanced collagen and integrin signaling, coupled with a high p-ERK1/2 expression, downstream. Besides, the involvement of DNA methylation in regulating the expression of cardiac genes i.e., Isl-1 and α-MHC was also observed. Overall, this study, for the first time, demonstrates that Aa-mediated cardiac enhancement is brought about, mechanistically, through the interplay of epigenetic changes in DNA methylation of cardiac genes (Isl-1 and α-MHC) and integrin signaling system.

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.

Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1.

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8(-/Y) ) model, we found that deletion of phf8 in ESCs 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 through a caspase 3-independent pathway during early ESC differentiation, 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. Furthermore, knockdown of pmaip1 mimicked the phenotype of ph8(-/Y) by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8(-/Y) ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells 2016;34:1527-1540.

Resveratrol Enhances Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells through Inhibiting Canonical WNT Signal Pathway and Enhancing Serum Response Factor-miR-1 Axis.

Resveratrol (trans-3,5,4′-trihydroxystilbene) (RSV) is a natural polyphenol with protective effects over cardiac tissues and can affect cell survival and differentiation in cardiac stem cells transplantation. However, whether this agent can affect cardiomyocytes (CMs) differentiation of induced pluripotent stem cells (iPSCs) is not yet clear. This study explored whether RSV can affect CMs differentiation of human iPSCs. Under embryoid bodies (EBs) condition, the effect of RSV on the change of pluripotent markers, endoderm markers, mesoderm markers, and ectoderm markers was measured using qRT-PCR. Under CM differentiation culture, the effect of RSV on CM specific markers was also measured. The regulative role of RSV over canonical Wnt signal pathway and serum response factor- (SRF-) miR-1 axis and the functions of these two axes were further studied. Results showed that RSV had no effect on the self-renewal of human iPSCs but could promote mesoderm differentiation. Under CM differentiation culture, RSV could promote CM differentiation of human iPSCs through suppressing canonical Wnt signal pathway and enhancing SRF-miR-1 axis.

Autologous serum enhances cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells in the presence of transforming growth factor-β1 (TGF-β1)

In spite of previous reports, the role of transforming growth factor-β1 (TGF-β1) on cardiomyocyte differentiation, especially in the present autologous serum (AS) in culture medium, is still unclear. So, the purpose of this study was to investigate the potential of rat bone marrow mesenchymal stem cells (rBMSCs) to proliferate and differentiate towards cardiomyocyte lineage with the use of AS. Most expansion protocols use a medium supplemented with fetal bovine serum (FBS) as nutritional supplement. FBS is an adverse additive to cells that are proliferated for therapeutic purposes in humans because the use of FBS carries the risk of transmitting viral and bacterial infections and proteins that may initiate xenogeneic immune responses. Therefore, bone marrow cells were cultured in a medium supplemented with 10% AS, 10% FBS, and serum free medium (SFM). Then, rBMSCs were cultured with TGF-β1 (10 ng/ml) for 2 wk. The number of viable cells in AS and FBS groups were measured with MTT assay. Beating areas frequency, up to fourth week after plating, were monitored and evaluated daily. The characteristics of cardiomyocytes were assessed by semi-quantitative reverse transcription polymerase chain reaction and western blot. MTT result indicated that rBMSCs in AS proliferated markedly faster than FBS and SFM. The number of beating areas significantly increased in AS compared to FBS medium. A noticeable increase in the cardiac genes expression was observed in AS. Moreover, western blot analysis confirmed that cardiac proteins were increased in the AS condition. In conclusion, the present study could be extended toward the safe culture of MSCs for the treatment of heart defects.

Spontaneous cardiomyocyte differentiation of mouse embryoid bodies regulated by hydrogel crosslink density.

Cellular therapies have great potential to provide alternative treatment options for those suffering from heart disease. In order to optimize cell delivery for therapeutic efficacy, a greater understanding of parameters that impact stem cell differentiation, survival, growth, and development are needed. In this study, we examine the role of hydrogel crosslink density on spontaneous cardiomyocyte (CM) differentiation of murine embryoid bodies (EBs). CM differentiation was accelerated in hydrogels of low crosslink density, where 100% of the hydrogels were positive for CM differentiation compared to only 53% in the high crosslink density group after 8 days of culture. DNA microarray data suggests that enhanced CM differentiation in the low crosslink density hydrogels was not tissue specific but rather a result of favoured EB development and cell proliferation. Additionally, enhanced EB growth and differentiation in low crosslink density hydrogels was independent of RGD ligand density and not a consequence of enhanced diffusion. We also demonstrate that matrix metalloproteinase activity is required for spontaneous CM differentiation in 3D hydrogels. Low hydrogel crosslink density regulates spontaneous EB differentiation by promoting EB growth and development. Elucidating the effects of microenvironmental cues on cell differentiation can aid in the optimization of stem cell-based therapies for tissue regeneration.

Controlling Embryonic Stem Cell Growth and Differentiation by Automation: Enhanced and More Reliable Differentiation for Drug Discovery

Despite significant use in basic research, embryonic stem cells have just begun to be used in the drug discovery process. Barriers to the adoption of embryonic stem cells in drug discovery include the difficulty in growing cells and inconsistent differentiation to the desired cellular phenotype. Embryonic stem cell cultures require consistent and frequent handling to maintain the cells in a pluripotent state. In addition, the preferred hanging drop method of embryoid body (EB) differentiation is not amenable to high-throughput methods, and suspension cultures of EBs show a high degree of variability. Murine embryonic stem cells passaged on an automated platform maintained ≥90% viability and pluripotency. We also developed a method of EB formation using 384-well microplates that form a single EB per well, with excellent uniformity across EBs. This format facilitated high-throughput differentiation and enabled screens to optimize directed differentiation into a desired cell type. Using this approach, we identified conditions that enhanced cardiomyocyte differentiation sevenfold. This optimized differentiation method showed excellent consistency for such a complex biological process. This automated approach to embryonic stem cell handling and differentiation can provide the high and consistent yields of differentiated cell types required for basic research, compound screens, and toxicity studies.

Eosinophil cationic protein enhances cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells by stimulating the FGF receptor signaling pathway

We investigated the functional role of eosinophil cationic protein (ECP) in regulating cardiomyogenesis using mouse P19CL6 embryonic carcinoma cells. ECP was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells by enhancing the rate and area size of beating of cardiomyocyte and by facilitating the expression of cardiomyocyte-specific genes, such as GATA4 and α-MHC. Since cardiomyocyte differentiation in vivo is considered to follow mesoderm induction, the induction of Brachyury, a marker of mesoderm, was assessed. Brachyury expression was found to be enhanced after the addition of ECP. This enhancement was due to the stimulation of extracellular signal-regulated kinase (ERK)1/2 phosphorylation by ECP. In this context, treatment with SU5402, an inhibitor of fibroblast growth factor (FGF) receptor 1, suppressed Brachyury expression, phosphorylation of ERK1/2, and cardiomyocyte differentiation induced by ECP. We concluded that ECP might induce mesoderm differentiation through FGF signaling pathway and enhance subsequent cardiomyocyte differentiation in concert with dimethyl sulfoxide in P19CL6 cells. ECP may be a novel factor for cardiomyocyte differentiation, which should be very useful to prepare adequate numbers of cardiomyocytes for therapeutic cell transplantation.

Ascorbic acid rescues cardiomyocyte development in Fgfr1−/− murine embryonic stem cells

Fibroblast growth factor receptor 1 (Fgfr1) gene knockout impairs cardiomyocyte differentiation in murine embryonic stem cells (mESC). Here, various chemical compounds able to enhance cardiomyocyte differentiation in mESC [including dimethylsulfoxide, ascorbic acid (vitC), free radicals and reactive oxygen species] were tested for their ability to rescue the cardiomyogenic potential of Fgfr1−/− mESC. Among them, only the reduced form of vitC, l-ascorbic acid, was able to recover beating cell differentiation in Fgfr1−/− mESC. The appearance of contracting cells was paralleled by the expression of early and late cardiac gene markers, thus suggesting their identity as cardiomyocytes. In the attempt to elucidate the mechanism of action of vitC on Fgfr1−/− mESC, we analyzed several parameters related to the intracellular redox state, such as reactive oxygen species content, Nox4 expression, and superoxide dismutase activity. The results did not show any relationship between the antioxidant capacity of vitC and cardiomyocyte differentiation in Fgfr1−/− mESC. No correlation was found also for the ability of vitC to modulate the expression of pluripotency genes. Then, we tested the hypothesis that vitC was acting as a prolyl hydroxylase cofactor by maintaining iron in a reduced state. We first analyze hypoxia inducible factor (HIF)-1α mRNA and protein levels that were found to be slightly upregulated in Fgfr1−/− cells. We treated mESC with Fe2+ or the HIF inhibitor CAY10585 during the first phases of the differentiation process and, similar to vitC, the two compounds were able to rescue cardiomyocyte formation in Fgfr1−/− mESC, thus implicating HIF-1α modulation in Fgfr1-dependent cardiomyogenesis.

Static magnetic fields increase cardiomyocyte differentiation of Flk-1+ cells derived from mouse embryonic stem cells via Ca2+ influx and ROS production

AimsTo investigate the effects of static magnetic fields (MFs) on cardiomyogenesis of mouse embryonic stem (ES) cell-derived embryoid bodies and Flk-1+ cardiac progenitor cells and to assess the impact of cytosolic calcium [Ca2+]c and reactive oxygen species (ROS). Methods and resultsEmbryoid bodies and ES cell-derived Flk-1+ cardiovascular progenitor cells were exposed to static MFs. The expression of cardiac genes was evaluated by RT-PCR; sarcomeric structures were assessed by immunohistochemistry; intracellular ROS and [Ca2+]c of ES cells were examined by H2DCF-DA- and fluo-4-based microfluorometry. Treatment of embryoid bodies with MFs dose-dependent increased the number of contracting foci and cardiac areas as well as mRNA expression of the cardiac genes MLC2a, MLC2v, α-MHC and β-MHC. In Flk-1+ cells MFs (1mT) elevated both [Ca2+]c and ROS, increased expression of the cardiogenic transcription factors Nkx-2.5 and GATA-4 as well as cardiac genes. This effect was due to Ca2+ influx, since extracellular Ca2+ chelation abrogated ROS production and MF-induced cardiomyogenesis. Furthermore absence of extracellular calcium impaired sarcomere structures. Neither the phospholipase C inhibitor U73122 nor thapsigargin inhibited MF-induced increase in [Ca2+]c excluding involvement of intracellular calcium stores. ROS were generated through NAD(P)H oxidase, since NOX-4 but not NOX-1 and NOX-2 mRNA was upregulated upon MF exposure. Ablation of NOX-4 by sh-RNA and treatment with the NAD(P)H oxidase inhibitor diphenylen iodonium (DPI) totally abolished MF-induced cardiomyogenesis. ConclusionThe ability of static MFs to enhance cardiomyocyte differentiation of ES cells allows high throughput generation of cardiomyocytes without pharmacological or genetic modification.

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 µm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.

Hydrogels with time-dependent material properties enhance cardiomyocyte differentiation in vitro

Tissue-specific elastic modulus ( E), or ‘stiffness,’ arises from developmental changes in the extracellular matrix (ECM) and suggests that progenitor cell differentiation may be optimal when physical conditions mimic tissue progression. For cardiomyocytes, maturing from mesoderm to adult myocardium results in a 9-fold stiffening originating in part from a change in collagen expression and localization. To mimic this temporal stiffness change in vitro, thiolated-hyaluronic acid (HA) hydrogels were crosslinked with poly(ethylene glycol) diacrylate, and their dynamics were modulated by changing crosslinker molecular weight. With the hydrogel appropriately tuned to stiffen as heart muscle does during development, pre-cardiac cells grown on collagen-coated HA hydrogels exhibit a 3-fold increase in mature cardiac specific markers and form up to 60% more maturing muscle fibers than they do when grown on compliant but static polyacrylamide hydrogels over 2 weeks. Though ester hydrolysis does not substantially alter hydrogel stiffening over 2 weeks in vitro, model predictions indicate that ester hydrolysis will eventually degrade the material with additional time, implying that this hydrogel may be appropriate for in vivo applications where temporally changing material properties enhance cell maturation prior to its replacement with host tissue.