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1Institut für Pharmakologie und Toxikologie, Medizinische Fakultät Carl Gustav Carus der TU Dresden, D-01307 Dresden, Germany 2 Medizinische Klinik und Poliklinik I, Medizinische Fakultät Carl Gustav Carus der TU Dresden, D-01307 Dresden, Germany
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Abstract |
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1C of the L-type calcium channel). Outward currents were recorded in almost all cells. The most abundant outward current rapidly activated at potentials positive to +20 mV. This current was identified as a large-conductance voltage- and Ca2+-activated K+ current, conducted by MaxiK channels, due to its high sensitivity to tetraethylammonium (IC50= 340 µM) and its inhibition by 100 nM iberiotoxin. A large fraction of cells also demonstrated a more slowly activating current at potentials positive to –30 mV. This current was selectively inhibited by clofilium (IC50= 0.8 µM). Ba2+ inward currents, stimulated by 1 µM BayK 8644 were found in a few cells, indicating the expression of functional L-type Ca2+ channels. Other inward currents such as sodium currents or inward rectifier currents were absent. We conclude that undifferentiated hMSC express a distinct pattern of ion channel mRNA and functional ion channels that might contribute to physiological cell function.
(Received 25 September 2003;
accepted after revision 23 October 2003;
first published online 24 October 2003)
Corresponding author U. Ravens: Institut für Pharmakologie und Toxikologie, Medizinische Fakultät Carl Gustav Carus der TU Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany. Email: ravens{at}rcs.urz.tu-dresden.de
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Introduction |
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There is evidence that hMSC can also obtain the characteristics of excitable cells. hMSC have the capability of differentiating into cells with a neurone-like phenotype both in vitro (Kim et al. 2002) and in vivo (Zhao et al. 2002), and implantation into the ischaemic brain of rats improved functional performance of the apoplectic animals (Zhao et al. 2002). In addition, hMSC might be a suitable cell source for cardiac tissue repair in patients with myocardial infarction or heart failure. It has been shown that hMSC injected into the ventricles of immunodeficient mice engrafted into the myocardium and appeared to differentiate into cardiomyocytes, albeit with low frequency (Toma et al. 2002). In a swine myocardial infarction model injection of hMSC into the region of myocardial infarction reduced the extent of wall thinning and improved contractile dysfunction (Shake et al. 2002). Based on these findings it was proposed that hMSC would be useful for cardiomyoplasty (Cahill et al. 2003).
Meanwhile, cell therapy for the treatment of ischaemic heart disease has advanced to early clinical studies. In patients with myocardial infarction, bone marrow cells were obtained and injected into the infarct-related coronary artery using a balloon catheter (Assmus et al. 2002; Strauer et al. 2002). Other groups implanted the cells into ischaemic myocardium by means of a catheter-directed intramural injection (Tse et al. 2003) or directly injected cells into the infarct border zone during open heart surgery (Stamm et al. 2003). All four studies report an improvement of myocardial perfusion or functional parameters. With the exception of one study (Stamm et al. 2003), the mononuclear bone marrow cell suspension employed for cardiac injection was highly heterogeneous, and the used cell populations varied greatly among these clinical studies.
Up to now, enhanced incidence of arrhythmias in patients who received cardiac implantation of bone marrow stem cells has not been noted, although such risk cannot be excluded (Al-Radi et al. 2003). Cardiomyocytes derived from human embryonic stem cells have the potential to generate arrhythmic action potentials under some conditions (Zhang et al. 2002). At present there is little information about the electrophysiological properties of human bone marrow stem cells. We therefore studied the population of hMSC isolated in our laboratory from bone marrow samples. Commercially available hMSC were used for comparison. We investigated whether undifferentiated hMSC express ion channel mRNA and whether the cells have functional ion currents.
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Methods |
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All parts of this study, especially isolation of human mesenchymal stem cells (hMS) were performed according to the Declaration of Helsinki. The study was approved by the local ethics committee and written informal consent was obtained from donors of bone marrow and cardiac samples. Bone marrow samples were collected from 16 healthy donors at the Mildred Scheel Bone Marrow Transplantation Center of the University Clinics, Dresden hMSC were isolated and cultured according to modifications of previously reported methods (Haynesworth et al. 1992; Pittenger et al. 1999). Briefly, an aliquot from bone marrow aspirate diluted with PBS–0.5% human serum albumin (HSA) was layered over a Percoll solution (d = 1.073 g ml-1, Biochrom, Germany) and centrifuged at 900 g for 30 min. Mononuclear cells at the interface were recovered, washed twice in PBS–HSA and seeded into 75 cm2 flasks containing Dulbecco's modified Eagle's medium (DMEM, low glucose) supplemented with 2 mM GlutaMAX, 10 U ml-1 penicillin, 100 µg ml-1 streptomycin (all from Gibco Invitrogen, UK) and 10% fetal calf serum (Biochrom). The medium was completely changed after 24 h. After automatic counting cells were maintained in a humidified atmosphere at 5% CO2 and 37°C until reaching 90% confluency. For subcultivation the cells were replated at a density of 5000 cells cm-2 (Bruder et al. 1997). Aliquots of different passages were used for flow-cytometric characterization of the cells (FACS calibor 3CS, Becton Dickinson).
In addition to hMSC isolated in our laboratory we investigated commercially available hMSC (Poietics, BioWhittaker, San Diego, CA, USA) for comparison. These hMSC originated from three Caucasian female donors aged 18, 19 and 26 years (lot numbers 0F2014, 1F0658 and 1F1061). The cells were obtained from the supplier in the 1st or 2nd passage and were certified by the following surface markers: CD29+, CD44+, CD105+, CD166+ (each > 95%) and CD14-, CD34-, CD45- (each < 1%). hMSC from BioWhittaker were cultured with the respective mesenchymal stem cell growth medium (hMSCGM; BioWhittaker). mRNA was extracted from subconfluent 4th passages and patch clamp experiments were performed on cells from the 3rd to 6th passage.
Electrophysiological recordings
Membrane currents were measured in the whole cell configuration of the patch clamp technique at 21–23°C (Hamill et al. 1981). Initially, we tried to record currents of hMSC attached to glass cover slips. However, electrophysiological analysis was not feasible under these conditions. We therefore detached subconfluent hMSC from small culture flasks (T25, Greiner, Frickenhausen, Germany) using trypsin–EDTA. After centrifugation at 88 g for 5 min cells were recovered in culture medium. The suspension was stored at room temperature and used within 6 h.
For electrophysiological recordings the cells were transferred to a small chamber (Warner Instruments, Hamden, CT, USA) and allowed to attach to the glass bottom for 15 min. Subsequently, the bath was perfused continuously at a rate of 1.8 ml min-1. Ion channel blockers were applied with the use of a magnet valve or with a rapid solution exchanger (DAD-12 superfusion system, ALA Scientific Instruments, Westbury, NY, USA). Membrane currents were measured with a List EPC-7 amplifier (List Medical Instruments, Darmstadt, Germany) under the control of pCLAMP 5.5 software (Axon Instruments, Foster City, CA, USA). Patch electrodes were pulled with a horizontal puller (Zeitz, München, Germany) from filamented borosilicate glass. The tip resistance was 1.5–4.0 M
, when filled with electrode solution. Membrane capacitance was measured with fast depolarizing ramp pulses (from –55 to –50 mV, duration 5 ms) at the beginning of each experiment as previously described (Heubach et al. 1999). Series resistance was routinely checked and compensated by 50–70%. Membrane currents were low-pass filtered at 2 kHz.
Outward currents were recorded with the following bath solution (mM): NaCl 150, KCl 5.4, CaCl2 2, MgCl2 2, glucose 11, Hepes 10 (pH 7.4 adjusted with NaOH). The pipette solution included (mM): NaCl 8, KCl 40, potassium aspartate 100, Tris-GTP 0.1, Mg-ATP 5, CaCl2 2, EGTA 5 (pH adjusted to 7.3 with KOH) resulting in a calculated free Ca2+ and Mg2+ concentration of 64 nM and 587 µM, respectively (Fabiato & Fabiato, 1979). Solutions containing different concentrations of tetraethylammonium chloride were prepared by mixing bath solution as described above with a bath solution in which 20 mM NaCl was replaced by 20 mM tetraethylammonium chloride, in order to keep osmolarity constant. All membrane potentials were corrected for a calculated liquid junction potential of 12.5 mV (JPCalc version 2.2; Barry, 1994). The stimulation frequency was 0.25 Hz. Current amplitude was determined at the end of individual depolarizing steps.
The presence of functional Ca2+ channels was assessed with Na+-free external solution supplemented with 2 mM Ca2+ or 10 mM Ba2+ under conditions previously described (Heubach et al. 2000). Inward rectifier currents were measured in 20 mM K+ solution with ramp pulses from -100 mV to +40 mV of 1250 ms duration from a holding potential of –80 mV as used for the recording of inward rectifier currents in human atrial myocytes (Dobrev et al. 2000). In order to enhance the ATP-sensitive potassium current (IK,ATP), if present, the intrapipette Mg-ATP concentration was reduced to 0.1 µM. The presence of hyperpolarization-activated currents was investigated as described for mouse ventricular myocytes (Graf et al. 2001). Membrane potential was hyperpolarized for 2 s by steps to –140 mV from a holding potential of –40 mV.
Isolation of RNA and polymerase chain reaction experiments
Total RNA (0.5 µg) isolated by the guanidinium method (Chomczynski & Sacchi, 1987) was reverse transcribed in a 21 µl reaction mixture that contained 75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM MgCl2, 0.5 mM of each dATP, dCTP, dGTP, dTTP, 600 ng of random hexamer primers, 10 mM DTT, 2 U of RNase inhibitor and 10 U of Superscript RNase H- (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. For PCR experiments 3 µl aliquots of total cDNA were amplified (Master Cycler, Eppendorf, Hamburg, Germany) in a 25 µl reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.2 mM of each dATP, dCTP, dGTP, dTTP, 25 pmol of each forward and reverse primer and 1.25 U of Taq polymerase (Applied Biosystems, Weiterstadt, Germany; for primers and reaction conditions see Table 1). The same single-stranded cDNA product was used to analyse the expression of all genes described. To ensure that amplification was in the exponential range, the progress of PCR was determined by amplifying identical reaction mixtures for ascending numbers of cycles. After the cited number of PCR cycles amplification rate was sufficient without reaching saturation for any of the amplicons. PCR products were analysed by agarose gel electrophoresis (2% agarose) and ethidium bromide staining. Bands imaged by a CCD camera (Biostep, Jahnsdorf, Germany) were analysed via optic densitometry with Phoretix 1D software (Biostep).
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All chemicals used were of analytical grade and were purchased from commercial suppliers (Sigma, Deisenhofen, Germany, and VWR, Darmstadt, Germany). 4-Aminopyridine, BayK 8644, tetraethylammonium chloride, linopirdine and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) were ordered from Sigma. Clofilium tosylate was from Lilly (Indianapolis, IN, USA), E-4031 was from Eisai Co. (Ibaraki, Japan), and HMR1556 was from Aventis (Frankfurt, Germany). Recombinant iberiotoxin was obtained from Calbiochem (San Diego, CA, USA) and recombinant ergtoxin was ordered from Alomone Laboratories (Jerusalem, Israel). Hanatoxin was a kind gift from Dr Kenton J. Swartz (National Institute of Neurological Disorders and Stroke, NIH Bethesda, MA, USA). Solutions containing peptide toxins were supplemented with 0.1% bovine serum albumin.
Data analysis and statistics
As shown below we detected two different outward currents (Ir and Is) in hMSC which variably contributed to total outward current. Differences in voltage dependence of the two currents allowed the classification of hMSC in cells with exclusive occurrence of Ir, Is or a mixture of both currents. We defined hMSC with exclusive prevalence of Ir current as cells where the ratio between current measured at +20 mV (I20) and current measured at +70 mV (I70) was below 0.25 (compare Fig. 1). A ratio of I20 and I70 of 0.25–0.5 defined cells with coexistence of both currents and I20/I70 above 0.5 defined cells with exclusive occurrence of Is. Significance of differences between means was tested using Student's paired or unpaired t test with a level of P < 0.05 taken to be statistically significant. ANOVA followed by Bonferroni's post hoc test was applied in most cases when several groups were compared.
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Human mesenchymal stem cells (hMSC) isolated by our group were characterized by means of flow cytometry. Cells from passages 0 and 1 were positive for CD29 (93 ± 2%), CD105 (92 ± 3%), and CD166 (95 ± 1%), and were negative for CD34 (0%), and CD45 (2 ± 1%) (n= 3–6 different donors). These levels remained constant during repeated subcultivation up to the last passage (5th) investigated by flow cytometry. The cultures demonstrated a characteristic growth pattern (Fig. 1A) of a homogeneous cell phenotype. Electrophysiological recordings were performed using ball-shaped cells (Fig. 1B) obtained after trypsin–EDTA treatment of the cultures shown in Fig. 1A.
Outward currents of hMSC
Almost all human mesenchymal stem cells (hMSC) investigated demonstrated outward currents (102 out of 118 cells). Distribution of current patterns and current amplitudes were independent of hMSC source (isolated by our group, n= 25 cells versus commercial supplier, n= 77 cells) and of passage number. Therefore, the pooling of the results for further analysis appeared to be justified.
The most frequently observed current was a rapidly activating outward current which we named Ir (Fig. 2, left). This current activated at potentials positive to +20 mV and only slightly inactivated at positive potentials during prolonged pulses of 1 s duration. Activation of Ir was associated with an increased noise of the current recordings (Fig. 2, left and middle). Tail currents were absent at 0 mV (Fig. 2, left) and also at more negative potentials (data not shown). Mean current density was 16.1 ± 1.8 pA pF-1 at +70 mV in 44 cells that exclusively demonstrated Ir. Forty-nine cells demonstrated an additional current (Fig. 2, middle) that activated at more negative potentials and with slower kinetics. This current is referred to as Is. Only nine cells exclusively demonstrated this Is current (Fig. 2, right). Is did not inactivate and saturated at +50 mV. Mean current density in these nine hMSC was 11.4 ± 1.8 pA pF-1 at +70 mV. Cell capacitance as a measure of cell size was similar in hMSC that exclusively demonstrated Ir(54.1 ± 2.9 pF, n= 44) or Is(54.8 ± 6.7 pF, n= 9) or the coexistence of both currents (55.2 ± 3.6 pF, n= 49). Membrane potential, however, was significantly more negative in cells demonstrating Is current as compared to cells lacking Is(–35.2 ± 1.6 mV, n= 29, versus–28.9 ± 2.1 mV, n= 17; P < 0.05).
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Characterization of the two currents depended on their appropriate separation. Investigation of Ir with pharmacological tools was performed on cells that exclusively demonstrated Ir and currents were analysed at a potential of +70 mV (compare Fig. 2, left). However, at +70 mV Is was contaminated by Ir in most cells (Fig. 2, middle). We therefore analysed Is at +20 mV, a potential where Is amplitude was indeed not at maximum, but free of Ir.
Ba2+ significantly blocked both currents, with 1 mM Ba2+ having a larger effect on Is than on Ir (Fig. 3A). 4-Aminopyridine, an unselective blocker of K+ currents (3 mM), reduced Ir to approximately 50% but only slightly inhibited Is. The selective blocker of cardiac delayed rectifier IKr, E-4031 (5 µM; Sanguinetti & Jurkiewicz, 1990) had no effect on Ir, whereas Is was reduced by 50%. The highly selective HERG channel blocker ergtoxin (100 nM; Gurrola et al. 1999) did not inhibit Is in three hMSC investigated (101 ± 4% of control; data not shown).
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Application of tetraethylammonium chloride markedly reduced the amplitudes of both currents (Fig. 3B). Is was blocked by tetraethylammonium with half-maximum inhibition at a concentration of 4.1 mM. Ir was found to be more sensitive to this blocker (IC50= 0.34 mM), and this, together with other characteristics, suggested that Ir was conducted by voltage- and Ca2+-activated K+ channels of large conductance (MaxiK).
To identify the channel molecule responsible for Ir we applied iberiotoxin, a selective blocker of MaxiK channels (Galvez et al. 1990; Fig. 4). Iberiotoxin (100 nM) inhibited current measured at +70 mV to 51 ± 1% of control amplitude in four cells and the extent of noise, typical for Ir, was clearly reduced. Is measured at +20 mV was largely unaffected by iberiotoxin. However, Is current at +20 mV was irreversibly blocked by clofilium (Castle, 1991) with little effect on Ir (Fig. 5). The IC50 value for the inhibition of Is by clofilium was 0.79 µM, as shown in Fig. 6.
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The conditions applied for recording outward currents were also suitable for detecting sodium inward currents, but sodium currents were absent in all cells investigated. In addition we tested for the presence of functional Ca2+ channels. At 2 mM external Ca2+ it was hard to identify clearly any inward current (Fig. 7A). However, after switching to 10 mM Ba2+, 10 out of 70 cells demonstrated inward currents that were stimulated by 1 µM BayK 8644 and completely blocked by 100 µM Cd2+ (Fig. 7B–D). The currents activated around –35 mV and peaked at –5 to 5 mV (Fig. 7E), consistent with Ba2+ currents conducted by L-type Ca2+ channels of other cell types. The capacitance of cells with Ba2+ currents (116 ± 17 pF) was significantly larger than the capacitance of cells where Ba2+ currents were below the limit of detection or absent (67.6 ± 3.8 pF; P < 0.05).
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mRNA expression of ion channel subunits in hMSC
Finally, we investigated the expression pattern for ion channel isoform mRNA in undifferentiated hMSC. Samples from human atrium and ventricle were taken as control tissues for comparison. hMSC isolated by our group and hMSC obtained from the commercial source exhibited a consistent pattern of ion channel mRNA. Figure 8 demonstrates mRNA expression for ion channel subunits associated with outward currents. There was clear mRNA amplification for Kv4.2 and Kv4.3, two ion channel subunits responsible for transitory outward currents, but transient outward currents were not observed in hMSC. However, the high levels of MaxiK mRNA were in line with the detection of an iberiotoxin-sensitive outward current (Ir) in most of the hMSC investigated. In addition we detected low levels of mRNA for Kv1.1, Kv1.4, Kv1.5, Kv2.1, Kv3.1, Kv7.2 and Kv7.3.
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1C subunit in all hMSC samples, whereas mRNA levels for other 1 subunits of the L-type Ca2+ channel (1D, 1S) or T-type Ca2+ channel (1G, 1H) were low or undetectable. As shown in the original gels of Figs 8–10, expression levels for several ion channels were in the same order of magnitude as in tissue samples from human heart. The expression patterns were similar in hMSC isolated by our group and in hMSC obtained from the commercial source.
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Discussion |
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Identification of outward currents
Most hMSC demonstrated outward currents that were inhibited by K+ channel blockers such as Ba2+ and 4-aminopyridine, but were not blocked by DIDS, a blocker of Cl- currents. This general finding suggests that both currents Ir and Is are carried by K+.
The most prevalent outward current of hMSC was Ir, a rapidly activating current which hardly demonstrated any inactivation. One clue towards the identification of its molecular nature is provided by the high sensitivity to tetraethylammonium. There are only a few K+ channels that are blocked with IC50 values around 0.3 mM, namely the Kv1.1 channel, channels of the Kv3 family, Kv7.2 channels and the voltage- and Ca2+-activated K+ channels of large conductance MaxiK (Rudy & McBain, 2001).
The electrophysiological properties and the pharmacological profiles of Kv1.1, Kv3, and Kv7.2 channels are incongruous with those of Ir current (Coetzee et al. 1999). We positively identified Ir as the large-conductance Ca2+-activated K+ current due to the following additional findings: (i) typical electrophysiological properties, (ii) noisy current traces, which we interpret to be due to large-conductance single channel openings, (iii) sensitivity to iberiotoxin, a specific blocker of MaxiK channels, and finally (iv) high expression levels of MaxiK mRNA in hMSC. MaxiK channels are expressed in many cells and tissues, including neurones, smooth muscle cells and secretory glands (Wallner et al. 1999). Moreover, Ca2+-activated K+ channels were found in osteoblasts (Westkamp et al. 2000), adipocytes (Pershadsingh et al. 1986), endothelial cells (Wiecha et al. 1998) and fibroblasts (Estacion, 1991), i.e. cell types that can be derived from undifferentiated hMSC.
Identification of Is based on a comparison of its electrophysiological and pharmacological properties with those of known channels is difficult. Slow kinetics of activation suggested some relation to cardiac delayed rectifier IKr, which is associated with HERG channels, and IKs, related to KCNQ1/minK. However, the IKr blocker E-4031 blocked Is to a lower extent than expected for the relatively high concentration of 5 µM. In addition, the peptide blocker of IKr, ergtoxin, had no effect, suggesting that Is does not flow through HERG channels. The blocker of cardiac IKs, HMR1556, only affected Is to a moderate extent. Based on slow activation kinetics and other electrophysiological parameters, Kv2.1, Kv7.2 and Kv7.3 were further candidate molecules for Is, but hanatoxin and linopirdine did not block this current. It was found that clofilium inhibited Is with relatively high potency and high selectivity over Ir, and hence can be used in functional experiments for selective suppression of Is.
Our failure to identify the molecular nature of Is could be due to the presence of ß subunits. The number of possible potassium channel constructs is extended by the presence of ß subunits that associate with several pore-forming potassium channel subunits. This association often results in a profound modification of electrophysiological or pharmacological behaviour of the respective current (Nerbonne, 2000). The involvement of one or more ß subunits in Is of hMSC is possible and renders the attribution to known potassium channel subunits even more difficult.
Ion channel mRNA, though expressed at high levels, is not necessarily translated into functionally active channel molecules. The discrepancy between the presence of mRNA but lack of the respective current is most striking for the channel subunits Kv4.2 and Kv4.3, associated with transient outward current Ito, but also for the hyperpolarization-activated and cyclic nucleotide-gated channel isoform 2 (HCN2), responsible for the If inward current. The reason for the lack of functional channel molecules remains unclear.
Functional role and heterogeneity of ion currents in hMSC
MaxiK channels are sensors of intracellular Ca2+. By this property they regulate membrane potential in a Ca2+-dependent manner (Kawano et al. 2003). The activity of MaxiK channels is modulated by phosphorylation via specific receptor-dependent signalling cascades, as recently shown for pathways involving the cellular proto-oncogene pp60 (c-Src; Alioua et al. 2002). The large-conductance Ca2+-activated K+ current could therefore be an effector of trophic factors within the body fluids or cell culture medium.
The functional role of the unidentified Is current is even more obscure. Is activates at much more negative potentials than the large-conductance Ca2+-activated K+ current. The presence of Is should therefore shift the membrane potential of hMSC to more negative potentials. In fact this was observed: hMSC with Is current had a significantly more negative membrane potential than hMSC without Is.
Regulation of intracellular calcium in hMSC depends on several mechanisms, and voltage-operated Ca2+ currents do not contribute much (Kawano et al. 2002; Kawano et al. 2003). Only 15% of hMSC demonstrated a small dihydropyridine-sensitive calcium current in the presence of a high external calcium concentration (Kawano et al. 2002). This dihydropyridine-sensitive Ca2+ current was probably due to the cardiac isoform of the L-type calcium channel, since we found high levels of mRNA for the pore-forming 1C subunit in hMSC. Our electrophysiological observations confirm the low frequency of hMSC with functional L-type Ca2+ channels. Ca2+ and Ba2+ currents were observed in cells that were significantly larger than cells without these currents. Given a fixed current density, the small current amplitudes would be more easily detectable in large cells. However, this does not completely explain the heterogeneity of Ca2+ and Ba2+ currents. Heterogeneity was also observed with outward currents, where currents of individual hMSC were not consistent, i.e. some of the cells exclusively showed the large-conductance Ca2+-activated K+ current, others expressed Is current or a mixture of both. Why do individual cells express different currents? There are several possible reasons. Despite the presence of consistent marker proteins on the cell surface and homogeneous morphology of the cultivated cells, the cells investigated possibly did not come from an absolutely homogeneous population of hMSC, but also included fractions of more or less committed progenitor cells (Minguell et al. 2001; Muraglia et al. 2000). These cell populations could slightly differ in their ion current patterns. Another possible reason is the dependence of ion current expression on cell function. For example, there is evidence that current expression varies at different phases of the cell cycle (Ouadid-Ahidouch et al. 2001; Chittajallu et al. 2002). Nevertheless, there is only a limited number of different currents observed in undifferentiated hMSC. The modification of present currents and the occurrence of new currents could be suitable markers of in vitro hMSC differentiation.
Consequences and perspectives
Preliminary clinical studies have shown that the injection of undifferentiated bone marrow stem cells into the infarcted heart can improve cardiac function. The fate of the implanted cells, whether they differentiate into cardiomyocytes, contribute to neoangiogenesis, or just perish, remains unclear. The injection transfers the undifferentiated cells, that can well include hMSC, directly into an electrically active environment. We have demonstrated that hMSC express a consistent pattern of ion channels and at least three different ion currents. Therefore, the cells have some bioelectrical activity, as also shown by others (Kawano et al. 2002, 2003). Based on our finding we cannot judge whether implantation of hMSC is safe or includes the risk of arrhythmia. However, careful monitoring of the patients will be necessary and will certainly be done, to rule out any pro-arrhythmogenic potential of the undifferentiated hMSC or of the hMSC-derived cardiomyocytes.
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