Cardiomyocyte Differentiation Of Embryonic Stem Cells Research Articles

Requirement of IP3 receptor 3 (IP3R3) in nitric oxide induced cardiomyocyte differentiation of mouse embryonic stem cells

Nitric oxide (NO) markedly induces cardiomyocyte (CM) differentiation of embryonic stem (ES) cells. Here we examined the role of the Ca2+ signaling in the NO-induced CM differentiation of mouse ES cells. We found that NO induced intracellular Ca2+ increases in ES cells in a dose-dependent manner, and application of IP3 pathway antagonists not only significantly inhibited this induced Ca2+ increase but also abolished NO-induced CM differentiation of ES cells. Subsequently, all 3 types of inositol 1, 4, 5-trisphosphate (IP3) receptors (IP3Rs) in mouse ES cells were individually or triply knocked down. Interestingly, only knockdown of type 3 IP3R (IP3R3) or triple-knockdown of three types of IP3Rs significantly inhibited the NO-induced Ca2+ increases. Consistently, IP3R3 knockdown blocked the NO-induced CM differentiation of ES cells. CMs derived from IP3R3 knockdown ES cells also showed both structural and functional defects. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO-induced CM differentiation of ES cells.

Inhibition of Cardiomyocytes Differentiation of Mouse Embryonic Stem Cells by CD38/cADPR/Ca2+ Signaling Pathway

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca(2+) mobilizing messenger that is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. Here we explored the role of CD38-cADPR-Ca(2+) in the cardiomyogenesis of mouse embryonic stem (ES) cells. We found that the mouse ES cells are responsive to cADPR and possess the key components of the cADPR signaling pathway. In vitro cardiomyocyte (CM) differentiation of mouse ES cells was initiated by embryoid body (EB) formation. Interestingly, beating cells appeared earlier and were more abundant in CD38 knockdown EBs than in control EBs. Real-time RT-PCR and Western blot analyses further showed that the expression of several cardiac markers, including GATA4, MEF2C, NKX2.5, and α-MLC, were increased markedly in CD38 knockdown EBs than those in control EBs. Similarly, FACS analysis showed that more cardiac Troponin T-positive CMs existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs compared with that in control EBs. On the other hand, overexpression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, CD38 knockdown ES cell-derived CMs possess the functional properties characteristic of normal ES cell-derived CMs. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2 cascade during CM differentiation of ES cells, and transiently inhibition of Erk1/2 blocked the enhanced effects of CD38 knockdown on the differentiation of CM from ES cells. Taken together, our data indicate that the CD38-cADPR-Ca(2+) signaling pathway antagonizes the CM differentiation of mouse ES cells.

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.

In This Issue

HomeCirculation ResearchVol. 109, No. 4In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published5 Aug 2011https://doi.org/10.1161/RES.0b013e31822ce912Circulation Research. 2011;109:349Wnt Inhibitors in hESC Cardiogenesis (p 360)Willems et al have identified a molecular booster of cardiomyocyte production.Download figureDownload PowerPointGiven the relative scarcity of hearts available for transplant, researchers are looking for ways to repair injured or diseased hearts by replacing lost myocytes. Much hope is placed in stem cell strategies, but whether such cells come from within the body or from an external source – such as embryonic stem (ES) cells – improvements in the understanding and efficiency of cardiomyocyte differentiation are needed. Willems et al tested 550 small molecules individually on ES-derived mesoderm cells to see which, if any, would improve cardiomyocyte differentiation. They found just one – a molecule called IWR, which stands for inhibitor of the Wnt signaling response. Wnt is a secreted signal responsible for the differentiation and development of a variety of tissue types. When added to ES-derived mesoderm cells, IWR promoted the expression of cardiac-specific genes and converted up to 30% of cells into cardiomyocytes – a whopping 200-fold increase over controls. Other, better-known Wnt inhibitors also improved cardiomyocyte differentiation, the team showed. Since IWR boosts cardiomyocyte differentiation of ES cells, it will be interesting to see whether it can do the same for the heart's own stem cells as well.Antibody Tagging for Imaging and Cell Delivery (p 365)Ta et al have a new technique for delivering molecules and cells to specific body locations. Their first target: blood clots.Download figureDownload PowerPointThe delivery of drugs, molecules, and therapeutic cells to particular body tissues, while often desired, is rarely easy. One strategy is to conjugate the molecule/cell of interest to an antibody that recognizes the target tissue. But such an approach often links the antibody to cargo in an array of conformations, some of which reduce antibody function. Ta et al have now developed a site-specific conjugation approach that creates just one antibody-cargo conformation, while maintaining antibody activity. To achieve this, the team used a bacterial enzyme that both recognizes and covalently links 2 peptide tag sequences. One tag was introduced into the antibody sequence – in this case an antibody that recognizes activated platelets. The other was added to the cargo –fluorescent proteins, magnetic particles, or cells. The fluorescence-conjugated antibodies bound activated platelets tightly in vitro, while in vivo the magnetic particle and cell-conjugated antibodies homed specifically to blood clots (which contain activated platelets). The new technique could be used to study, and potentially treat, thrombotic, atherosclerotic, and inflammatory diseases, say the team, but could also be adapted to target molecules or cells to any desired tissue.CD34+ Cell Therapy for Refactory Angina (p 428)There is new hope for patients with refractory angina, say Losordo et al.Therapies for angina include lifestyle changes, such as weight loss and smoking cessation, medications such as anti-platelet drugs, beta-blockers, calcium channel blockers and nitrates, as well as surgical interventions. There are some patients, however – currently 850,000 in the U.S. – that have exhausted these options, and suffer from so-called refractory angina. Recent pre-clinical and early clinical data suggest that CD34+ cells might help. Although CD34+ cells are hematopoietic stem cells, they can also give rise to endothelial cells, secrete pro-angiogenic factors, and promote neovascularization in ischemic heart tissue. Losordo et al now report data from a phase II double-blind trial in which 167 refractory angina patients had either their own CD34+ cells or placebo injected into their hearts. After six months, patients that received CD34+ cells had fewer episodes of angina and showed an increased tolerance to exercise compared with patients injected with placebo. Mortality was also lower. Interestingly, a lower dose of CD34+ cells (100,000) worked better than a higher dose (500,000). The researchers suggest that perhaps the high dose exceeds the optimum cell number for efficient paracrine effects. Whatever the case, the good news is that refractory angina patients could have access to another treatment option after all.Written by Ruth Williams Previous Back to top Next FiguresReferencesRelatedDetails August 5, 2011Vol 109, Issue 4 Advertisement Article InformationMetrics © 2011 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31822ce912 Originally publishedAugust 5, 2011 PDF download Advertisement

Extrinsic regulation of cardiomyocyte differentiation of embryonic stem cells

Cardiovascular disease is one of leading causes of death throughout the U.S. and the world. The damage of cardiomyocytes resulting from ischemic injury is irreversible and leads to the development of progressive heart failure, which is characterized by the loss of functional cardiomyocytes. Because cardiomyocytes are unable to regenerate in the adult heart, cell-based therapy of transplantation provides a potential alternative approach to replace damaged myocardial tissue and restore cardiac function. A major roadblock toward this goal is the lack of donor cells; therefore, it is urgent to identify the cardiovascular cells that are necessary for achieving cardiac muscle regeneration. Pluripotent embryonic stem (ES) cells have enormous potential as a source of therapeutic tissues, including cardiovascular cells; however, the regulatory elements mediating ES cell differentiation to cardiomyocytes are largely unknown. In this review, we will focus on extrinsic factors that play a role in regulating different stages of cardiomyocyte differentiation of ES cells.

Arginine Vasopressin-mediated Cardiac Differentiation: INSIGHTS INTO THE ROLE OF ITS RECEPTORS AND NITRIC OXIDE SIGNALING

Despite the existence of a functional arginine vasopressin (AVP) system in the adult heart and evidence that AVP induces myogenesis, its significance in cardiomyogenesis is currently unknown. In the present study, we hypothesized a role for AVP in cardiac differentiation of D3 and lineage-specific embryonic stem (ES) cells expressing green fluorescent protein under the control of atrial natriuretic peptide (Anp) or myosin light chain-2V (Mlc-2V) promoters. Furthermore, we investigated the nitric oxide (NO) involvement in AVP-mediated pathways. AVP exposure increased the number of beating embryoid bodies, fluorescent cells, and expression of Gata-4 and other cardiac genes. V1a and V2 receptors (V1aR and V2R) differentially mediated these effects in transgenic ES cells, and exhibited a distinct developmentally regulated mRNA expression pattern. A NO synthase inhibitor, L-NAME, powerfully antagonized the AVP-induced effects on cardiogenic differentiation, implicating NO signaling in AVP-mediated pathways. Indeed, AVP elevated the mRNA and protein levels of endothelial NO synthase (eNOS) through V2R stimulation. Remarkably, increased beating activity was found in AVP-treated ES cells with down-regulated eNOS expression, indicating the significant involvement of additional pathways in cardiomyogenic effects of AVP. Finally, patch clamp recordings revealed specific AVP-induced changes of action potentials and increased L-type Ca2+ (ICa,L) current densities in differentiated ventricular phenotypes. Thus, AVP promotes cardiomyocyte differentiation of ES cells and involves Gata-4 and NO signaling. AVP-induced action potential prolongation appears likely to be linked to the increased ICa,L current in ventricular cells. In conclusion, this report provides new evidence for the essential role of the AVP system in ES cell-derived cardiomyogenesis.

Efficient cardiomyocyte differentiation of embryonic stem cells by bone morphogenetic protein-2 combined with visceral endoderm-like cells

As the signals required for cardiomyocyte differentiation and functional regulation are complex and only partly understood, the mechanisms prompting the differentiation and specification of pluripotential embryonic stem (ES) cells into cardiomyocytes remain unclear. We hypothesized that a combined technology system, cocultured with a visceral endoderm (VE) - like cell line, END-2, and added cytokine BMP-2, would induce high percentage conversion of murine ES-D3 cell line into cardiomyocytes, and derived cardiomyocytes in this system would exhibit more mature characteristics. It was observed that 92% (P<0.01) ES cell-derived aggregates in this system exhibited rhythmic contractions, and the contractile areas were greater. By contrast, in ES cells cultured alone, on the feeder layer of END-2 cells, or with added BMP-2, the total percentage of beating aggregates was 19, 69 (P<0.01) and 44% (P<0.01), respectively. All the rhythmically contractile cells derived from ES cells expressed cardiac-specific proteins for troponin T. Among them, the combined system resulted in significantly increased cardiac-specific genes (NKx2.5, alpha-MHC). Transmission electron microscopy (TEM) revealed varying degrees of myofibrillar organization, and the combined system resulted in a more mature phenotype such as Z bands, nascent intercalated discs and gap junctions. Before shifting to the cardiomyocyte phenotype, this system could accelerate apoptosis of the cell population (P<0.01). The inductive efficacy of this system can provide an opportunity to facilitate cardiomyocyte differentiation of ES cells. The inducible effects of this system may depend on increasing cardiac-specific gene expression and the induction of apoptosis in cells that are not committed to cardiac differentiation.