Cardiac-specific Gene Expression Research Articles

Transcriptional Abundance of Myosin Light Chain 2 Gene in Cardiac Differentiated Canine Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are promising cell source for cardiac tissue engineering and cell based therapies for heart repair as they can be expanded in vitro and differentiated into most cardiovascular cell types, including cardiomyocytes. During embryonic heart development, this differentiation occurs under the influence of internal and external stimuli that guide cells to go down the cardiac lineage. The aim of this study was to characterize the cardiac differentiation potential of a canine iPS cell. With the use of a standard embryoid body-based differentiation protocol for iPS cells were differentiated for 24 days. In vitro differentiations of canine iPSCs via embryoid bodies (EBs) were produced by ‘Hanging Drop’ method. EB's were differentiated using 5-azacytidine (5-Aza). During differentiation, EBs were collected on day 4, 6, 8, 12, 16, 20 and 24 to evaluate the expression of cardiomyocyte specific marker. Analyses on molecular, structural, and functional levels demonstrated that iPS cell-derived cardiomyocytes show typical features of ES cell-derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes. The differentiated cells expressed cardiac-specific gene myosin light chain 2 (MYL2) which started from day 8 of differentiation and highest expression was observed on day 16. Immunocytochemistry and relative expression of cardiac specific genes revealed that iPS cells differentiate into functional cardiomyocytes and allow to derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.

Circulating re-entrant waves promote maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring

Directed differentiation methods allow acquisition of high-purity cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes has been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived cardiomyocytes can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, reentrant waves (ReWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with ReWs show higher maturation including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen-consumption rate, and enhanced contractile force. We subsequently use a mathematical model to interpret the origination, propagation, and long-term behavior of the ReWs within the SOTRs.

Design and characterization of an electroconductive scaffold for cardiomyocytes based biomedical assays.

Cardiovascular diseases (CVD) are a major cause of mortality worldwide. Accessibility to heart tissue is limited due to sampling issues and lack of appropriate culture conditions. In addition, animal models are not an ideal choice for physiological, pharmacological, and fundamental evaluations in the cardiovascular field due to interspecies differences. Hence, there is an inevitable need for functional in vitro cardiac models. In this study, we have synthesized a novel electroconductive scaffold comprised of cardiac extracellular matrix (ECM) derived pre-cardiogel (pCG) blended with polypyrrole (Ppy). Our data revealed that 2.5% (w/v) pyrrole (Py) had the highest possible Py ratio that provided pCG-Ppy gel formation. The prepared mixture was fabricated into a scaffold by using the freeze-dried method. The scaffolds had open interconnected pores that ranged from 55±24μm for the cardiogel (CG)-Ppy to 74±26μm for the CG scaffolds, with no alterations in vital ECM components of collagen, polysaccharides, and glycosaminoglycans (GAGs). Incorporation of Ppy increased the CG stiffness with a final complex modulus from 80 pa to 140 pa. The CG-Ppy group had significantly greater electrical conductivity than the CG group. Scaffolds supported neonatal mouse cardiomyocyte (NMCM) adhesion, viability, cardiac-specific gene expression, and spontaneous beating up to 14days after seeding. Among the fabricated hydrogels, the CG-Ppy group resulted in the synchronous beating of cardiomyocyte clusters and upregulation of cardiac genes involved in cardiac muscle contraction (cardiac troponin T [cTNT]) and cardiomyocyte electrical coupling (connexin 43 [Cx43]). Thus, this ECM-based electro-conductive scaffold might provide a promising substrate for constructing in vitro cardiac models for drug testing, disease modeling, developmental studies, and cardiac regenerative approaches.

Expression of Cardiac Specific Cell Marker in Ex Vivo Differentiated Canine iPSC

Induced Pluripotent stem cells (iPSC) have a high ability to renew and differentiate themselves into various lineages and as vehicles of cell based therapy. Stem cell can differentiate under appropriate in vitro and in vivo conditions into different cell types. This study described the establishment of condition for in vitro expression of alpha MHC gene in cardiac differentiated canine iPSC (ciPSC). In vitro differentiation of canine iPSCs via embryoid bodies (EBs) were produced by ‘Hanging Drop’ method. EB’s were differentiated by using IMDM differentiation media: FBS – 10%, NEAA (100X) – 0.5%, Â-Mercaptoethanol- 100mM, Gentamycin- 5µg/ml supplemented with Azacytidine- 0.5µM. During differentiation, EBs were collected on day 4, 6, 8, 12, 16, 20 and 24 for characterization of cardiomyocytes specific marker expression. Total RNA from EBs were extracted by using Trizol method and subsequently cDNA were synthesized. The differentiated cells expressed cardiac specific gene (Alpha MHC) which started from day 6 of differentiation upto day 24 Immunocytochemistry and relative expression of cardiac specific genes revealed that ciPSC have the potential to differentiate into cardiomyocytes which can be used for cardiac tissue regeneration and as disease models for pharmaceutical testing.

P6376The role of circulating lymphocytes on hypertensive heart disease

Abstract Aims and background Hypertension is a disease accompanied by moderate inflammation. Physical activity and pharmacological intervention protect the organisms against hypertension-dependent end-organ damage. They should therefore interfere with the mobilization of lymphocytes. Different subsets of lymphocytes should specifically interfere with vascular and cardiac remodeling. In this study we correlated the amount of circulating lymphocytes with the expression of vascular and cardiac specific genes in the aorta and left ventricle to identify the tissue-specific effects of these cells. Methods 50 female spontaneously hypertensive rats (SHR) and 6 female normotensive Wistar rats (Wis) were analyzed. They were grouped in 7 subgroups exposed to different treatment regimes (modification of activity, type of activity, aldosterone blockade, and a combination thereof. Indicator genes of mal-adaptive vascular and cardiac remodeling were quantified by qRT-PCR. Results SHRs had an increased number of circulating NK cells and monocytes, but lower numbers of T cells. T cells inversely correlated with the vascular expression of p22phox, a member of the NADPH oxidase complex. Thus, lower number of T-cells favors the vascular expression of p22phox and thereby oxidative stress. In the left ventricle, T cells are inversely correlated with the expression of arginase-1 and UCP-2 but positively correlated with Glut-4 expression. The lower number of T cells in SHR therefore favors the expression of proteins often associated with oxidative stress and glucose metabolism. Monocytes are positively correlated with key markers of left ventricular hypertrophy (such as ANP, collagen-1, and β-MHC). Thus, the increased number of monocytes in SHRs supports cardiac hypertrophy. NK cells are inversely correlated with the expression of anti-inflammatory cytokines in the aorta (IL-10, IL-15). Thus, high number of circulating NK cells seems to favor a pro-inflammatory phenotype in the vasculature. Conclusion In a standard model of essential hypertension the profile of circulating lymphocytes differs from that in normotensive rats. The different subsets of lymphocytes specifically interfere with specific processes in the ventricle and vessel. Collectively, the difference between hypertensive and normotensive rats (less T cells, more NK cells and monocytes) supports a mal-adaptive role under hypertensive conditions.

Mitochondrial Fusion by M1 Promotes Embryoid Body Cardiac Differentiation of Human Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) can be differentiated in vitro into bona fide cardiomyocytes for disease modelling and personalized medicine. Mitochondrial morphology and metabolism change dramatically as iPSCs differentiate into mesodermal cardiac lineages. Inhibiting mitochondrial fission has been shown to promote cardiac differentiation of iPSCs. However, the effect of hydrazone M1, a small molecule that promotes mitochondrial fusion, on cardiac mesodermal commitment of human iPSCs is unknown. Here, we demonstrate that treatment with M1 promoted mitochondrial fusion in human iPSCs. Treatment of iPSCs with M1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. The pro-fusion and pro-cardiogenic effects of M1 were not associated with changes in expression of the α and β subunits of adenosine triphosphate (ATP) synthase. Our findings demonstrate for the first time that hydrazone M1 is capable of promoting cardiac differentiation of human iPSCs, highlighting the important role of mitochondrial dynamics in cardiac mesoderm lineage specification and cardiac development. M1 and other mitochondrial fusion promoters emerge as promising molecular targets to generate lineages of the heart from human iPSCs for patient-specific regenerative medicine.

Role of somatic cell sources in the maturation degree of human induced pluripotent stem cell-derived cardiomyocytes

BackgroundInduced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) are a unique source of human cardiomyocytes for cardiac disease modeling. Incomplete functional maturation remains a major limitation, however. One of the determinants of iPSC-CM maturation is somatic cell origin. We therefore compared iPSC-CMs derived from different somatic cell sources. MethodsCardiac-derived mesenchymal progenitor cells (CPCs), bone marrow-derived mesenchymal stem cells (BMCs), and human dermal fibroblasts (HDFs) from same patients were reprogrammed into iPSCs and differentiated into iPSC-CMs. Expression of cardiac-specific genes, caffeine-responsive cells, and electrophysiological properties of differentiated cells were analyzed. To assess the contribution of epigenetic memory toward differences in gene expression observed during cardiac differentiation, DNA methylation patterns were determined in the early mesodermal cardiac promoter NKX2–5 and KCNQ1, which encodes for the pore-forming α-subunit of the slow component of delayed-rectifier potassium current (IKs). ResultsCardiac genes (MYH6, TNNI3, KCNQ1, KCNE1) were upregulated in CPC-vs. BMC- and HDF-iPSC-CMs. At early differentiation stages, CPC-iPSC-CMs displayed higher numbers of caffeine-responsive cells than BMC- and HDF-iPSC-CMs. The hERG1 (KV11.1) blocker, E4031, followed by the IKs blocker, JNJ303, increased extracellular field potential duration in CPC-iPSC-CMs to a greater extent than in BMC- and HDF-iPSC-CMs. The promoter region of NKX2–5 was more highly methylated in BMCs and HDFs compared to CPCs, and to a lesser extent in BMC-iPSCs compared to CPC-iPSCs. ConclusionsThese results suggest that human iPSCs from cardiac somatic cell sources may display enhanced capacity toward cardiac re-differentiation compared to non-cardiac cell sources, and that epigenetic mechanisms may play a role in this regard.

DNA methylation microarray uncovers a permissive methylome for cardiomyocyte differentiation in human mesenchymal stem cells

Differentiation of Wharton's Jelly-Mesenchymal Stem cells (WJ-MSCs) into cardiomyocytes (CMs) in vitro has been reported widely although contradictions remain regarding the maturation of differentiated MSCs into fully functioning CMs. Studies suggest that use of epigenetic modifiers like 5′Azacytidine (5-AC) in MSCs de-methylates DNA and results in expression of cardiac-specific genes (CSGs). However, only partial expression of the CSG set leads to incomplete differentiation of WJ-MSCs to CMs. We used the Agilent 180 K human DNA methylation microarray on WJ-MSCs, 5-AC treated WJ-MSCs and human cardiac tissue (hCT) to analyze differential DNA methylation profiles which were then validated by bisulfite sequencing PCR (BSP). BSP confirmed that only a limited number of CSGs were de-methylated by 5-AC in WJ-MSCs. It also revealed that hCT displays a methylation profile similar to promoter regions of CSG in untreated WJ-MSCs. Thus, the presence of hypo-methylated CSGs indicates that WJ-MSCs are ideal cell types for cardiomyogenic differentiation.

Polyethylene Terephthalate Textiles Enhance the Structural Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to serve as a model for human cardiomyocytes. However, hiPSC-CMs are still considered immature. CMs differentiated from hiPSCs more resemble fetal than adult cardiomyocytes. Putative factors enhancing maturation include in vitro culture duration, culture surface topography, and mechanical, chemical, and electrical stimulation. Stem cell-derived cardiomyocytes are traditionally cultured on glass surfaces coated with extracellular matrix derivatives such as gelatin. hiPSC-CMs are flat and round and their sarcomeres are randomly distributed and unorganized. Morphology can be enhanced by culturing cells on surfaces providing topographical cues to the cells. In this study, a textile based-culturing method used to enhance the maturation status of hiPSC-CMs is presented. Gelatin-coated polyethylene terephthalate (PET)-based textiles were used as the culturing surface for hiPSC-CMs and the effects of the textiles on the maturation status of the hiPSC-CMs were assessed. The hiPSC-CMs were characterized by analyzing their morphology, sarcomere organization, expression of cardiac specific genes, and calcium handling. We show that the topographical cues improve the structure of the hiPSC-CMs in vitro. Human iPSC-CMs grown on PET textiles demonstrated improved structural properties such as rod-shape structure and increased sarcomere orientation.

Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models.

Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.

Engineering human ventricular heart tissue based on macroporous iron oxide scaffolds

Myocardial infarction (MI) is a primary cardiovascular disease threatening human health and quality of life worldwide. The development of engineered heart tissues (EHTs) as a transplantable artificial myocardium provides a promising therapy for MI. Since most MIs occur at the ventricle, engineering ventricular-specific myocardium is therefore more desirable for future applications. Here, by combining a new macroporous 3D iron oxide scaffold (IOS) with a fixed ratio of human pluripotent stem cell (hPSC)-derived ventricular-specific cardiomyocytes and human umbilical cord-derived mesenchymal stem cells, we constructed a new type of engineered human ventricular-specific heart tissue (EhVHT). The EhVHT promoted expression of cardiac-specific genes, ion exchange, and exhibited a better Ca2+ handling behaviors and normal electrophysiological activity in vitro. Furthermore, when patched on the infarcted area, the EhVHT effectively promoted repair of heart tissues in vivo and facilitated the restoration of damaged heart function of rats with acute MI. Our results show that it is feasible to generate functional human ventricular heart tissue based on hPSC-derived ventricular myocytes for the treatment of ventricular-specific myocardium damage. Statement of significanceWe successfully generated highly purified homogenous human ventricular myocytes and developed a method to generate human ventricular-specific heart tissue (EhVHT) based on three-dimensional iron oxide scaffolds. The EhVHT promoted expression of cardiac-specific genes, ion exchange, and exhibited a better Ca2+ handling behaviors and normal electrophysiological activity in vitro. Patching the EhVHT on the infarct area significantly improved cardiac function in rat acute MI models. This EhVHT has a great potential to meet the specific requirements for ventricular damages in most MI cases and for screening drugs specifically targeting ventricular myocardium.

Modelling cardiac fibrosis using three-dimensional cardiac microtissues derived from human embryonic stem cells

BackgroundCardiac fibrosis is the most common pathway of many cardiac diseases. To date, there has been no suitable in vitro cardiac fibrosis model that could sufficiently mimic the complex environment of the human heart. Here, a three-dimensional (3D) cardiac sphere platform of contractile cardiac microtissue, composed of human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) and mesenchymal stem cells (MSCs), is presented to better recapitulate the human heart.ResultsWe hypothesized that MSCs would develop an in vitro fibrotic reaction in response to treatment with transforming growth factor-β1 (TGF-β1), a primary inducer of cardiac fibrosis. The addition of MSCs improved sarcomeric organization, electrophysiological properties, and the expression of cardiac-specific genes, suggesting their physiological relevance in the generation of human cardiac microtissue model in vitro. MSCs could also generate fibroblasts within 3D cardiac microtissues and, subsequently, these fibroblasts were transdifferentiated into myofibroblasts by the exogenous addition of TGF-β1. Cardiac microtissues displayed fibrotic features such as the deposition of collagen, the presence of numerous apoptotic CMs and the dissolution of mitochondrial networks. Furthermore, treatment with pro-fibrotic substances demonstrated that this model could reproduce key molecular and cellular fibrotic events.ConclusionsThis highlights the potential of our 3D cardiac microtissues as a valuable tool for manifesting and evaluating the pro-fibrotic effects of various agents, thereby representing an important step forward towards an in vitro system for the prediction of drug-induced cardiac fibrosis and the study of the pathological changes in human cardiac fibrosis.

Preparation of an Electrically Conductive Graphene Oxide/Chitosan Scaffold for Cardiac Tissue Engineering.

Cardiac tissue engineering is of great importance for therapeutic and pharmaceutical applications. The scaffolds that can provide electrical conductivity and structural organization will be highly beneficial for cardiac tissue engineering. Here, we developed conductive scaffolds with electrical conductivity and porous structure composed of chitosan (CS) blending with graphene oxide (GO) for cardiac tissue engineering. Our results showed that the swelling, porosity, and conductive properties of GO/CS scaffolds could be modulated via adjusting the ratio of GO to CS. More importantly, GO/CS scaffolds had a swelling ratio ranging from 23.20 to 27.38 (1000%) and their conductivity (0.134S/m) fell in the range of reported conductivities for native cardiac tissue. Furthermore, we assessed their biological activity by seeding heart H9C2 cells in GO/CS scaffolds. Our data showed that these GO/CS scaffolds exhibited good cell viability, promotion of cell attachment and intercellular network formation, and upregulation of the cardiac-specific gene and protein expression involved in muscle conduction of electrical signals (Connexin-43). Overall, it is concluded that the GO/CS scaffolds promote the properties of cardiac tissue constructs. Our findings provide a new strategy and insight in developing new scaffolds for cardiac tissue engineering.

Chitosan/Calcium Silicate Cardiac Patch Stimulates Cardiomyocyte Activity and Myocardial Performance after Infarction by Synergistic Effect of Bioactive Ions and Aligned Nanostructure.

Cardiac tissue engineering (CTE) remains a great challenge to construct a cell-inductive scaffold that has positive effects on cardiac cell behaviors and cardiac tissue repair. In this study, we for the first time demonstrated that Si ions evidently stimulated the expression of cardiac-specific genes and proliferation of neonatal rat cardiomyocytes (NRCMs) at concentration ranges of 0.13-10.78 ppm. Accordingly, the optimized concentrations of calcium silicate (CS) were incorporated into the controllable aligned chitosan electrospun nanofibers, constructing the composite cardiac patch scaffolds. These scaffolds showed synergistic effect of bioactive chemical and structural signals on both cardiomyocytes and endothelial cells with aligned cell morphology and enhanced viability and function characterized by upregulated expressions of cardiac and angiogenic specific markers, improved myofilament structure, and better Ca2+ transients of NRCMs as compared to the scaffolds free of CS component or with disordered structures. The in vivo studies further demonstrated that the NRCM-seeded aligned CS/chitosan cardiac patch evidently improved cardiac function via limiting the scar area and promoting angiogenesis in postmyocardial infarction rats. Conclusively, our study highlights the potential application of bioactive ions and nanostructured biomaterials in CTE, and the CS/chitosan composite cardiac patch may be a promising scaffold for repair of infarcted myocardium.

Gene expression changes in human iPSC-derived cardiomyocytes after X-ray irradiation

Purpose: Radiation-induced heart disease caused by cardiac exposure to ionizing radiation comprises a variety of cardiovascular effects. Research in this field has been hampered by limited availability of clinical samples and appropriate test models. In this study, we wanted to elucidate the molecular mechanisms underlying electrophysiological changes, which we have observed in a previous study.Materials and methods: We employed RNA deep-sequencing of human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) 48 h after 5 Gy X-ray irradiation. By comparison to public data from hiPSC-CMs and human myocardium, we verified the expression of cardiac-specific genes in hiPSC-CMs. Results were validated by qRT-PCR.Results: Differentially gene expression analysis identified 39 and 481 significantly up- and down-regulated genes after irradiation, respectively. Besides, a large fraction of genes associated with cell cycle processes, we identified genes implicated in cardiac calcium homeostasis (PDE3B), oxidative stress response (FDXR and SPATA18) and the etiology of cardiomyopathy (SGCD, BBC3 and GDF15).Conclusions: Notably, observed gene expression characteristics specific to hiPSC-CMs might be relevant regarding further investigations of the response to external stressors like radiation. The genes and biological processes highlighted in our study present promising starting points for functional follow-up studies for which hiPSC-CMs could pose an appropriate cell model when cell type specific peculiarities are taken into account.

Pentraxin 3 promotes cardiac differentiation of mouse embryonic stem cells through JNK signaling pathway.

Cardiovascular disease is a leading cause of death worldwide, requiring the development of new therapeutic strategies including stem cell therapy. Pentraxins (PTXs) are a superfamily of proteins highly involved in different myocardial disorders, and thus this study aimed to identify the modulation of long pentraxin 3 (PTX3) in the differentiation of mouse embryonic stem cells (mESCs) toward cardiomyocytes. Cell toxicity of PTX3 was detected by MTT and LDH assays in mESCs. Embryoid bodies (EBs) were differentiated using hanging drop method, and the beating was observed under microscope. Expressional levels of early cardiac progenitor marker genes were assessed by qRT-PCR. Expression of marker proteins in early myocardial development and the activation of JNK signaling pathway was evaluated by Western blot. PTX3 treatment at 50 ng/mL significantly promoted the expression of cardiac-specific marker genes including Nkx2.5, Mef2c, Tbx5, dHand, and αMHC, and increased the expression of cardiac maturity indicative markers including connexin 43 and troponin C1. PTX3 enhanced the phosphorylation of JNK across the incubation duration, whereas the activation of p38 remained the same as control group. Co-treatment of JNK signaling pathway inhibitor SP600125 impaired the PTX3-promoted transcription of Nkx2.5, Mef2c, Tbx5, dHand, and αMHC. This study revealed the promotion of PTX3 in the differentiation of mESCs into cardiomyocytes and the underlying mechanism.

Efficient generation of functional cardiomyocytes from human umbilical cord-derived virus-free induced pluripotent stem cells.

We have previously demonstrated that human umbilical cord-derived mesenchymal stem cells (UC-MSCs) can differentiate into cardiomyocyte-like cells. However, no contracting cells were observed during differentiation. In this study, we generated induced pluripotent stem cells (iPSCs) from UC-MSCs using mRNA reprogramming and focused on the differentiation of reprogrammed iPSCs into functional cardiomyocytes. For cardiac differentiation, the spontaneously contracting cell clusters were present on day 8 of differentiation. Immunostaining studies and cardiac-specific gene expression confirmed the cardiomyocyte phenotype of the differentiated cells. Electrophysiology studies indicated that iPSCs derived from UC-MSCs had a capacity for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics, and the derived cardiomyocytes exhibited responsiveness to β-adrenergic and muscarinic stimulations. Moreover, the derived cardiomyocytes displayed spontaneous intracellular Ca2+ transients. These results demonstrate that functional cardiomyocytes can be generated from reprogrammed UC-MSCs, and the methodology described here will serve as a useful protocol to obtain functional cardiomyocytes from human mesenchymal stem cells.

Comparative effectiveness of three-dimensional scaffold, differentiation media and co-culture with native cardiomyocytes to trigger in vitro cardiogenic differentiation of menstrual blood and bone marrow stem cells

The main purpose of this study was to find effectiveness of 3D silk fibroin scaffold in comparison with co-culturing in presence of native cardiomyocytes on cardiac differentiation propensity of menstural blood(MenSCs)-versus bone marrow-derived stem-cells (BMSCs). We showed that both 3D fibroin scaffold and co-culture system supported efficient cardiomyogenic differentiation of MenSCs and BMSCs, as judged by the expression of cardiac-specific genes and proteins, Connexin-43, Connexin-40, alpha Actinin (ACTN-2), Tropomyosin1 (TPM1) and Cardiac Troponin T (TNNT2). No significant difference (except for higher expression of ACTN-2 in co-cultured MenSCs) was found between differentiation potential of the cells cultured in 3D fibroin scaffold and co-culture system. Collectively, our results imply that inductive signals served by biological factors of native cardiomyocytes to trigger cardiogenic differentiation of stem-cells may be efficiently provided by natural and biocompatible 3D fibroin scaffold suggesting the usefulness of this construct for cardiac tissue engineering.

Chimeric Adeno-Associated Virus-Mediated Cardiovascular Reprogramming for Ischemic Heart Disease.

Here, we demonstrated chimeric adeno-associated virus (chimeric AAV), AAV-DJ-mediated cardiovascular reprogramming strategy to generate new cardiomyocytes and limit collagen deposition in cardiac fibroblasts by inducing synergism of chimeric AAV-expressing Gata4, Mef2c, Tbx5 (AAV-GMT)-mediated heart reprogramming and chimeric AAV-expressing thymosin β4 (AAV-Tβ4)-mediated heart regeneration. AAV-GMT promoted a gradual increase in expression of cardiac-specific genes, including Actc1, Gja1, Myh6, Ryr2, and cTnT, with a gradual decrease in expression of a fibrosis-specific gene, procollagen type I and here AAV-Tβ4 help to induce GMT expression, providing a chimeric AAV-mediated therapeutic cell reprogramming strategy for ischemic heart diseases.

Downregulation of MiR-199b-5p Inducing Differentiation of Bone-Marrow Mesenchymal Stem Cells (BMSCs) Toward Cardiomyocyte-Like Cells via HSF1/HSP70 Pathway.

BackgroundBone-marrow mesenchymal stem cells (BMSCs) are pluripotent stem cells with potent self-renewal and differentiation ability that are widely used in transplantation of cell therapy. But the mechanism on microRNA (miRNA) regulating stem cell differentiation is complicated and unclear. The aim of this study was to investigate whether miR-199b-5p is involved in differentiation of cardiomyocyte-like cells and identify potential signal pathways in BMSCs.Material/MethodsMouse BMSCs were treated with 5-azacytidine and transfected by miR-199b-5p mimic and inhibitor, respectively. qRT-PCR was used to detect the expression of miR-199b-5p in BMSCs, 5-azacytidine treated BMSCs, and neonatal murine cardiomyocytes. The expression of cardiac specific genes and the HSF1/HSP70 signal pathway were examined by qRT-PCR or western blotting. The proliferation and migration of BMSCs were evaluated by CCK-8 assay and wound-healing assay.ResultsThe expression of miR-199b-5p decreased gradually in the process of differentiation of BMSCs toward cardiomyocyte-like cells. The expression of cardiac specific genes and HSF1/HSP70 were increased in the miR-199b-5p inhibitor group; however, the miR-199b-5p mimic group presented an opposite result. Both the miR-199b-5p inhibitor group and the miR-199b-5p mimic group had no influence on BMSCs proliferation and migration. Using lentivirus vectors bearing HSF1 shRNA to silence HSF1 and HSP70, the anticipated elevated expression effect of cardiac specific genes induced by miR-199b-5p inhibitor was suppressed.ConclusionsDownregulation of miR-199b-5p induced differentiation of BMSCs toward cardiomyocyte-like cells partly via the HSF1/HSP70 signaling pathway, and had no influence on BMSCs proliferation and migration.