Tobias Hannes

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.

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.

Fibroblasts Facilitate the Engraftment of Embryonic Stem Cell-Derived Cardiomyocytes on Three-Dimensional Collagen Matrices and Aggregation in Hanging Drops

There is growing interest in the use of cardiomyocytes purified from embryonic stem (ES) cells for tissue engineering and cardiomyoplasty. However, most transplanted cells are lost shortly after transplantation due to the lack of integration into the host tissue and subsequent apoptosis. Here we examine whether murine embryonic fibroblasts (MEFs) can support the integration of purified murine ES cell-derived cardiomyocytes in a 3-dimensional tissue culture model based on a freezed-dryed collagen matrix with tubular structure. Collagen matrix was seeded either with cardiomyocytes alone or in combination with MEFs. The collagen sponges that were transplanted with cardiomyocytes alone showed neither morphological nor functional integration of viable cells. Cardiomyocytes also did not appear to be capable of attaching quantitatively to any of 16 different 2-dimensional biomaterials. However, cardiomyocytes co-cultured with MEFs formed fiber-like structures of rod-shaped cells with organized sarcomeric structure that contracted spontaneously. Electrical coupling between cardiomyocytes was suggested by strong expression of connexin 43. In addition, MEFs as well as cardiac fibroblasts supported re-aggregation of dissociated cardiomyocytes in hanging drops in the absence of collagen matrix. We conclude that fibroblasts promote cardiomyocyte engraftment and formation of functional 3-dimensional tissue in vitro. Elucidation of the mechanism of this phenomenon may help improve the integration of cardiomyocytes in vivo.

Induced Pluripotent Stem Cells: A New Approach for Physiological Research

The generation of induced pluripotent stem (iPS) cells by controlled delivery of reprogramming factors enables the derivation of pluripotent cells from a variety of somatic cell types. Patient-tailored iPS cells remove the major roadblock of immune rejection for clinical applications associated with the use of human embryonic stem (hES) cells. Beside therapeutic issues, iPS cell technology opens the door for broader research on human pluripotent cells because ethical limitations are lifted with iPS cells compared to hES cells. Scientists are now able to generate iPS cells for disease modelling and use them in basic research of physiological and pathophysiological models. In this concise review, we discuss the state of the art in the field of iPS cell induction by cell fusion or defined factors. Techniques to derive pluripotent cells from somatic sources are introduced and discussed, as well as some biological factors that influence the generation of iPS cells. We compare ES and iPS cells to answer the question whether these cells are identical, and we finish with an outlook on clinical research with iPS cells with a focus on cardiovascular medicine.

Physiological Differences Between Transplanted and Host Tissue Cause Functional Decoupling after in vitro Transplantation of Human Embryonic Stem Cell-Derived Cardiomyocytes

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) might provide cells to repopulate injured myocardium. Electrical coupling of these cells to the host myocardium is a prerequisite for improved functionality. The aim of this study was to investigate electrical interaction of hESC-CMs with myocardial tissue and to identify factors challenging functional integration. Beating clusters containing hESC-CMs were cocultured in vitro with viable slices of late-stage embryonic murine ventricles. Field potentials recorded with micro-electrode arrays and video data were analyzed. The effects of heptanol, electrical pacing, beta-adrenergic, and muscarinic stimulation on coupling were studied. Beating clusters integrated morphologically and functionally resulting in a synchronized beating pattern after two to four days of coculture. Heptanol-induced conduction block between transplanted cells and host tissue and immunoreactivity for connexin43 suggested electrical coupling via gap junctions. Beta-adrenergic or muscarinic stimulation induced uncoupling and arrhythmias probably due to genetically determined differences of hormonal modulation of spontaneous beating rates of transplanted cells and host tissue. HESC-CMs can integrate functionally and develop synchronized beating. Interventions unraveling the different electrophysiological properties of transplanted and host tissue induce functional disintegration. Successful cellular replacement has to improve coupling but should also aim to transplant cardiomyocytes with similar electrophysiological properties as the host tissue.

Fibroblasts support functional integration of purified embryonic stem cell-derived cardiomyocytes into avital myocardial tissue.

Transplantation of purified pluripotent stem cell-derived cardiomyocytes into damaged myocardium might become a therapy to improve contractile function after myocardial infarction. However, engraftment remains problematic. Aim of this study was to investigate whether murine embryonic fibroblasts (MEFs) support the functional integration of purified embryonic stem cell-derived cardiomyocytes (ES-CMs). Neonatal murine ventricular tissue slices were subjected to oxygen and glucose deprivation to simulate irreversible ischemia. Vital tissue slices served as control. Vital and avital tissue slices were cultured with or without MEFs before coculturing with clusters of puromycin-selected ES-CMs. Integration of ES-CM clusters was assessed morphologically, motility by long-term microscopy, and functional integration by isometric force measurements. We observed a good morphological integration into vital but a poor integration into avital slices. Adding MEFs improved morphological integration into irreversibly damaged slices and enabled purified ES-CMs to migrate and to confer force. We conclude that noncardiomyocytes like MEFs support morphological integration and force transmission of purified ES-CMs by enabling adhesion and migration.

Biological pacemakers: characterization in an in vitro coculture model

BACKGROUND Biological pacemakers could be an alternative or complement to electronic pacemakers. Embryonic stem cells (ESCs) can be differentiated in vitro to spontaneously active cells. Although numerous studies show that ESC-derived cardiomyocytes (ESC-CMs) and other cell types are capable to exert pacemaker function in vivo, detailed analyses of pattern and safety of conduction on a tissue level are rare. METHODS Murine ESCs (mESCs) expressing enhanced green fluorescent protein and puromycin resistance under control of the promoter of alpha-myosin (heavy chain) were differentiated to cardiomyocytes (mESC-CMs) and purified by negative antibiotic selection. Ventricles of mouse embryonic hearts (embryonic day 16.5) were embedded in agarose and sliced along the short axis. Clusters of mESC-CMs and the murine, vital heart slices were cocultured on multielectrode arrays for 4 days. Field potentials and videos were recorded daily to investigate beating behavior and excitation spreading within the slice. RESULTS On the first day of coculture, the mean beating rate of the tissue slices cocultured with mESC-CMs (n = 19) did not differ significantly from the beating rate of control slices (n = 19) (37 +/- 10 versus 19 +/- 7 bpm, P = .133). After 4 days of coculture, beating rates were significantly higher in cocultures than in control slices (154 +/- 22 versus 49 +/- 8 bpm, P < .001). On day 4, 1:1 coupling could be found in 1 of 19 preparations; 2:1, 3:1, or 4:1 coupling in another 4 of 19 preparations; 14 of 19 propagation patterns were irregular. CONCLUSION In this in vitro model, the increase of the beating rate suggests that purified mESC-CMs can pace native heart tissue, albeit with low efficiency.

Electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes are cell line-dependent.

Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines. Methods: Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD) as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

Time-course of the electrophysiological maturation and integration of transplanted cardiomyocytes

Electrophysiological maturation and integration of transplanted cardiomyocytes are essential to enhance safety and efficiency of cell replacement therapy. Yet, little is known about these important processes. The aim of our study was to perform a detailed analysis of electrophysiological maturation and integration of transplanted cardiomyocytes. Fetal cardiomyocytes expressing enhanced green fluorescent protein were transplanted into cryoinjured mouse hearts. At 6, 9 and 12 days after transplantation, viable slices of recipient hearts were prepared and action potentials of transplanted and host cardiomyocytes within the slices were recorded by microelectrodes. In transplanted cells embedded in healthy host myocardium, action potential duration at 50% repolarization (APD50) decreased from 32.2 ± 3.3 ms at day 6 to 27.9 ± 2.6 ms at day 9 and 19.6 ± 1.6 ms at day 12. The latter value matched the APD50 of host cells (20.5 ± 3.2 ms, P=0.78). Integration improved in the course of time: 26% of cells at day 6 and 53% at day 12 revealed no conduction blocks up to a stimulation frequency of 10 Hz. APD50 was inversely correlated to the quality of electrical integration. In transplanted cells embedded into the cryoinjury, which showed no electrical integration, APD50 was 49.2 ± 4.3 ms at day 12. Fetal cardiomyocytes transplanted into healthy myocardium integrate electrically and mature after transplantation, their action potential properties after 12 days are comparable to those of host cardiomyocytes. Quality of electrical integration improves over time, but conduction blocks still occur at day 12 after transplantation. The pace of maturation correlates with the quality of electrical integration. Transplanted cells embedded in cryoinjured tissue still possess immature electrophysiological properties after 12 days.