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Functional cardiomyocytes derived from human induced pluripotent stem cells.

ABSTRACT: Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines because of residual transgene expression. Proliferation of iPS and ES cell-derived cardiomyocytes based on 5-bromodeoxyuridine labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to beta-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.

SUBMITTER: Zhang J

PROVIDER: S-EPMC2741334 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Functional cardiomyocytes derived from human induced pluripotent stem cells.

Zhang Jianhua J Wilson Gisela F GF Soerens Andrew G AG Koonce Chad H CH Yu Junying J Palecek Sean P SP Thomson James A JA Kamp Timothy J TJ

Circulation research 20090212 4

Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body ( ...[more]

PMID: 19213953

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Project description:Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including ?-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.

Project description:BACKGROUND:Circular RNAs (circRNAs) are regarded as a novel class of noncoding RNA regulators. Although a number of circRNAs have been identified by bioinformatics analysis of RNA-seq data, tissue and disease-specific circRNAs are still to be uncovered to promote their application in basic research and clinical practice. The purpose of this study was to explore the circRNA profiles in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived cardiomyocytes (hiPSC-CMs), and to identify cardiac or disease-specific circRNAs. METHODS:hiPSCs were generated from fibroblasts, and then further differentiated to hiPSC-CMs by modulating WNT signaling in RPMI+B27 medium. Following high-throughput RNA sequencing, circRNAs were extracted and quantified by a combined strategy known as CIRCexplorer. Integrative analysis was performed to illuminate the correlation between circRNAs and their parental linear isoforms. Cardiac and disease-specific expression of circRNAs was confirmed by quantitative reverse-transcription PCR. RESULTS:In this study, a total of 5602 circRNAs were identified in hiPSCs and hiPSC-CMs. Our data indicated, for the first time, more enriched expression of circRNAs in differentiated cardiomyocytes than in undifferentiated hiPSCs. In addition to the host gene-dependent expression, our integrative analysis also identified a number of circRNAs showing host gene-independent expression in hiPSCs and hiPSC-CMs. CircRNAs including circSLC8A1, circCACNA1D, circSPHKAP and circALPK2 showed cardiac-selective expression during cardiac differentiation and human heart-specific enrichment in fetal tissues. Furthermore, circSLC8A1 abnormally increased in heart tissues from patients suffering from dilated cardiomyopathy. CONCLUSIONS:CircRNAs are highly enriched in hiPSC-differentiated CMs, and cardiac-specific circRNAs such as circSLC8A1, circCACNA1D, circSPHKAP and circALPK2 may serve as biomarkers of CMs. Detection of the excessive expression of circSLC8A1 provides a potential approach for pathological status indication of heart disease.

Project description:RationaleCardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling.ObjectiveWe investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs).Methods and resultsWe differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs.ConclusionsThe results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts.

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Functional cardiomyocytes derived from human induced pluripotent stem cells.

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Functional cardiomyocytes derived from human induced pluripotent stem cells.

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