Dataset Information

Generation and characterization of functional cardiomyocytes derived from human T cell-derived induced pluripotent stem cells.

ABSTRACT: Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR) gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.

SUBMITTER: Seki T

PROVIDER: S-EPMC3897468 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Generation and characterization of functional cardiomyocytes derived from human T cell-derived induced pluripotent stem cells.

Seki Tomohisa T Yuasa Shinsuke S Kusumoto Dai D Kunitomi Akira A Saito Yuki Y Tohyama Shugo S Yae Kojiro K Kishino Yoshikazu Y Okada Marina M Hashimoto Hisayuki H Takei Makoto M Egashira Toru T Kodaira Masaki M Kuroda Yusuke Y Tanaka Atsushi A Okata Shinichiro S Suzuki Tomoyuki T Murata Mitsushige M Fujita Jun J Fukuda Keiichi K

PloS one 20140121 1

Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements ...[more]

PMID: 24465630

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Project description:Endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) provide a new opportunity for mechanistic research on vascular regeneration and drug screening. However, functions of hiPSC-ECs still need to be characterized. The objective of this study was to investigate electrophysiological and functional properties of hiPSC-ECs compared with primary human cardiac microvascular endothelial cells (HCMECs), mainly focusing on ion channels and membrane receptor signaling, as well as specific cell functions. HiPSC-ECs were derived from hiPS cells that were generated from human skin fibroblasts of three independent healthy donors. Phenotypic and functional comparison to HCMECs was performed by flow cytometry, immunofluorescence staining, quantitative reverse-transcription polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), tube formation, LDL uptake, exosome release assays and, importantly, patch clamp techniques. HiPSC-ECs were successfully generated from hiPS cells and were identified by endothelial markers. The mRNA levels of KCNN2, KCNN4, KCNMA1, TRPV2, and SLC8A1 in hiPSC-ECs were significantly higher than HCMECs. AT1 receptor mRNA level in hiPSC-ECs was higher than in HCMECs. AT2 receptor mRNA level was the highest among all receptors. Adrenoceptor ADRA2 expression in hiPSC-ECs was lower than in HCMECs, while ADRA1, ADRB1, ADRB2, and G-protein GNA11 and Gai expression were similar in both cell types. The expression level of muscarinic and dopamine receptors CHRM3, DRD2, DRD3, and DRD4 in hiPSC-ECs were significantly lower than in HCMECs. The functional characteristics of endothelial cells, such as tube formation and LDL uptake assay, were not statistically different between hiPSC-ECs and HCMECs. Phenylephrine similarly increased the release of the vasoconstrictor endothelin-1 (ET-1) in hiPSC-ECs and HCMECs. Acetylcholine also similarly increased nitric oxide generation in hiPSC-ECs and HCMECs. The resting potentials (RPs), ISK1-3, ISK4 and IK1 were similar in hiPSC-ECs and HCMECs. IBK was larger and IKATP was smaller in hiPSC-ECs. In addition, we also noted a higher expression level of exosomes marker CD81 in hiPSC-ECs and a higher expression of CD9 and CD63 in HCMECs. However, the numbers of exosomes extracted from both types of cells did not differ significantly. The study demonstrates that hiPSC-ECs are similar to native endothelial cells in ion channel function and membrane receptor-coupled signaling and physiological cell functions, although some differences exist. This information may be helpful for research using hiPSC-ECs.

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.

Dataset Information

Generation and characterization of functional cardiomyocytes derived from human T cell-derived induced pluripotent stem cells.

Publications

Generation and characterization of functional cardiomyocytes derived from human T cell-derived induced pluripotent stem cells.

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