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First published online March 16, 2006
Stem Cells Vol. 24 No. 6 June 2006, pp. 1423 -1432
doi:10.1634/stemcells.2005-0380; www.StemCells.com
© 2006 AlphaMed Press

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EMBRYONIC STEM CELLS

Generation and Characterization of Functional Cardiomyocytes from Rhesus Monkey Embryonic Stem Cells

Kristin Schwankea, Stephanie Wunderlicha, Michael Reppelb, Monica E. Winklera, Matthias Matzkiesb, Stephanie Groosc, Joseph Itskovitz-Eldord, André R. Simona, Jürgen Heschelerb, Axel Havericha, Ulrich Martina

a Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO) and Department of Thoracic and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany;
b Institute of Neurophysiology, University of Cologne, Cologne, Germany;
c Department of Cell Biology; Hannover Medical School, Hannover, Germany;
d Department of Obstetrics and Gynecology, Rambam Medical Center, Faculty of Medicine, The Technion, Haifa, Israel

Key Words. Embryonic stem cells • Differentiation • Cardiomyocytes • Primates

Correspondence: Ulrich Martin, Ph.D., Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Podbielskistr. 380, 30659 Hannover, Germany. Telephone: +49 (511) 532-8820; Fax: +49 (511) 532-8819; e-mail: martin.ulrich{at}mh-hannover.de

Received August 9, 2005; accepted for publication March 6, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
Embryonic stem cells (ESCs) from mice and humans (hESCs) have been shown to be able to efficiently differentiate toward cardiomyocytes (CMs). Because murine ESCs and hESCs do not allow for establishment of pre-clinical allogeneic transplantation models, the aim of our study was to generate functional CMs from rhesus monkey ESCs (rESCs). Although formation of ectodermal and neuronal/glial cells appears to be the default pathway of the rESC line R366.4, we were able to change this commitment and to direct generation of endodermal/mesodermal cells and further differentiation toward CMs. Differentiation of rESCs resulted in an average of 18% of spontaneously contracting embryoid bodies (EBs) from rESCs. Semiquantitative reverse transcription-polymerase chain reaction analyses demonstrated expression of marker genes typical for endoderm, mesoderm, cardiac mesoderm, and CMs, including brachyury, goosecoid, Tbx-5, Tbx-20, Mesp1, Nkx2.5, GATA-4, FOG-2, Mlc2a, MLC2v, ANF, and {alpha}-MHC in rESC-derived CMs. Immunohistological and ultrastructural studies showed expression of CM-typical proteins, including sarcomeric actinin, troponin T, titin, connexin 43, and cross-striated muscle fibrils. Electrophysiological studies by means of multielectrode arrays revealed evidence of functionality, electrical coupling, and ß-adrenergic signaling of the generated CMs. This is the first study demonstrating generation of functional CMs derived from rESCs. In contrast to hESCs, rESCs allow for establishment of pre-clinical allogeneic transplantation models. Moreover, rESC-derived CMs represent a cell source for the development of high-throughput assays for cardiac safety pharmacology.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
Cardiac diseases, including coronary artery disease, are among the most frequent in the industrialized nations. Existing therapies are still unsatisfactory, and cell-based approaches may result in dramatically improved therapeutical options. Because postnatal cardiomyocytes (CMs) have lost their mitotic potential [1, 2], injury of the myocardium due to the ischemic loss of CMs is to a great extent irreversible. Although a subpopulation of CMs appears to retain a limited potential for proliferation [3] and recent studies suggest the existence of cardiogenic stem cell populations in the heart [4–7], it remains controversial whether these cellular populations with mitotic or cardiogenic activity can effect significant regeneration in the normal adult heart or will allow for therapeutic restoration of destroyed myocardium.

Given that embryonic stem cells (ESCs), including those of human origin (hESCs), have been shown to undergo robust differentiation [8–13], these cells may well represent the most promising cell sources for cellular cardiomyoplasty. However, although combining many advantageous properties, including the potential of unlimited differentiation and proliferation as well as high levels of genetic stability compared with adult cells, ESCs as cell source for myocardial transplantation may also pose exorbitant risks, including their arrhythmogenic potential [14], the development of teratoma [15], and possible transmission of pathogens from mouse feeder cells [16]. To assess such risks, it will be indispensable to perform preclinical transplantation experiments.

Obviously, mouse models are of limited value to address many of the actually unsolved preclinical questions and concerns. In view of the major distinctions in embryonic development and underlying molecular regulation between mouse and primates, it is not surprising that mouse ESCs are quite different from human and nonhuman primate ESCs. Moreover, due to the size of the murine organ, it is difficult to measure improvements of heart function reliably in a mouse model. Therefore, transplantation of ESC-derived cells into larger animals, including nonhuman primates, will be necessary to fully assess functional improvements after implantation of ESCs.

For several reasons, nonhuman primate ESCs, in particular of cynomolgus [17, 18] or rhesus monkey origin [19], represent the most suitable model to perform such studies [20]. First, no true ESCs have been isolated from rat or other larger animals, including swine. Second, although xenogeneic transplantation of hESCs into swine has been reported [10], additional challenges involved with cross-species transplantation may change the results considerably when compared with an allogeneic setting. In particular, potential molecular species incompatibilities [21] and the necessary high levels of pharmacological immunosuppression may have profound effects on cell survival rates, immunological attack of the cell transplant, in vivo differentiation, and functional integration.

As a first step to establish a preclinical myocardial cell transplantation model, we have now generated functional CMs from rhesus monkey ESCs (rESCs) using an embryoid body (EB)-based approach. Analysis of mRNA expression, immunohistology, and ultrastructural and electrophysiological studies revealed expression patterns and functional characteristics comparable with hESC-derived CMs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosures
 References
 
Culture of Undifferentiated rESCs
The rESC line R366.4 (passages 41–76) was cultured on a mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer (40,000 cells/cm2). For inactivation, 10 µg/ml mitomycin C was used (Sigma-Aldrich, Taufkirchen, Germany, http://www.sigmaaldrich.com). The culture medium consisted of 80% knockout-Dulbecco’s modified Eagle’s medium (ko-DMEM) supplemented with 20% knockout serum replacement, 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol, 1% nonessential amino acid stock, and 4 ng/ml basic fibroblast growth factor (all from Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com). Cells were cultured in six-well tissue culture plates (Nunc, Wiesbaden, Germany, http://www.nuncbrand.com) and passaged every 4–6 days using 1 mg/ml type IV collagenase (Invitrogen Corporation).

Differentiation of rESCs
To induce differentiation, ESCs were dispersed into small clumps using 1 mg/ml type IV collagenase. Approximately 1 x 106 rESCs were transferred to one 12-well plate (Nunc), resulting in an average formation of 70 EBs. To avoid attachment and allow formation of EBs, plates were coated with 1% agarose. The rESCs/EBs were cultured in these plates in suspension for 7 days in differentiation medium, which consisted of 80% Iscove’s modified Dulbecco’s medium (or ko-DMEM; Invitrogen Corporation) supplemented with 20% fetal calf serum (FCS), 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol, and 1% nonessential amino acid stock. Different lots of FCS from Invitrogen Corporation, PAA Laboratories (Cölbe, Germany, http://www.paa.at), and HyClone (Logan, UT, http://www.hyclone.com) were tested; for final experiments, FCS from HyClone was used. Subsequently, approximately 20 EBs per well were plated on six-well tissue culture plates coated with 0.1% gelatin, and were checked daily for beating areas.

Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was prepared using TriZol (Sigma-Aldrich) according to the manufacturer’s instructions. Integrity of total RNA was controlled using an Agilent 2100 Bioanalyzer. Contaminating DNA was digested by DNAse I (Stratagene, La Jolla, CA, http://www.stratagene.com) for 15 minutes at 37°C followed by phenol/chloroform extraction. After precipitation, 750 ng of RNA was used for Oligo(dT)12–18-primed cDNA synthesis with Superscript II, a modified moloney murine leukemia virus reverse transcriptase (Invitrogen Corporation). One microliter of cDNA was amplified by polymerase chain reaction (PCR) with 1.25 U of REDTaq DNA-Polymerase (Sigma-Aldrich) in RED-Taq 10x reaction buffer, using 0.4 µM of each primer and 0.4 µM dNTPs in a 25-µl reaction. PCR conditions included denaturation at 94°C for 1 minute, annealing at the suitable annealing temperature (TA, supplemental online Table 1) for 1 minute, and polymerization at 72°C for 1 minute 30 seconds; after 35 cycles, an extension step of 10 minutes at 72°C was added. Sequences and specifications of primers are shown in supplemental online Table 1. Reactions without reverse transcription (RT) were performed in parallel to control for remaining contaminations of genomic DNA. Species specificity was controlled using RNA of adult mouse and, because no heart samples of rhesus monkeys were available, human heart tissue. Human tissue samples were obtained from patients who underwent cardiac transplantation for terminal heart failure after informed consent according to the legal requirements.

Immunohistological Staining
After plating of EBs and further differentiation, beating areas were mechanically removed, plated on culture slides (BD Biosciences, Heidelberg, Germany, http://www.bdbiosciences.com), and cultivated for an additional 4–5 days. Cells were fixed for immunohistological staining in 4% paraformaldehyde for 20 minutes at room temperature. The following primary antibodies were used: a mouse immunoglobulin (Ig) G2a monoclonal anti-troponin T antibody (clone CT3, diluted 1:10; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww), a mouse IgM monoclonal anti-titin antibody (clone MAB1553, diluted 1:100; Chemicon, Temecula, CA, http://www.chemicon.com), a rabbit IgG poly-clonal anti-connexin 43 antibody (diluted 1:25; Chemicon), a mouse IgG1 monoclonal anti-sarcomeric {alpha}-actinin antibody (clone EA-53, diluted 1:800; Sigma-Aldrich), a mouse IgG1 monoclonal anti-myosin light chain two ventricular (MLC2v) antibody (clone F109.3E1, diluted 1:10; Biocytex, Marseille, France, http://www.biocytex.fr), and a polyclonal rabbit IgG anti-atrial natriuretic factor (ANF) antibody (clone T-4011, diluted 1:500; Bachem, Weil am Rhein, Germany, http://www.bachem.com).

The culture slides were incubated for 1 hour at room temperature with the primary antibody diluted in Tris-buffered saline (TBS) with 0.05% Tween 20 (Sigma-Aldrich) and then rinsed three times for 5 minutes each time with standard phosphate-buffered saline (PBS). Further incubation was performed with the appropriate secondary antibodies CyTM2-labeled donkey anti-mouse IgG (1:500; Dianova, Hamburg, Germany, http://www.dianova.de), CyTM3-labeled donkey anti-mouse IgM (1:500; Dianova) and CyTM2-labeled goat anti-rabbit IgG (Dianova, 1:100), each diluted in PBS with 0.05% Tween 20 for 60 minutes at room temperature. The slides were rinsed once more, counterstained with DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich), and after mounting with IMMU-MOUNT (Shandon Lipshaw, Pittsburgh, http://www.shandon.com) analyzed with a TE300 fluorescence microscope (Nikon, Tokyo, http://nikon.com).

Electron Microscopy
After 21 days of differentiation, contracting areas were excised, pelleted, washed three times in PBS, and immersed in a fixative solution composed of 3% glutaraldehyde and 0.1 M sodium cacodylate/HCl buffer pH 7.3 for at least 4 hours at 4°C. After washing overnight in 0.1 M sodium cacodylate/HCl buffer (pH 7.3), the pellets were postfixed for 90 minutes in 2% OsO4 in the same buffer, dehydrated in ascending concentrations of ethanol, and embedded in epoxy resin (Serva, Heidelberg, Germany, http://www.serva.de). Thin sections (approximately 70 nm thick) were cut with an ultramicrotome Reichert Ultracut R (Reichert-Jung, Nußloch, Germany, http://www.reichertms.com), collected on formvar-coated copper slot grids, stained with uranylacetate and lead citrate, and examined in an electron microscope Zeiss EM 10 CR (Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com) at an acceleration voltage of 80 kV.

Microelectrode Mapping
To characterize functional properties of rESC-derived CMs, extracellular recording of field potentials (FPs) was performed using a multielectrode array (MEA) data acquisition system (Multi Channel Systems, Reutlingen, Germany, http://www.multichannelsystems.com) [22–24]. For this aim, beating areas were mechanically removed and plated on MEA plates. The MEA system allows measurement of frequency and activity of different ion channels involved in cardiac action potential generation, as well as of propagation in three-dimensional cardiac tissue in parallel, and therefore serves as an ideal system to characterize specific functional properties of rESC-derived CMs [24]. The standard substrate-integrated MEA culture dish contains 60 titanium nitride-coated gold electrodes (30 µm diameter) arranged in an 8 x 8 electrode grid with an interelectrode distance of 200 µm, allowing simultaneous recording of FPs from all electrodes at a sampling rate of 1–50 kHz with the MEA amplifier system. Standard measurements were performed at 5 kHz in serum-free medium. (±)-Isoproterenol hydrochloride (ISO), used as standard stimulator of the ß-adrenergic signaling cascade, was dissolved in serum-free medium and stored according to the manufacturer’s guidelines. During recordings, the temperature was kept at 39.0°C. Data were analyzed offline with a customized toolbox programmed with MATLAB (The MathWorks, Natick, MA, http://www.mathworks.com) to detect and characterize FPs as has been described previously [12, 25]. The frequency of contractions was calculated by measuring the distance between respective FP minima (FPMIN) and denoted inter-spike interval (ISI). FPMIN in general represents the activity of Na+ channels. A second slowly occurring negative FP deflection (FPSLOW), representing the activity of voltage-dependent Ca2+ channels [24], was analyzed to allow characterization of pharmacological modulation of CM cultures originating from rESCs. For mapping of the distribution of extracellular FPs, the MEA toolbox was used [26].


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosures
 References
 
Differentiation of rESCs Toward Spontaneously Contracting CMs
Based on experimental conditions reported to result in differentiation of hESCs into CMs, we developed a protocol aimed at differentiating rESCs. Using an EB-based approach, effective differentiation comparable with that with hESCs was achieved (Fig. 1Go). Critical to this process was the culture medium used: The addition of knockout serum replacement totally prevented formation of CMs. Only one of six tested lots of FCS resulted in efficient formation of CMs; most others supported differentiation of neuronal/glial cell types (supplemental online Fig. 1Go). In this context, it is of interest to note that lots, very efficiently directing cardiac differentiation of mouse ESCs, did not result in prominent differentiation of rESCs toward CMs (data not shown). Importantly, no agents such as 5-azacytidine that rather nonspecifically modify cellular expression through alteration of the methylation/acetylation status have been applied.


Figure 1
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  		Figure 1. Induction of differentiation in rhesus monkey embryonic stem cells (rESCs). Morphology of rESCs during induction of differentiation: (A): Undifferentiated rESC colonies grown on mouse embryonic fibroblasts. (B): Day-2 embryoid body (EB) in suspension. (C): Outgrowth of day-9 EB (7 days in suspension, 2 days after plating). Arrow indicates contracting area. Scale bars = 100 µm.

 
Formation of the first contracting regions was usually observed at day 8 of differentiation (7 days in suspension in dishes coated with agarose followed by 1 day of outgrowth in gelatin-coated dishes; supplemental online movie 1). The proportion of EBs with contracting areas increased from day 8 (1 day after plating), reaching a maximal percentage of contractile EBs on day 15 of differentiation (7 days after plating). The first beating was very irregular but became consistent a few days after showing first contractions, with EBs beating at an average frequency of 42 ± 23 beats per minute (n = 6). Contractile activity of rESC-derived CMs was very sensitive to temperature when compared with mouse ESCs and decreased during microscopical observation (data not shown). Differentiated cultures were observed for up to 31 days. During the whole observation period, no decrease in the proportion of contracting EBs was noticed (supplemental online Fig. 2Go). Most EBs had only one contracting area, and in some cases up to three beating regions/EB were detected, differing considerably in size and morphology. Some contractile areas seemed to be located in cystic structures, whereas others were located in the periphery of EB outgrowths.


Figure 2
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  		Figure 2. Differentiation of rESCs induces mRNA expression of markers for endoderm, mesoderm, cardiac mesoderm, and cardiomyocytes. Semiquantitative RT-polymerase chain reaction (PCR) was performed from undifferentiated rESCs, rESCs at different stages after induction of differentiation, and without RT to exclude false-positive results based on contaminations with genomic DNA. Mouse embryonic fibroblasts served as control for primer species specificity. (A): Expression of markers for endoderm. (B): Mesoderm, cardiac mesoderm, and cardiomyocytes. Abbreviations: AFP, {alpha}-feto protein; ANF, atrial natriuretic factor; bp, base pairs; FOG-2, friend of GATA 2; HNF3ß, hepatocyte nuclear factor 3ß; HNF4, hepatocyte nuclear factor 4; MESP1, mesoderm posterior factor 1; MHC, myosin heavy chain; MLC, myosin light chain; RhEB, rhesus embryonic stem cell-derived embryoid body; RT, reverse transcriptase; SPARC, secreted protein acidic and rich in cysteine; SP-D, surfactant protein D; Ttr, transthyretin.

 
RT-PCR Analyses of rESC-Derived EBs Reveal Expression of Marker Genes Typical for Endoderm, Mesoderm, Cardiac Mesoderm, and Mature CMs
To characterize the differentiation pathway of undifferentiated rESCs into functional CMs, semiquantitative RT-PCR analyses were performed. To exclude false-positive results based on contaminating MEFs, all primer pairs were designed to mismatch the corresponding murine sequences. In cases in which no sequence information on the Macaca mulatta genes was available, primers were designed based on available sequences of Homo sapiens, Macaca fascicularis, Pan troglodytes, Sus scrofa, Canis familiaris, and Bos taurus (supplemental online Table 1). The choice of target genes was based on the fact that early mesendodermal progenitors give rise to mesoderm, cardiac mesoderm, and early CMs. Moreover, endodermal marker genes were included because there is evidence that endoderm supplies important molecular signals for these differentiation steps. To control for the presence of different cellular intermediates and key regulators as well as for more mature CMs, we have included a series of marker genes in the study: for early mesendoderm, we chose brachyury and goosecoid; mesoderm posterior factor 1 (Mesp1) was selected for mesoderm and hepatocyte nuclear factor 3-ß (HNF3-ß), hepatocyte nuclear factor 4 (HNF4), transthyretin (Ttr), secreted protein acidic and rich in cysteine (SPARC), {alpha}-feto protein (AFP), and surfactant protein D (SP-D) as endodermal markers. Friend of GATA 2 (FOG-2), GATA-4, Nkx2.5, Tbx5, and Tbx20 were included as markers for cardiac mesoderm. In addition, atrial natriuretic factor (ANF), myosin light chain 2 (atrial and ventricular transcripts) (Mlc2a and Mlc2v), and {alpha}-myosin heavy chain ({alpha}-MHC) served as markers for CMs. Our expression analysis demonstrated that even in "undifferentiated" rESCs, some endodermal and mesodermal marker genes, including SPARC, AFP, HNF3-ß, Ttr, brachyury, goosecoid, Mesp1, GATA-4, FOG-2, and Mlc2a and MLC2v, could be detected (Fig. 2Go). This is in correlation with the observation that cultures of undifferentiated rESCs, similar to hESCs, usually contain a low proportion of colonies with somewhat "differentiated" morphology (supplemental online Fig. 3Go). Analysis of rESCs after 2 days of differentiation in suspension cultures yielded similar results, although a reproducible slight increase in band intensity may indicate increased AFP, Ttr, and brachyury expression (Fig. 2Go). Although statements on differences in expression levels that are based on semiquantitative RT-PCR should be judged with caution, our data suggest an increase of expression of several endodermal and mesodermal marker genes (SPARC, AFP, HNF3-ß, Ttr, brachyury, Mesp1, FOG-2, GATA-4, and Mlc2a) during ongoing differentiation. Goosecoid expression seemed to decrease from day 4 on while other markers for endoderm, cardiac mesoderm, and CMs appeared (HNF4, SP-D, Nkx2.5, Tbx5, Tbx20, ANF, and {alpha}-MHC) (Fig. 2Go).


Figure 3
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  		Figure 3. Demonstration of cardiomyocyte markers in rESC-derived CMs by immunofluorescence staining. rESC-derived cardiomyocytes at day 23 of differentiation. Corresponding immunofluorescence (top) and phase-contrast (bottom) images are shown. Cells were stained with (A) anti-sarcomeric {alpha}-actinin (red), (B) anti-titin (red), (C) anti-troponin T (green), and (D) anti-myosin light chain ventricular (green); nuclei are stained with DAPI (blue). Scale bars = 50 µm. Abbreviations: CM, cardiomyocyte; DAPI, 4,6-diamidino-2-phenylindole; rESC, rhesus monkey embryonic stem cell.

 
Immunohistological Analyses Demonstrate the Presence of Different Phenotypes of CM Precursors/CMs
Figure 3Go shows immunofluorescence staining of rESC-derived CMs (day 23 of differentiation) specific for {alpha}-sarcomeric actinin, titin, troponin T, and MLC2v. Whereas Figure 3Go shows well-organized typical cross-striated muscle filaments visible in CMs, Figure 4Go shows different types of CM precursors and CMs. Depicted in Figure 4AGo are relatively large cells with a predominantly compact round shape without marked cross-striations, which may represent CM precursors or immature CMs. In Figure 4B and 4CGo, smaller and apparently more mature CMs with visible cross-striations and more elongated, triangular, or quadrangular shape are shown. Notably, levels of connexin 43 expression appeared significantly lower in the well-organized CMs (data not shown). Although RT-PCR demonstrated relatively low levels of ANF expression, no ANF-specific antibody staining was detected.


Figure 4
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  		Figure 4. Immunofluorescence staining of morphologically different types of cardiomyocyte precursors/cardiomyocytes expressing connexin 43. rESC-derived cardiomyocytes at day 23 of differentiation. (A): CM precursors/immature CMs. (B, C): Cells displaying a more mature CM phenotype. Corresponding immunofluorescence (top and middle panels) and phase-contrast (bottom panel) images are shown; top panel: anti-sarcomeric {alpha}-actinin (red), anti-connexin 43 (green), DAPI (blue); middle panel: anti-connexin 43 (green), DAPI (blue). Scale bars = 50 µm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; rESC, rhesus monkey embryonic stem cell.

 
Ultrastructural Evidence for Ongoing Formation of Functional Myofibrils
Thin sections of the specimens containing contractile cells showed different cell types: fibroblast-like cells, cells containing several electron dense granules with a diameter of up to 1.75 µm (presumably containing lipids), and cells with large amounts of glycogen particles. The latter cells were identified as CMs apparently in gradual stages of differentiation (supplemental online Figs. 4Go and 5Go). In general, these cells displayed an "embryonic" appearance. Their cytoplasm was comparatively electron-lucent, and the ribosomes were arranged in a polyribosomal fashion rather than associated with the endoplasmic reticulum. The cells were usually surrounded by a basement membrane, which was discontinuous at places (supplemental online Fig. 5BGo). Frequently, electron-dense structures lining the plasma membrane and reminiscent of hemidesmosomes were visible at such sites (supplemental online Fig. 5BGo). Between adjacent cells, junctions of the adherens type were regularly observed. Cytoskeletal elements, mainly microfilaments (approximately 6 nm in diameter) and filaments of approximately 12 nm in diameter (presumably myosin), in different stages of structural organization converged toward these junctions and appeared to be attached to them (supplemental online Fig. 4CGo). In a subpopulation of cells, aggregates of the myofilaments were already aligned as myofibrils (supplemental online Fig. 5Go). These were predominantly located in the cellular periphery. Longitudinal sections of these structures revealed an arrangement in sarcomers formed by distinct Z-lines, narrow I-bands, and wide A-bands, indicating a contracted state of the myofibrils (supplemental online Fig. 5BGo).


Figure 5
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  		Figure 5. Detection of spontaneous and rhythmical electric activity by means of MEAs. (A): An 8 x 8 overview plot of original voltage traces recorded from 60 MEA electrodes. The spontaneous extracellular electrical activity coincided with spontaneous and regular contractions across the recording area. (B): Representative FP waveform. FPPRE, FPMIN, FPSLOW, FPMAX, and FPDUR indicate characteristic components that can be distinguished in the FP (see Materials and Methods). Abbreviations: FP, field potential; FPDUR, FP duration; FPMAX, FP maxima; FPMIN, FP minima; FPPRE, FP pre-spike; FPSLOW, slowly occurring negative FP deflection; MEA, multielectrode array.

 
Electrophysiological Studies by Means of MEAs Demonstrate Functionality, ß-Adrenergic Signaling, and Electrical Coupling of rESC-Derived CMs
To determine basic functional properties of rESC-derived CMs, the MEA system was used as a standard tool for the measurement of extracellular electrical activity. In line with previous reports using hESCs and native embryonic CMs [12, 24, 25], spontaneous contractions paralleled by extracellular electrical activity as well as typical cardiac FP morphologies could be shown (Fig. 5A, 5BGo). To test three-dimensional rESC-derived cardiac clusters for generation and kinetics of electrical propagation as a typical feature of cardiac cells, more complex cardiac clusters were generated. Plating of two beating areas on the MEA led to development of conductive narrow tissue strands in some cases. Figure 6Go shows a typical example of MEA recordings (Fig. 6AGo) performed in a complex cardiac structure with two beating clusters connected by a conduction permitting tissue bridge (Fig. 6BGo, left). These recordings revealed FP morphologic properties that are comparable with the P wave and the QRS complex of electrocardiograms (ECGs). In fact, a small P-like spike (Fig. 6AGo [1]), representing the beating area around electrode 34, was driving a QRS-like complex (Fig. 6AGo [2]), illustrating the beating area in the vicinity of electrode 65. Most interestingly, the latency between these different FP complexes was found to be constant under control conditions (0.16 ± 0.005 seconds, n = 4). These findings indicate that the first beating area may serve as a primary pacemaker, continuously driving the activity of the second cluster. Mapping of the FP amplitude distribution is shown in Figure 6BGo (right) using the original recordings depicted for electrode 34 and 65 in Figure 6CGo as its basis. Another finding, typical for cardiac cells, is the positive chronotropic response to ß-adrenergic agonists. Accordingly, application of ISO at a concentration of 1 µM increased the beating frequency as shown in Figure 7AGo. In supplemental online Figure 6Go, the corresponding statistical analysis is shown. This original recording, as well as the time-dependent decrease of the ISI (Fig. 7BGo), indicates a stable increase of beating frequency until washout of ISO was performed. Figure 7CGo depicts original recordings at higher magnification of control conditions (left), after application of ISO (middle) and after washout (right), respectively. As previously reported [24], FPSLOW can be taken as a direct readout for L-type Ca2+ channel activity. In addition, we performed experiments applying 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)pyridine-3-carboxylic acid methyl ester (Bay-K 8644) (10 µM), an L-type Ca2+ channel agonist, in cardiac clusters derived from rESCs and studied effects on FPSLOW amplitude. As would be expected, FPSLOW amplitude was significantly increased after application of Bay-K 8644 (supplemental online Fig. 7Go). Because it was expected to find an enhancement of L-type Ca2+ channel activity and increased conduction velocities after stimulation of CMs derived from the rESC system with ß-adrenergic agonists, the FPSLOW amplitude and conduction was analyzed. For the respective experiment shown in Figure 7A–7CGo, FPSLOW amplitude was found to be significantly increased under influence of ISO (Fig. 7DGo). The conduction time between the two cardiac clusters shown in Figure 6BGo was strongly reduced after ß-adrenergic stimulation (0.144 ± 0.001 seconds, n = 3, p < .05).


Figure 6
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  		Figure 6. Electrical coupling of isolated beating areas via formation of a conductive tissue strand. (A): Coupling between beating areas. Plating of more than one contracting cluster on the MEA leads to the development of FPs that are comparable with the P wave and the QRS complex of electrocardiograms; that is, the population FP consisted of a small (P-like [1]) spike followed by a large (QRS-like, [2]) complex. The latency between the P-like and the QRS-like spike was found to be constant under control conditions, indicating that the P-like spike represents the activity of the primary pacemaker area and the QRS-like spike the activity of the driven area. (B): Mapping of rESC activity. Distribution of the FP amplitude of an rESC cluster containing two spontaneously active areas (around electrode #34 and #65) connected by a narrow tissue strand (left). The image was taken at the beginning of phase II as indicated in (C). Original single FP recordings of #34 and #65. Arrows indicate time point displayed in (B) (right). Abbreviations: FP, field potential; ISI, inter-spike interval; MEA, multielectrode array; rESC, rhesus monkey embryonic stem cell.

 

Figure 7
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  		Figure 7. ß-Adrenergic signaling in rESC-derived cardiac clusters. (A): Original recording of FPs derived from a representative rESC cluster under the influence of 1 µM ISO. After application of ISO, spontaneous beating and FP frequency were significantly increased, whereas the washout procedure restored the frequency of contractions to basal conditions. (B): Time trace of the ISI. The time trace depicts the decrease of the ISI, indicating a significant increase of the beating and FP frequency. After washout, the FP frequency declined to control conditions. (C): Original FPs recorded in (A) at higher magnification. Left: control conditions; middle: maximum of ISO response; right: washout. (D): Statistical analysis of the FPSLOW amplitude. The FPSLOW amplitude, taken as an indirect readout for voltage-dependent Ca2+ channel activity, was significantly increased after application of ISO (1 µM). For statistical evaluation, ANOVA was performed. Abbreviations: ANOVA, analysis of variance; FP, field potential; FPSLOW, slowly occurring negative FP deflection; ISI, interspike interval; ISO, isoproterenol; rESC, rhesus monkey embryonic stem cell.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosures
 References
 
hESCs may represent an ideal cell sources for clinical restoration of myocardial injuries [11, 27, 28]. Whereas questions of optimized expansion, specific in vitro differentiation, and purification of ESC-derived CMs prior to transplantation can be addressed directly by using hESCs [9, 11, 13], other issues such as postimplantation functionality, ESC transplantation-related immunological processes [29, 30], or formation of teratoma [31] have to be investigated in allogeneic large animal models prior to clinical use.

According to the (still very limited) available data, one can expect rESCs to be very similar to hESCs in many aspects, including expression of marker genes, pluripotency, proliferation potential, differentiation potential, and genetic stability [32]. Therefore, rESCs and rESC-derived CMs represent a suitable model to assess mechanistic issues, therapeutic effects, and risks of cell therapy applying CM precursors/CMs generated from hESCs.

Similar to many hESC lines, the rESC line R366.4, which has been used in this study, seems already somewhat committed in its potential for differentiation. In particular, formation of neuronal/glial cells appears to represent a default pathway [33]. In our hands and in good correlation with other reports [33], the majority of differentiation protocols reproducibly resulted in prominent formation of ectoderm and efficient generation of cells with neuronal/glial phenotype (supplemental online Fig. 1Go). Nevertheless, using our adapted new protocol, we were able to successfully generate functional CMs from rESCs with efficiencies comparable with hESCs, thereby demonstrating that existing commitments of ESC lines are still reversible by providing the appropriate stimuli and environment. One of the crucial factors to achieve efficient differentiation toward mesoderm/endoderm and toward CMs was the choice of FCS. Whereas five different lots of FCS resulted in prominent formation of neuronal/glial cells, only one batch of FCS provided by HyClone was able to efficiently drive cardiac differentiation. Certainly, identification of factors responsible for these effects will be one of the most important tasks for the future.

Morphologically, undifferentiated ESCs as well as EBs were similar to hESCs. Although at this point, the efficiency of CM formation is relatively low in comparison with murine ESCs, the establishment of our differentiation system allows for systematic investigation of molecular pathways and improvement of cardiac differentiation. During differentiation of rESCs, expression of most of the analyzed markers typical for endoderm, mesoderm, cardiac mesoderm, and CMs developed as was expected from corresponding data of mouse ESCs and hESCs. In contrast to other cardiac markers, Mlc2a and MLC2v were already present in "undifferentiated" rESC cultures. This result was unexpected but was confirmed in both cases by a second independent primer pair (data not shown). At least for Mlc2a, this has also been reported in the human system [11].

To extend the characterization of the generated CMs to structural features, contracting regions were stained with different antibodies specific for components of the contractile muscle filaments. As depicted in Figure 3Go, the generated CMs were positive for sarcomeric {alpha}-actinin, troponin T, titin, and MLC2v. Notably, in all cases except for MLC2v, cross-striations of the filaments were visible. Although low levels of ANF expression have been demonstrated on the mRNA level, immunohistology did not confirm this finding. It is currently not known whether the cellular ANF expression level was too low to result in detectable antibody staining or whether a low frequency of ANF-expressing CMs prevented immunohistological staining.

Besides the phenotypes depicted in Figures 3Go and 4B and 4CGo, cells positive for sarcomeric {alpha}-actinin were observed, which had quite different appearance (Fig. 4AGo): relatively large cells with a predominantly compact round shape without marked cross-striations. Most probably, these different phenotypes represent different developmental stages. This assumption is strongly supported by our ultrastructural observations (supplemental online Figs. 4Go and 5Go). In general, the comparatively electron-lucent cytoplasm and the polyribosomal arrangement of ribosomes with a majority not associated with the endoplasmic reticulum are typical for an embryonic phenotype. In correspondence with the phenotype shown in Figure 4AGo, a proportion of cells showed beginning formation of myofilaments, partially converging toward junctions (supplemental online Fig. 4Go). In such cells, similar to the immunohistological staining (Fig. 4AGo), no myofibrils were observed. Although those primate ESC-derived cells, different than typical murine cardiac precursors, display a relatively small nucleus/cytoplasm ratio, and although we are currently not able to prove this assumption, we assume that the cells shown in Figure 4AGo and supplemental online Figure 4Go represent early CMs/cardiac precursors and not different subtypes of myocytes.

Apparently more mature CMs already contained cytoskeletal filaments arranged as myofibrils and sarcomeric structures containing Z-lines, broad A-bands, and narrow I-bands (supplemental online Fig. 5Go). The morphologies of these cells are similar to morphologies that can be observed after cardiac differentiation of mouse ESCs [34].

In view of the observed synchronous contraction of the generated CMs, it was not surprising to detect expression of connexin 43, one of the major gap junction components. Although not demonstrated in Figure 4Go, it was striking that connexin 43 expression appeared significantly lower in the well-organized CMs than in the relatively large cells without clear cross-striation. Further detailed analysis will be necessary to uncover the basis for this observation.

Electrophysiological studies have been performed to obtain further proof for the functionality of the generated CMs. Using the MEA system, we were able to record spontaneous extracellular activity in beating cell clusters. One advantage of the MEA system is that areas with electric activity can be localized and isolated, because only those electrodes located nearby active CMs will detect FPs (Fig. 5AGo). Although the recorded FP is dependent not only on the functional characteristics of the adjacent cells but also on the distance of the CM membrane to the electrode, details of the recorded FPs principally allow the identification of different CM subtypes. In our case, specific statements on the measured cell types were not possible, because adult or fetal rhesus monkey-derived CMs were not available as control groups. In good correlation with the connexin 43 expression was the finding that independent beating cell clusters were able to establish physical connections and to electrically communicate after plating on MEAs. Plating complex EBs on MEAs, we were even able to demonstrate ECG-like recordings in rESCs (Fig. 6Go). Furthermore, ß-adrenergic signaling as a specific feature of CMs versus other muscle cell types, including skeletal muscle and smooth muscle cells, was investigated by stimulation with the ß-adrenergic agonist ISO. Very similar to hESC-derived CMs, rESC-derived CMs responded with an increased beating and FP frequency (Fig. 7A–7CGo) until washout of ISO was performed. Moreover, and also very typical, the FPSLOW amplitude as an indirect readout for voltage-dependent Ca2+ channel activity was significantly increased after application of ISO (Fig. 7DGo). Nevertheless, we cannot exclude that our cultures contained a certain portion of skeletal myoblasts.

Thus, rESCs display typical functional characteristics of embryonic CMs: (a) development of spontaneous electrical activity and beating, (b) electrical propagation, (c) positive chronotropy, (d) increased L-type Ca2+ channel activity, and (e) enhanced conduction after application of ß-adrenergic agonists.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosures
 References
 
This is the first study demonstrating successful differentiation of rESCs toward functional CMs. Strikingly, this differentiation was possible, although the applied rESC line R366.4 is already committed to a certain extent in its potential for differentiation and differentiates toward ectoderm and cells of the neuronal/ glial lineage as a default pathway. rESCs represent a valuable tool to investigate cell survival, electrical coupling, functional integration, and functional improvement as well as the risk of teratoma formation after myocardial transplantation in allogeneic preclinical nonhuman primate models. Moreover, rESC-derived CMs in combination with MEA technology should allow the development of a cell-based high-throughput pharmacological test system for cardiac drug profiling [35].


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
This work has been supported by the Brauckmann-Wittenberg Foundation, the Niedersachsen Foundation/CORTISS Foundation, the BMBF (Network for Development of a Clinical Applicable Human Stem Cell-Based Cardiac Tissue Engineering Technology), and a grant of the Lower Saxony Ministry for Education and Science to fund joint Lower Saxony-Israel research projects. S.W. is supported by a scholarship of the Aventis Foundation. We are grateful to Michal Amit for assistance with stem cell biology techniques. We also thank Aret Bekcioglu for his technical assistance.


   REFERENCES
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 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 

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