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Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells.

ABSTRACT: The purpose of this study is to establish a murine embryonic stem cell (mESC) line for isolation of functional ventricular cardiomyocytes (VCMs) and then to characterize the derived VCMs. By crossing the myosin light chain 2v (Mlc2v)-Cre mouse line with the reporter strain Rosa26-yellow fluorescent protein (YFP), we generated mESC lines from these double transgenic mice, in which Cre-mediated removal of a stop sequence results in the expression of YFP under the control of the ubiquitously active Rosa26 promoter specifically in the VCM. After induction of differentiation via embryoid body (EB) formation, contracting YFP(+) cells were detected within EBs and isolated by fluorescence-activated cell sorting. N-cadherin, the cadherin expressed in cardiomyocytes, and the major cardiac connexin (Cx) isoform, Cx43, were detected in the respective adherens and gap junctions in these VCMs. Using current clamp recordings we demonstrated that mESC-derived VCMs exhibited action potential characteristics comparable to those of neonatal mouse VCMs. Real-time intracellular calcium [Ca(2+)](i) imaging showed rhythmic intracellular calcium transients in these VCMs. The amplitude and frequency of calcium transients were increased by isoproterenol stimulation, suggesting the existence of functional ?-adrenergic signaling. Moreover, [Ca(2+)](i) oscillations responded to increasing frequencies of external electrical stimulation, indicating that VCMs have functional excitation-contraction coupling, a key factor for the ultimate cardiac contractile performance. The present study makes possible the production of homogeneous and functional VCMs for basic research as well as for cardiac repair and regeneration.

SUBMITTER: Lee MY

PROVIDER: S-EPMC3222859 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells.

Lee Min Young MY Sun Baonan B Schliffke Simon S Yue Zhichao Z Ye Mingyu M Paavola Jere J Bozkulak Esra Cagavi EC Amos Peter J PJ Ren Yongming Y Ju Rong R Jung Yong Woo YW Ge Xin X Yue Lixia L Ehrlich Barbara E BE Qyang Yibing Y

Stem cell research 20110822 1

The purpose of this study is to establish a murine embryonic stem cell (mESC) line for isolation of functional ventricular cardiomyocytes (VCMs) and then to characterize the derived VCMs. By crossing the myosin light chain 2v (Mlc2v)-Cre mouse line with the reporter strain Rosa26-yellow fluorescent protein (YFP), we generated mESC lines from these double transgenic mice, in which Cre-mediated removal of a stop sequence results in the expression of YFP under the control of the ubiquitously active ...[more]

PMID: 22099020

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Project description:BACKGROUND: Characterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes. RESULTS: A transgenic alpha-myosin heavy chain (MHC) embryonic stem cell lineage was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter. A puromycin-resistant, EGFP-positive, alpha-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of alpha-MHC-positive cardiomyocytes in comparison with undifferentiated alpha-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in alpha-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the alpha-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway. CONCLUSION: The specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.

Project description:BackgroundMicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive.Methods and resultsWe hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p<0.05) and contractile protein expression without affecting their electrophysiological properties (p>0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased I(to), I(Ks) and I(Kr), and decreased I(f) (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca(2+) transient amplitude and kinetics. Molecular pathway analyses were performed for further insights.ConclusionWe conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.

Dataset Information

Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells.

Publications

Derivation of functional ventricular cardiomyocytes using endogenous promoter sequence from murine embryonic stem cells.

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