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(Circulation. 2003;107:2733.)
© 2003 American Heart Association, Inc.
Basic Science Reports |
From the Hubrecht Laboratory (C.M., D.W.v.O., R.S., S.v.d.B., R.H., R.P., L.T.); the Departments of Medical Physiology (M.v.d.H., T.O.), Cardiothoracic Surgery (R.H., A.B.d.l.R.), and Cardiology (P.D.), University Medical Center Utrecht; and the Interuniversity Cardiology Institute of the Netherlands (C.M., P.D., R.P.), Utrecht, Netherlands; and the Monash Institute of Reproduction and Development, Melbourne Australia (M.P.).
Correspondence to Christine Mummery, Hubrecht Laboratory, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands. E-mail christin{at}niob.knaw.nl
| Abstract |
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Methods and Results hES cells were cocultured with visceral-endoderm (VE)like cells from the mouse. This initiated differentiation to beating muscle. Sarcomeric marker proteins, chronotropic responses, and ion channel expression and function were typical of cardiomyocytes. Electrophysiology demonstrated that most cells resembled human fetal ventricular cells. Real-time intracellular calcium measurements, Lucifer yellow injection, and connexin 43 expression demonstrated that fetal and hES-derived cardiomyocytes are coupled by gap junctions in culture. Inhibition of electrical responses by verapamil demonstrated the presence of functional
1c-calcium ion channels.
Conclusions This is the first demonstration of induction of cardiomyocyte differentiation in hES cells that do not undergo spontaneous cardiogenesis. It provides a model for the study of human cardiomyocytes in culture and could be a step forward in the development of cardiomyocyte transplantation therapies.
Key Words: electrophysiology myocytes stem cells
| Introduction |
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See p 2638
Molecular pathways leading to specification and terminal differentiation of cardiomyocytes from embryonic mesoderm during development are still unclear. Data derived from chick and amphibian suggested that cardiac progenitors require interaction with anterior endoderm and possibly the organizer for myocardial differentiation to take place.57 More recently, primitive streak and visceral embryonic endoderm were shown to be important for the multistep induction through which cardiac progenitor cells acquired the competence to complete terminal differentiation at day 7.5 of gestation in mice.8
Here, we demonstrate that coculture of pluripotent human ES (hES) cell lines with END-2 cells induces extensive differentiation to 2 distinctive cell types from different lineages. One is epithelial; it forms large cystic structures that stain positively for
-fetoprotein and is presumably extraembryonic VE; the others are grouped in areas of high local density and beat spontaneously. We show that these beating cells are cardiomyocytes. Although differentiation of hES cells to cardiomyocytes has been described previously,911 the hES cell lines used differentiate spontaneously to somatic derivatives in embryoid bodies, reminiscent of those formed by mES cells.12 The present work is thus the first describing induction of cardiomyocyte differentiation in hES cells, which do not undergo cardiogenesis spontaneously, even at high local cell densities, and is the first direct electrophysiological comparison of hES-derived cardiomyocytes with primary human fetal cardiomyocytes in culture.
| Methods |
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Immunohistochemistry
Cells were fixed with 3.0% paraformaldehyde, then permeabilized with 0.1% Triton X-100. Undifferentiated hES colonies were stained overnight at 4°C with anti-oct4 (Sigma) and visualized by use of the avidin-biotin complex/horseradish peroxidase kit (DAKO) and the Fast 3,3'-diaminobenzidine tablet set (Sigma). For immunofluorescence antibodies against
-actinin, tropomyosin, and pan-cadherin (Sigma), myosin light chain (MLC)-2a and -2v (from Dr K. Chien, San Diego Institute of Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, Calif),
1c and Cav1.2a (Alomone Laboratories), connexin 43 (Cx43) (Transduction Laboratories), and phalloidin-Cy3 (Sigma) were used in combination with fluorescence-conjugated secondary antibodies (Jackson Laboratories). Confocal images (Leica Systems) were made (63x objective) from 2D projected Z series.
Primary Human Adult and Fetal Cardiomyocytes
Primary tissue was obtained during cardiac surgery or after abortion after individual permission had been obtained by use of standard informed consent procedures and approval of the ethics committee of the University Medical Center, Utrecht. Adult cardiomyocytes were isolated and cultured as reported previously.3 Fetal cardiomyocytes were isolated from fetal hearts (16 to 17 weeks) perfused by Langendorffs method and cultured on glass coverslips. For patch-clamp electrophysiology, cells were collected in Tyrodes buffer with low Ca2+.15
Reverse TranscriptionPolymerase Chain Reaction
RNA was isolated by use of Ultraspec (Biotecx Laboratories) and reverse transcribed (RT; 500 ng total RNA) as described previously.16 Primer sequences and conditions for polymerase chain reaction (PCR) are given in Table 1. Products were analyzed on ethidium bromidestained 1.5% agarose gel. ß-Actin or ß-tubulin was used as RNA input control.
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Electrophysiology
Data were recorded from cells at 33°C in spontaneously beating areas by use of an Axopatch 200B amplifier (Axon Instruments Inc). Cell-attached patches were made in the whole-cell voltage-clamp mode. The pipette offset, series resistance, and transient cancellation were compensated; subsequent action potentials were recorded by switching to the current-clamp mode of the 200B amplifier. Output signals were digitized at 4 kHz by use of a Pentium III equipped with an AD/DAC LAB PC+ acquisition board (National Instruments). Patch pipettes with a resistance between 1 and 3 M
were used. Bath medium was (mmol/L) 140 NaCl, 5 KCl, 2 CaCl2, and 10 HEPES, adjusted to pH 7.45 with NaOH. Pipette composition (mmol/L) was 145 KCl, 5 NaCl, 2 CaCl2, 4 EGTA, 2 MgCl2, and 10 HEPES, adjusted to pH 7.30 with KOH. Verapamil was used at 5 µmol/L.
Calcium Measurements
Cells were labeled for 15 minutes at 37°C with 10 µmol/L fura 2-AM. The light from 2 excitation monochromators (SPEX fluorolog, SPEX Industries) was rapidly alternated between 340 (slit width: 8) and 380 (slit width: 8) nm and coupled into a microscope via a UV-optic fiber. Fluorescence intensity images were recorded from living cells at a maximal rate of 120 ms/pair and corrected for background fluorescence. Calibration used the minimal ratio (Rmin) after addition of 5 µg/mL ionomycin and 4 mmol/L EGTA (pH 8) to the cells and the maximal ratio (Rmax) after addition of 5 µg/mL ionomycin and 10 mmol/L CaCl2. The calcium concentration was calculated as follows: (R-Rmin)/(Rmax-R)xsf2/sb2xKd, where sf2 indicates the free dye concentration at 380 nm at saturating calcium conditions and sb2 is the calcium-bound dye concentration at 380 nm at saturating calcium conditions.17
Dye Coupling
A filtered solution of 3% wt/vol Lucifer yellow lithium salt (Molecular Probes) in 150 mmol/L LiCl was microinjected through Quickfill glass microelectrodes (Clark Electromedical Instruments). Dye was injected into one of a group of spontaneously beating cells by a 1-Hz square pulse (50% duty cycle), amplitude of 5x10-9 A. Directly after injection, confocal laser scanning microscope images were made of the injected areas.
| Results |
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60% showed nuclear staining for oct-4; flattened cells were negative (Figure 1B). Oct-4 expression thus correlated with phenotypic characteristics of undifferentiated cells. hES cells were subcultured by transferring small clumps of undifferentiated cells onto either new MEFs or END-2 cells. After
5 days, epithelial cells appeared, which gradually become fluid-filled cysts (Figure 1C). These stained for
-fetoprotein (Figure 1H), suggesting that they represent extraembryonic VE. By 10 days, areas of rhythmically contracting cells in more solid aggregates became evident in the hESEND-2 cocultures (Figure 1C, arrow) with a variety of overall morphologies (Figure 1D). In a 12-well plate, 35±10% of the wells (n=30) contained beating areas, each of which could be dissociated and replated to yield up to 12 new colonies of beating cells with a 2D rather than 3D morphology (Figure 1G); this facilitated access to the cells for electrophysiology. Each beating area consisted of 10 to 200 cardiomyocytes. Control cultures on MEFs showed no evidence of beating muscle or extensive cyst formation but had formed very large colonies with many flattened cells at the edges (not shown). Conversely, hES on HepG2 cells did form areas of beating muscle, usually attached to HepG2 cell colonies.
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Before and after dissociation, hES-derived cardiomyocytes beat 35 to 90 times per minute (Table 2). Cardiomyocyte colonies could be frozen and sometimes resumed beating on thawing. To characterize the cardiomyocytes further, we carried out immunofluorescent staining for sarcomeric proteins, used BIDOPY-ryanodine as a vital stain for ryanodine receptors in the sarcoplasmic reticulum, and analyzed the expression of ion channels by RT-PCR. In each case, we used primary human fetal and adult atrial and ventricular tissue as controls. The data showed that hES-derived cardiomyocytes exhibited sarcomeric striations when stained with
-actinin (Figure 2A), organized in separated bundles. These were reminiscent of the bundles observed in human fetal cardiomyocytes (Figure 2, B and C), although the individual sarcomeres were less well defined. The morphology was different from the highly organized, parallel bundles in cells from biopsies of adult human heart (Figure 2, G and H). hES-derived cardiomyocytes also stained with MLC-2a, MLC-2v (not shown), and tropomyosin (Figure 2D); again, the sarcomeres were less evident than in human fetal and adult cardiomyocytes (Figure 2, E, F, and H).
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Expression of Cardiac Ion Channels and Stem Cell/Sarcomere Markers in hES/END-2 Cocultures
Expression of cardiac-specific ion channels was determined in undifferentiated hES cells and at 8 and 15 days after initiation of coculture with END-2 cells (Figure 2K). As shown previously by others,10 areas of beating hES-derived cardiomyocytes express atrial natriuretic factor. Expression of the
-subunits of the cardiac-specific L-type calcium channel (
1c) and the transient outward potassium channel (Kv4.3) was also detected, the expression of Kv4.3 preceding onset of beating by several days. RNA for the delayed rectifier potassium channel KvLQT1 was found in undifferentiated cells, but transcripts disappeared during early differentiation and reappeared later.
Over a similar time course, expression of oct-4 was reduced, whereas transcripts for
-actinin, MLC-2a, and MLC-2v became detectable (Figure 2K), reflecting the results of antibody staining.
Electrophysiology
Patch-clamp electrophysiology on dissociated hES cardiomyocytes showed that different electrical phenotypes were present (Figure 3A). Ventricular-like action potentials predominated (28 of 33; Table 2), but atrial-like (n=2), pacemaker-like (n=1), and vascular smooth musclelike cells (n=2) were also found. In areas in which the cells were not beating but had adopted morphologies indistinguishable from those of beating areas (Figure 1F), current injection was sufficient to induce repeated action potentials and sustained synchronous rhythmic contractions. Transcripts for MLC-2v were also detected by RT-PCR in nonbeating, myocyte-like areas (not shown); scoring beating muscle may thus underestimate the number of cardiomyocytes present in culture. The upstroke velocities (Volts/s) for the ventricular-like cells were low (8 Volts/s) but comparable to those in cultured human fetal ventricular cardiomyocytes, although incidental peak values were found (Table 2).
1-Adrenoceptors, ß1-adrenoceptors (regulated via a cAMP-dependent mechanism), and nicotinic acetylcholine receptors are known to influence cardiac function. Chronotropic responses of dissociated hES cardiomyocytes were also compared with human fetal ventricular cells (Figure 3C). Addition of carbachol decreased the beating rate of hES-derived cardiomyocytes and human fetal ventricular cells, whereas phenylephrine and isoprenaline increased the rate in both cell types. Similar effects were reported in mES-derived cardiomyocytes18 and mouse fetal cells.19
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[Ca2+]i Transients in Differentiated hES Cells
Calcium oscillations were recorded in dissociated groups of spontaneously beating hES cardiomyocytes (Figure 4). The continuous character of the repetitive line scans in Figure 4B in the left-to-right direction, compared with the vertical lines in 4C, shows that the action potential in Figure 3A propagates in a top-down direction and indicated tightly developed cell-to-cell coupling in this synchronously contracting group of cells. Regular repetitive oscillations in [Ca2+]i are found in single hES cardiomyocytes (Figure 4E). Coupling between cells was confirmed by Lucifer yellow injection into single cells; the dye spread within minutes to other cells within the group in both hES-derived (Figure 5E) and primary fetal cardiomyocytes (not shown). Cx43 staining (Figure 5, B and D) indicated the presence of gap junctions. Staining with a pan-cadherin antibody also indicated the presence of adherens junctions between cells in fetal and hES-derived cardiomyocytes (Figure 5, A and C).
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L-type calcium channels compose the predominant route for calcium entry into cardiac myocytes and are key components in excitation-contraction coupling. A specific
1C antibody stained cardiomyocytes in both differentiated hES cultures (Figure 4F) and human fetal ventricular cells (Figure 4G), in agreement with the RT-PCR data (Figure 2K).
| Discussion |
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Staining for junctional proteins showed that the hES-derived cardiomyocytes were very immature, although real-time determination of intracellular Ca2+ concentrations clearly showed that the cells were electrically coupled. Kehat et al9 recently reported similar findings in independently derived hES-cardiomyocytes. It will be of interest to subject these hES-derived cardiomyocytes to oscillating stress to see whether the sarcomeric structure matures to the adult phenotype.
In the adult mammalian myocardium, cellular Ca2+ entry is regulated by the sympathetic nervous system. L-type Ca2+ channel currents are markedly increased by ß-adrenergic agonists, which contribute to changes in rate and contractile activity of the heart. Exactly how this Ca2+ regulatory system is established in development is not yet clear. Our data indicate that the L-type Ca2+ channels in hES-derived cardiomyocytes and fetal cardiomyocytes responded to adrenergic stimuli, indicating a fully developed and connected downstream pathway. Verapamil, which specifically blocks L-type Ca2+ channels, inhibited action potentials in fetal and hES-derived cardiomyocytes, as expected. This contrasts with mouse fetal myocytes and mES-derived cardiomyocytes, in which early cells were nonresponsive despite the presence of L-type Ca2+ channels. Here, the lack of cAMP-dependent protein kinase appeared to be the limiting factor.12,19 Thus, although hES-derived and early human fetal cardiomyocytes show some features in common with early mouse cardiomyocytes, their calcium channel modulation resembles that in the adult mouse. hES cells may thus represent an excellent system for studying changes in calcium channel function during early human development, which appears to differ significantly from that in mice. Furthermore, the appropriate calcium handling makes the cells more suitable for transplantation. Interesting was the observation of cells with plateau- and nonplateau-type action potentials in the fetal atrial cultures. These have been described dispersed throughout the atrium of intact fetal hearts28 and have been considered a possible index of specialization of an atrial fiber, although their significance is not clear. The nonplateau type was not observed among the hES-derived cardiomyocytes.
Finally, vital fluorescent staining with ryanodine or antibodies against cell surface
1c ion channels allowed cardiomyocytes to be identified in mixed cultures. This may provide a means of isolating cells for transplantation without genetic manipulation or compromising viability.
| Acknowledgments |
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| Footnotes |
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Received December 9, 2002; revision received March 4, 2003; accepted March 5, 2003.
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