Expression Of Cardiac-specific Markers Research Articles

The Effects of Mechanical Loading Variations on the Hypertrophic, Anti-Apoptotic, and Anti-Inflammatory Responses of Differentiated Cardiomyocyte-like H9C2 Cells.

Cardiomyocytes possess the ability to respond to mechanical stimuli by adapting their biological functions. This study investigated cellular and molecular events in cardiomyocyte-like H9C2 cells during differentiation as well as the signalling and gene expression responses of the differentiated cells under various mechanical stretching protocols in vitro. Immunofluorescence was used to monitor MyHC expression and structural changes during cardiomyoblast differentiation. Moreover, alterations in the expression of cardiac-specific markers, cell cycle regulatory factors, MRFs, hypertrophic, apoptotic, atrophy and inflammatory factors, as well as the activation of major intracellular signalling pathways were evaluated during differentiation and under mechanical stretching of the differentiated H9C2 cells. Compared to undifferentiated cells, advanced-differentiation cardiomyoblasts exhibited increased expression of cardiac-specific markers, MyHC, MRFs, and IGF-1 isoforms. Moreover, differentiated cells that underwent a low strain/frequency mechanical loading protocol of intermediate duration showed enhanced expression of MRFs and hypertrophic factors, along with a decreased expression of apoptotic, atrophy, and inflammatory factors compared to both high-strain/frequency loading protocols and to unloaded cells. These findings suggest that altering the strain and frequency of mechanical loading applied on differentiated H9C2 cardiomyoblasts can regulate their anabolic/survival program, with a low-strain/frequency stretching being, overall, most effective at inducing beneficial responses.

Injectable hydrogel for co-delivery of 5-azacytidine in zein protein nanoparticles with stem cells for cardiac function restoration

Heart failure is major cause of mortality associated with mostly Myocardial infarction (MI). Transplanting mesenchymal stem cells (MSC) have exhibited potential role in myocardial regeneration. Secretion of immune-modulatory cytokines and various growth factors after transplantation plays significant role in remodelling process of MI region. However, low retention, higher shear stress during administration and rejection at host infarct environment hinders therapeutic efficacy. Myocardial regeneration demands for accurate spatio-temporal delivery of MSCs with supportive vascular network that leads to improvement of cardiac function. In this study, injectable alginate based microporous hydrogel has been used to deliver 5-Azacytidine (5-Aza) in zein protein nanoparticle with MSCs for attenuating adverse cardiac remodelling after MI. Zein nanoparticles loaded with 5-Aza were prepared by liquid–liquid dispersion, and it was found that 35% of drug was released in 7 days supported with mathematical modelling. The presence of 5-Aza and zein in developed hydrogel supported in vitro MSC proliferation, migration and angiogenesis. Significant increased expression of cardiac specific markers, GATA4, MEF2C, MLC, SERCA and NKX2.5 was observed in vitro. 5-Aza loaded protein nanoparticle with MSCs encapsulated hydrogels in rat MI model also exhibited substantial improvement of functional cardiac parameters such as cardiac output and ejection fraction. Histopathological analysis showed reduced fibrosis, attenuated infarct expansion and cardiac tissue restoration and angiogenesis. In brief, we developed nanocarrier-hydrogel system a promising strategy for co-delivering 5-Aza as cardiac differentiation cue with MSCs to achieve higher cell retention and enhanced improvement in myocardial regeneration after MI.

Label-Free Isolation and Single Cell Biophysical Phenotyping Analysis of Primary Cardiomyocytes Using Inertial Microfluidics.

To advance the understanding of cardiomyocyte (CM) identity and function, appropriate tools to isolate pure primary CMs are needed. A label-free method to purify viable CMs from mouse neonatal hearts is developed using a simple particle size-based inertial microfluidics biochip achieving purities of over 90%. Purified CMs are viable and retained their identity and function as depicted by the expression of cardiac-specific markers and contractility. The physico-mechanical properties of sorted cells are evaluated using downstream real-time deformability cytometry. CMs exhibited different physico-mechanical properties when compared with non-CMs. Taken together, this CM isolation and phenotyping method could serve as a valuable tool to progress the understanding of CM identity and function, and ultimately benefit cell therapy and diagnostic applications.

Cardiac Differentiation of Adipose Tissue-Derived Stem Cells Is Driven by BMP4 and bFGF but Counteracted by 5-Azacytidine and Valproic Acid.

Objective Bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) play important roles in embryonic heart development. Also, two epigenetic modifying molecules, 5ˊ-azacytidine (5ˊ-Aza) and valproic acid (VPA) induce cardiomyogenesis in the infarcted heart. In this study, we first evaluated the role of BMP4 and bFGF in cardiac trans-differentiation and then the effectiveness of 5´-Aza and VPA in reprogramming and cardiac differentiation of human adipose tissue-derived stem cells (ADSCs). Materials and Methods In this experimental study, human ADSCs were isolated by collagenase I digestion. For cardiac differentiation, third to fifth-passaged ADSCs were treated with BMP4 alone or a combination of BMP4 and bFGF with or without 5ˊ-Aza and VPA pre-treatment. After 21 days, the expression of cardiac-specific markers was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, immunocytochemistry, flow cytometry and western blot analyses. Results BMP4 and more prominently a combination of BMP4 and bFGF induced cardiac differentiation of human ADSCs. Epigenetic modification of the ADSCs by 5ˊ-Aza and VPA significantly upregulated the expression of OCT4A, SOX2, NANOG, Brachyury/T and GATA4 but downregulated GSC and NES mRNAs. Furthermore, pre-treatment with 5ˊ-Aza and VPA upregulated the expression of TBX5, ANF, CX43 and CXCR4 mRNAs in three-week differentiated ADSCs but downregulated the expression of some cardiac-specific genes and decreased the population of cardiac troponin I-expressing cells. ConclusionOur findings demonstrated the inductive role of BMP4 and especially BMP4 and bFGF combination in cardiac trans-differentiation of human ADSCs. Treatment with 5ˊ-Aza and VPA reprogrammed ADSCs toward a more pluripotent state and increased tendency of the ADSCs for mesodermal differentiation. Although pre-treatment with 5ˊ-Aza and VPA counteracted the cardiogenic effects of BMP4 and bFGF, it may be in favor of migration, engraftment and survival of the ADSCs after transplantation.

Adipose tissue derived mesenchymal stem cells are better respondents to TGFβ1 for in vitro generation of cardiomyocyte-like cells.

Mesenchymal stem cells (MSCs) are multipotent cells which hold immense potential in translational research as a novel treatment modality. In recent years, MSCs isolated from various tissues have been used in several clinical trials for the treatment of cardiac injury caused by permanent myocardial loss. However, a better MSCs source and an optimum inducer for in vitro cardiac differentiation are still far reaching and unexplored. The aim of the study was to compare the ability and efficiency of differentiation of MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ADSC) into cardiomyocyte-like cells to aid translational research. To fulfill this aim, freshly isolated BM-MSCs and ADSCs were differentiated into cardiomyocytes using 5-Azacytidine (6μM) and TGF-β1 (25ng/ml). These two differentiation protocols were compared on the basis of morphological, transcriptional, translational and functionality analysis. Both tissue specific MSCs, ADSCs and BM-MSCs, have similar surface marker profile and population doubling time. In both the treatment regimes, BM-MSCs and ADSCs showed morphological changes like flattening of cells and myotube formation in concurrence with structure and multinucleation, with early sign of differentiation in ADSCs. Further, the expression of cardiac specific markers including myosin light chain-2v (Mlc-2v), cardiac troponin I (cTnI), and sarco/endoplasmic reticulum Ca2+-ATPase (SerCa2) were higher in AD-TGFβ1 group, both at transcriptional and translational level. During functionality analysis by KCl stimulation, increased intracellular calcium fluorescence was observed in AD- TGFβ1 group as compared to others. Thus, ADSCs proved to be a better choice for stem cell therapy in cardiovascular diseases when induced with TGF-β1.

Novel preparation of Au nanoparticles loaded Laponite nanoparticles/ECM injectable hydrogel on cardiac differentiation of resident cardiac stem cells to cardiomyocytes

Cardiac tissue engineering offers a facile biomedical technology to develop a cardiac tissue regeneration or tissue repair by involving combinations of effective biomaterials and nanomaterials with engineering strategies. The cardiac regenerative materials were fabricated by electrically active nanoparticles onto the biocompatible matrix to inspire low-resistance electrical signal in the native heart tissues. In the current report, we investigated on the improvement of cardiac functionally inspired conductive injectable hydrogel fabricated from the electroactive gold (Au) and laponite (Lap) nanoparticles loaded myocardial extracellular matrix (ECM) to enhance the functional and biological properties of cardiomyocytes. The incorporation of nanoformulations into the ECM was maintained the functionality behaviors and maintenance of electrical conductivity into cardiomyocytes. The effects of nanoparticles onto the ECM were provided decreasing porous structure and interconnected pores in the hydrogel structure for the favorable environment of cardiac tissues. The biological analysis of cell survival and immunostaining studies of cardiomyocytes established that Au loaded Lap/ECM hydrogel improve cell compatibility and phenotypes maturation of cardiac specific proteins. The combination of electrically active nanoformulations and biologically active ECM was enhanced the cell expression of cardiac specific markers (SAC, cTnl and Cx43), indicating the potential role of nanoparticles loaded ECM hydrogel as an appropriate cardiac regenerative material for the repair of infarcted myocardium.

Strategic design of cardiac mimetic core-shell nanofibrous scaffold impregnated with Salvianolic acid B and Magnesium l-ascorbic acid 2 phosphate for myoblast differentiation

The major loss of myocardial tissue extracellular matrix after infarction is a serious complication that leads to heart failure. Regeneration and integration of damaged cardiac tissue is challenging since the functional restoration of the injured myocardium is an incredible task. The injured micro environment of myocardium fails to regenerate spontaneously. The emergence of nano-biomaterials would be a promising approach to regenerate such a damaged cardiomyocytes tissue. Here, we have fabricated a dual bioactive embedded nanofibrous cardiac patch via coaxial electrospinning technique, to mimic the topographical and chemical cues of the natural cardiac tissue. The proportion and the concentration of the polymers were optimized for tailored delivery of bioactives from a spatio-temporally designed scaffold. The functionalization of polymeric core shell nanofibrous scaffold with dual bioactives enhanced the physico-chemical and bio-mechanical properties of the scaffolds that has resulted in a 3-dimensional topography mimicking the natural cardiac like extracellular matrix. The sustained delivery of bioactive signals, improved cell adhesion, proliferation, migration and differentiation could be attributed to its highly interconnected nanofibrous matrix with good extended morphology. Further, the expression of cardiac specific markers were found to increase on investigation of mRNA by real time PCR studies and proteins by immunofluorescence and western blotting techniques, confirming cell - biomaterial interactions. Flow cytometry analysis authenticated a potent mitochondrial membrane potential of cells treated with nanocomposite. In addition, in ovo studies in chicken chorioallantoic membrane assay confirm the efficacy of the developed scaffold in inducing angiogenesis required for maintaining its viability after transplantation onto the infarcted zone. These promising results demonstrate the potential of the composite nanofibrous scaffold as an effective biomaterial substrate for cardiac regeneration providing cues for development of novel cardiac therapeutics.

Synergistic role of 5-azacytidine and ascorbic acid in directing cardiosphere derived cells to cardiomyocytes in vitro by downregulating Wnt signaling pathway via phosphorylation of β-catenin.

BackgroundCardiosphere derived cells (CDCs) represent a valuable source in stem cell based therapy for cardiovascular diseases, yet poor differentiation rate hinders the transplantation efficiency. The aim of this study is to check the ability of 5-Azacytidine (Aza) alone and in combination with ascorbic acid (Aza+AA) in delineating CDCs to cardiomyogenesis and the underlying Wnt signaling mechanism in induced differentiation.MethodsCDCs were treated with Aza and Aza+AA for a period of 14 days to examine the expression of cardiac specific markers and Wnt downstream regulators by immunofluorescence, real time PCR and western blot.ResultsResults revealed that Aza+AA induced efficient commitment of CDCs to cardiomyogenic lineage. Immunofluorescence analysis showed significant augment for Nkx 2.5, GATA 4 and α-Sarcomeric actinin markers in Aza+AA group than control group (p = 0.0118, p = 0.009 and p = 0.0091, respectively). Relative upregulation of cardiac markers, Nkx 2.5 (p = 0.0156), GATA 4 (p = 0.0087) and down regulation of Wnt markers, β-catenin (p = 0.0107) and Cyclin D1 (p = 0. 0116) in Aza+AA group was revealed by RNA expression analysis. Moreover, the Aza+AA induced prominent expression of GATA 4, α-Sarcomeric actinin and phospho β-catenin while non phospho β-catenin and Cyclin D1 expression was significantly suppressed as displayed in protein expression analysis. Generation of spontaneous beating in Aza+AA treated CDCs further reinforced that Aza+AA accelerates the cardiomyogenic potential of CDCs.ConclusionCombined treatment of Aza along with AA implicit in inducing cardiomyogenic potential of CDCs and is associated with down regulating Wnt signaling pathway. Altogether, CDCs represent a valuable tool for the treatment of cardiovascular disorders.

Involment of RAS/ERK1/2 signaling and MEF2C in miR-155-3p inhibition-triggered cardiomyocyte differentiation of embryonic stem cell.

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate post-transcriptional gene expression by targeting messenger RNAs (mRNAs) for cleavage or translational repression. Growing evidence indicates that miR-155 expression changes with the development of heart and plays an important role in heart physiopathology. However, the role of miR-155 in cardiac cells differentiation is unclear. Using the well-established embryonic stem cell (ESC), we demonstrated that miR-155-3p expression was down-regulated during cardiogenesis from mouse ESC. By contrast, the myogenic enhance factor 2C (MEF2C), a predicted target gene of miR-155-3p, was up-regulated. We further demonstrated that miR-155-3p inhibition increased the percentage of embryoid bodies (EB) beating and up-regulated the expression of cardiac specific markers, GATA4, Nkx2.5, and cTnT mRNA and protein. Notably, miR-155-3p inhibition caused upregulation of MEF2C, KRAS and ERK1/2. ERK1/2 inhibitor, PD98059 significantly decreased the expression of MEF2C protein. These findings indicate that miR-155-3p inhibition promotes cardiogenesis, and its mechanisms are involved in the RAS-ERK1/2 signaling and MEF2C.

Comparison of Cardiomyocyte Differentiation Potential Between Type 1 Diabetic Donor- and Nondiabetic Donor-Derived Induced Pluripotent Stem Cells.

Type 1 diabetes mellitus (T1DM) is the most common type of diabetes in children and adolescents. Diabetic subjects are more likely to experience a myocardial infarction compared to nondiabetic subjects. In recent years, induced pluripotent stem cells (iPSCs) have received increasing attention from basic scientists and clinicians and hold promise for myocardial regeneration due to their unlimited proliferation potential and differentiation capacity. However, cardiomyogenesis of type 1 diabetic donor-derived iPSCs (T1DM-iPSCs) has not been investigated yet. The aim of the study was to comparatively analyze cardiomyocyte (CM) differentiation capacity of nondiabetic donor-derived iPSCs (N-iPSCs) and T1DM-iPSCs. The differentiated CMs were confirmed by both expression of cardiac-specific markers and presence of cardiac action potential. Since mitochondrial bioenergetics is vital to every aspect of CM function, extracellular acidification rates and oxygen consumption rates were measured using Seahorse extracellular flux analyzer. The results showed that N-iPSCs and T1DM-iPSCs demonstrated similar capacity of differentiation into spontaneously contracting CMs exhibiting nodal-, atrial-, or ventricular-like action potentials. Differentiation efficiency was up to 90%. In addition, the CMs differentiated from N-iPSCs and T1DM-iPSCs (N-iPSC-CMs and T1DM-iPSC-CMs, respectively) showed 1) well-regulated glucose utilization at the level of glycolysis and mitochondrial oxidative phosphorylation and 2) the ability to switch metabolic pathways independent of extracellular glucose concentration. Collectively, we demonstrate for the first time that T1DM-iPSCs can differentiate into functional CMs with well-regulated glucose utilization as shown in N-iPSCs, suggesting that T1DM-iPSC-CMs might be a promising autologous cell source for myocardial regeneration in type 1 diabetes patients.

Dynamic Support Culture of Murine Skeletal Muscle-Derived Stem Cells Improves Their Cardiogenic Potential In Vitro.

Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.

Oxytocin improves the expression of cardiac specific markers in porcine bone marrow stem cells differentiation

Bone marrow stem cells (BMSCs) treated with 5-azacytidine possess myogenic differentiation potential. Oxytocin (OT) induces cardiomyogenesis in murine embryonic and cardiac stem cells. We attempted to isolate, characterize, and induce OT-mediated cardiomyogenic differentiation of porcine pBMSCs. Cells were treated as: control, OT, and 5-azacytidine groups. During early passages, transcripts of Oct4, GATA4, OT receptor, and phospholamban were expressed. RT-PCR showed upregulation of GATA4 in OT and 5-azacytidine-induced groups. Immunocytochemistry revealed higher expressions of cardiac troponin T and myosin heavy chain in OT than in 5-azacytidine-induced groups (p < 0.01). Western blot analysis showed upregulation of cardiac troponin I in OT-induced pBMSCs (p < 0.01). We infer pBMSCs should be induced during early passages, when expressing transcription factors related to pluripotency and cardiomyogenesis, as well as OT receptor. The more abundant expression of cardiac specific proteins in OT-treated pBMSCs suggests OT could be a more potent cardiomyogenic inducer of pBMSC.

Promotion of cardiac differentiation of brown adipose derived stem cells by chitosan hydrogel for repair after myocardial infarction

The ability to restore heart function by replacement of diseased myocardium is one of the great challenges in biomaterials and regenerative medicine. Brown adipose derived stem cells (BADSCs) present a new source of cardiomyocytes to regenerate the myocardium after infarction. In this study, we explored an injectable tissue engineering strategy to repair damaged myocardium, in which chitosan hydrogels were investigated as a carrier for BADSCs. In vitro, the effect and mechanism of chitosan components on the cardiac differentiation of BADSCs were investigated. In vivo, BADSCs carrying double-fusion reporter gene (firefly luciferase and monomeric red fluorescent protein (fluc-mRFP)) were transplanted into infarcted rat hearts with or without chitosan hydrogel. Multi-techniques were used to assess the effects of treatments. We observed that chitosan components significantly enhanced cardiac differentiation of BADSCs, which was assessed by percentages of cTnT+ cells and expression of cardiac-specific markers, including GATA-4, Nkx2.5, Myl7, Myh6, cTnI, and Cacna1a. Treatment with collagen synthesis inhibitors, cis-4-hydroxy-d-proline (CIS), significantly inhibited the chitosan-enhanced cardiac differentiation, indicating that the enhanced collagen synthesis by chitosan accounts for its promotive role in cardiac differentiation of BADSCs. Longitudinal in vivo bioluminescence imaging and histological staining revealed that chitosan enhanced the survival of engrafted BADSCs and significantly increased the differentiation rate of BADSCs into cardiomyocytes in vivo. Furthermore, BADSCs delivered by chitosan hydrogel prevented adverse matrix remodeling, increased angiogenesis, and preserved heart function. These results suggested that the injectable cardiac tissue engineering based on chitosan hydrogel and BADSCs is a useful strategy for myocardium regeneration.

Study of Adipose Tissue-Derived Mesenchymal Stem Cells Transplantation for Rats with Dilated Cardiomyopathy

Increasing evidences indicated that adipose-derived mesenchymal stem cells (ADMSCs) can stay survive, then gradually proliferate and differentiate into myocardial cells after transplanted into damaged areas and improve function of heart. In this article, ADMSCs were isolated from adipose tissue of Wistar rats and cultured. When treated with 5-azacytidine (5-aza), ADMSCs were differentiated into myocardial cells, then we implant these cells into myocardium of rats of DCM to observe cell population and differentiation and compare cardiac function and hemodynamics changes before and after transplantation. The expression of Cardiac-specific markers indicated that ADMSCs which were isolated from adipose tissue of Wistar rats can differentiate into various cell types. Meanwhile, the treatment group displayed a higher level of LVESP, left ventricular intraventricular pressure (+dP/dt max), left ventricular intraventricular pressure (-dP/dt max) and left ventricular EF (%) than the control group. Altogether, these results indicate that heart systolic and diastolic function of rats of DCM was significantly improved meanwhile ventricular dilatation remodeling was inhibited after ADMSCs transplantation. Therefore, this research provides an experimental basis for further clinical application of ADMSCs transplantation for the treatment of DCM and non-ischemic HF.

GW24-e0516 Study of Adipose Tissue- Derived Mesenchymal Stem Cells Transplantation for rats with dilated cardiomyopathy

ObjectivesIncreasing evidences indicated that adipose-derived mesenchymal stem cells (ADMSCs) can stay survive, then gradually proliferate and differentiate into myocardial cells after transplanted into damaged areas and improve function of heart.MethodsIn...

MIR-499 INDUCES CARDIAC DIFFERENTIATION OF RAT BONE MARROW-DERIVED MESENCHYMAL STEM CELLS

ObjectivesTo test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells.MethodsThe fourth passage of rat BM-MSCs were infected with...

Phorbol myristate acetate differentiates human adipose-derived mesenchymal stem cells into functional cardiogenic cells

To achieve effective regeneration of injured myocardium, it is important to find physiological way of improving the cardiogenic differentiation of stem cells. Previous studies demonstrated that cardiomyocytes from bone marrow-derived mesenchymal stem cells (BMSCs) activated with phorbolmyristate acetate (PMA), a protein kinase C (PKC) activator, restore electromechanical function in infarcted rat hearts. In this study, we investigated the effect of PMA on cardiogenic differentiation of adipose-derived MSCs (ASCs) for clinical applications. To confirm the effect of PMA, ASCs treated with 1μM PMA were grown for nine days. The expression of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain) in PMA-treated MSCs was demonstrated by immunocytochemistry. Alhough few α1A receptors exist in ASCs, α1-adrenergic receptor subtypes were preferentially expressed in PMA-treated ASCs. Moreover, expression of the β-adrenergic and muscarinic receptors increased in PMA-treated ASCs compared to normal cells. The mRNA levels of Ca2+-related factors (SERCA 2a; sarcoplasmic reticulum Ca2+-ATPase, LTCC; L-type Ca2+ channel) in treated ASCs were similar to the levels in cardiomyocytes. Following the transplantation of chemically activated cardiogenic ASCs into infarcted myocardium, histological analysis showed that infarct size, interstitial fibrosis, and apoptotic index were markedly decreased and cardiac function was restored. In conclusion, PMA might induce the cardiogenic differentiation of human ASCs as well as BMSCs. This result suggests successful use of human ASCs in cardiac regeneration therapy.

MiR-499 induces cardiac differentiation of rat mesenchymal stem cells through wnt/β-catenin signaling pathway

ObjectiveTo test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells through the wnt/β-catenin signaling pathway. MethodsRat BM-MSCs were infected with lentiviral vectors bearing miR-499. The expression of cardiac-specific markers, NKx2.5, GATA4, MEF2C, and cTnI in these cells were examined by rtPCR or Western blot analysis and the activity of the wnt/β-catenin signaling pathway was evaluated by measuring the phosphorylation status of β-catenin. ResultsOver-expression of miR-499 in rat BM-MSCs increased the expression of cardiac-specific genes, such as NKx2.5, GATA4, MEF2C, and cTnI and decreased the ratio of phosphorylated/dephosphorylated β-catenin in the wnt/β-catenin signaling pathway, thus activating the pathway. Knocking down the expression of Dvl, an adaptor molecule in the wnt/β-catenin signaling, partially blocked the role of the miR-499 and decreased those cardiac-specific genes. ConclusionOver-expression of miR-499 in rat BM-MSCs induces them toward cardiac differentiation through the activating the wnt/β-catenin signal pathway.

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.

Combinatorial Activin Receptor-Like Kinase/Smad and Basic Fibroblast Growth Factor Signals Stimulate the Differentiation of Human Embryonic Stem Cells into the Cardiac Lineage

The transforming growth factor beta/bone morphogenetic protein-activated Smad signaling pathway plays a complicated role in the maintenance of human embryonic stem cell (hESC) pluripotency and in the cell fate decision of hESCs. Here, we report that sustained inhibition of the transforming growth factor beta type I receptor (also termed activin receptor-like kinase or ALK) using a chemical inhibitor selective for ALK4/5/7 (ALKi) leads to the cardiac differentiation of hESCs under feeder-free and serum-free conditions. Treatment with ALKi reduced Smad2/3 phosphorylation and increased Smad1/5/8 phosphorylation in hESCs, suggesting a requirement for active Smad1/5/8 signaling for cardiac induction in these cells when ALK/Smad2/3 is inhibited. Importantly, active basic fibroblast growth factor (bFGF) signaling was also required for ALKi-mediated cardiac differentiation of monolayer-cultured hESCs. The FGF receptor inhibitor SU5402 blocked ALKi-mediated cardiac induction in hESCs, whereas bone morphogenetic protein-4 enhanced the ALKi-induced increase in phospho-Smad1/5/8 levels but failed to induce the cardiac differentiation of hESCs and instead promoted trophoblastic differentiation. We also confirmed that ALKi potentially enhanced the cardiac differentiation of human embryoid bodies, as determined by expression of cardiac-specific markers, increased beating areas, and action potential recorded from beating areas. These results demonstrate that an ALKi could be used as a potential cardiac-inducing agent and that the development of culture conditions that provide an appropriate balance between ALK/Smad and bFGF signaling is necessary to direct the fate of hESCs into the cardiac lineage.