Kurt Pfannkuche

Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro

Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c‐Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS‐cell‐based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RTPCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, αMHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac‐specific voltage‐gated Na+, Ca2+, and K+ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels;CMs generated from iPS cells have a ventricular phenotype;and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC‐derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.—Kuzmenkin, A., Liang, H., Xu, G., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H., Saric, T., Wernig, M., Jaenisch, R., Hescheler, J. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB J. 23, 4168‐4180 (2009). www.fasebj.org

Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC‐CMs) and iPS cells (iPS‐CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5–7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force‐frequency relationship (FFR), effects of Ca2+, Ni2+, nifedipine, ryanodine, β‐adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC‐CMs and iPS‐CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca2+ and decreased with nifedipine. Force was reduced by Ni2+. Isoproterenol (1 µM) increased the force most pronounced in embryonic tissue (207±31%, n=7;ESC‐CMs: 123±5%, n=4; iPS‐CMs: 120 ±4%, n=8). EC50 values were similar. Contractile properties of iPS‐CMs and ESC‐CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the β‐adrenergic response of iPS‐CMs and ESC‐CMs.—Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel, M., Šarić, T., Wernig, M., Jaenisch, R., Brockmeier, K., Hescheler, J., Pillekamp, F. Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells. FASEB J. 24, 2739–2751 (2010). www.fasebj.org

Cardiac myocytes derived from murine reprogrammed fibroblasts: intact hormonal regulation, cardiac ion channel expression and development of contractility.

Aims: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. Methods and Results: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to β-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of INa, If, INCX, and ICaL, respectively, and suggested their involvement in cardiac pacemaking, with ICaL being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to β-adrenergic stimulation. Conclusions: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty.

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

BackgroundFunctional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells.ResultsHierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes.ConclusionsThese data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.

Electrophysiological Maturation and Integration of Murine Fetal Cardiomyocytes After Transplantation

In the present study, we investigated the electrophysiological maturation and integration of immature cardiomyocytes after transplantation; maturation and integration are essential to achieve the cardiac regeneration. Murine fetal cardiomyocytes (FCMs) (d12.5-d15.5) expressing enhanced green fluorescent protein under the control of the &agr;-actin promoter were injected into cryoinjured areas and adjacent myocardium of cryoinjured mouse ventricles. Viable short axis tissue slices (thickness, 150 &mgr;m) of the ventricles were prepared 5 to 6 days after transplantation. Glass microelectrodes were used for measurements of action potentials in transplanted FCMs and host cardiomyocytes within the slices. Stimulation at frequencies of up to 10 Hz was performed via a unipolar electrode placed in viable host tissue. Transplanted FCMs could be distinguished clearly from host tissue by their green fluorescence and their electrophysiological properties: maximal upstroke velocity (Vmax) was significantly lower and action potential duration at 50% repolarization (APD50) was significantly longer compared with values of adult cardiomyocytes. Transplanted FCMs surrounded by cryoinjured tissue showed spontaneous electrical and contractile activity, which was in no case synchronous with host tissue. Vmax and APD50 of these nonintegrated cells matched values of cultivated dissociated FCMs. In contrast, 82% of transplanted FCMs surrounded by viable host tissue were electrically integrated; ie, electrical and contractile activity was synchronous with host tissue and these cells had more mature action potential parameters (significantly higher Vmax and shorter APD50) compared with nonintegrated FCMs. In conclusion, electrophysiological maturation and integration of transplanted FCMs depend on an embedment in viable host myocardium. FCMs surrounded by cryoinjured tissue maintain physiological but immature AP properties.

The High Mobility Group Protein HMGA2: A Co-Regulator of Chromatin Structure and Pluripotency in Stem Cells?

The small, chromatin-associated HMGA proteins contain three separate DNA binding domains, so-called AT hooks, which bind preferentially to short AT-rich sequences. These proteins are abundant in pluripotent embryonic stem (ES) cells and most malignant human tumors, but are not detectable in normal somatic cells. They act both as activator and repressor of gene expression, and most likely facilitate DNA architectural changes during formation of specialized nucleoprotein structures at selected promoter regions. For example, HMGA2 is involved in transcriptional activation of certain cell proliferation genes, which likely contributes to its well-established oncogenic potential during tumor formation. However, surprisingly little is known about how HMGA proteins bind DNA packaged in chromatin and how this affects the chromatin structure at a larger scale. Experimental evidence suggests that HMGA2 competes with binding of histone H1 in the chromatin fiber. This could substantially alter chromatin domain structures in ES cells and contribute to the activation of certain transcription networks. HMGA2 also seems capable of recruiting enzymes directly involved in histone modifications to trigger gene expression. Furthermore, it was shown that multiple HMGA2 molecules bind stably to a single nucleosome core particle whose structure is known. How these features of HMGA2 impinge on chromatin organization inside a living cell is unknown. In this commentary, we propose that HMGA2, through the action of three independent DNA binding domains, substantially contributes to the plasticity of ES cell chromatin and is involved in the maintenance of a un-differentiated cell state.

Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes

BackgroundCharacterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes.ResultsA transgenic α-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 α-MHC promoter. A puromycin-resistant, EGFP-positive, α-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of α-MHC-positive cardiomyocytes in comparison with undifferentiated α-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 α-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 α-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.ConclusionThe 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.

Contractile Properties of Early Human Embryonic Stem Cell-Derived Cardiomyocytes: Beta-Adrenergic Stimulation Induces Positive Chronotropy and Lusitropy but Not Inotropy

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide the unique opportunity to study the very early development of the human heart. The aim of this study was to investigate the effect of calcium and beta-adrenergic stimulation on the contractile properties of early hESC-CMs. Beating clusters containing hESC-CMs were co-cultured in vitro with noncontractile slices of neonatal murine ventricles. After 5-7 days, when beating clusters had integrated morphologically into the damaged tissue, isometric force measurements were performed during spontaneous beating as well as during electrical field stimulation. Spontaneous beating stopped when extracellular calcium ([Ca²⁺](ec)) was removed or after administration of the Ca²⁺ channel blocker nifedipine. During field stimulation at a constant rate, the developed force increased with incremental concentrations of [Ca²⁺](ec). During spontaneous beating, rising [Ca²⁺](ec) increased beating rate and developed force up to a [Ca²⁺](ec) of 2.5 mM. When [Ca²⁺](ec) was increased further, spontaneous beating rate decreased, whereas the developed force continued to increase. The beta-adrenergic agonist isoproterenol induced a dose-dependent increase of the frequency of spontaneous beating; however, it did not significantly change the developed force during spontaneous contractions or during electrical stimulation at a constant rate. Force developed by early hESC-CMs depends on [Ca²⁺](ec) and on the L-type Ca²⁺ channel. The lack of an inotropic reaction despite a pronounced chronotropic response after beta-adrenergic stimulation most likely indicates immaturity of the sarcoplasmic reticulum. For cell-replacement strategies, further maturation of cardiac cells has to be achieved either in vitro before or in vivo after transplantation.

Force Measurements of Human Embryonic Stem Cell-Derived Cardiomyocytes in an In Vitro Transplantation Model

Human embryonic stem cell (hESC)‐derived cardiomyocytes have been suggested for cardiac cell replacement therapy. However, there are no data on loaded contractions developed by these cells and the regulation thereof. We developed a novel in vitro transplantation model in which beating cardiomyocytes derived from hESCs (line H1) were isolated and transplanted onto noncontractile, ischemically damaged ventricular slices of murine hearts. After 2–3 days, transplanted cells started to integrate mechanically into the existing matrix, resulting in spontaneous movements of the whole preparation. Preparations showed a length‐dependent increase of active tension. In transplanted early beating hESC‐derived cardiomyocytes, frequency modulation by field stimulation was limited to a small range around their spontaneous beating rate. Our data demonstrate that this novel in vitro transplantation model is well suited to assess the mechanical properties and functional integration of cells suggested for cardiac replacement strategies.

In vitro Model for Assessing Arrhythmogenic Properties of Drugs Based on High-resolution Impedance Measurements

Background/Aims: Cardiac dysfunction is one of the main cause of drug candidate failures in the preclinical and/or clinical studies and responsible for the retraction of large number of drugs from the market. The prediction of arrhythmic risk based on preclinical trials during drug development remains limited despite intensive and costly investigation. Moreover, methods for analyzing beating behavior of cardiomyocytes (CMs) in culture to diagnose arrhythmias are not well developed. Methods: In this study, we combined two emerging technologies, induced pluripotent stem (iPS) cell-derived CMs and impedance-based real-time (xCELLigence RTCA Cardio Instrument) monitoring of CM electrical activity, to assess the effect of drugs known affect cardiac activity such as isoproterenol, carbachol, terfenadine, sotalol and doxorubicin. Cells were exposed to a drug in a single dose or repeated dose scenarios and data were analyzed using RTCA Cardio software, Poincaré plot and detrended fluctuation analysis. Results: The results revealed significant changes in beating parameters of iPS-CMs induced by reference compounds. Heptanol, gap junction blocker, completely disrupted the synchronous beating pattern of iPS-CMs. Decrease of beating rate, amplitude and beat-to-beat signal variations of iPS-CMs monolayer observed in the presence of doxorubicin revealed severe abnormality detected by the system. Additionally, the irregular beating rhythms recorded in the presence of Terfenadine and Sotalol at high concentration, reflect abnormalities in cell contraction and/or relaxation which may lead to arrhythmia. Conclusions: All these results indicated that xCELLigence RTCA Cardio system combined with iPS cells, has the potential to be an attractive high-throughput tool for studying CMs during prolonged culture times and to screen potential drugs for cardiotoxic side effects.