Bone marrow mesenchymal stem cells (MSCs) are used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not fully understood. The present study was to investigate the functional ionic channels in undifferentiated mouse bone marrow MSCs using whole cell patch-voltage clamp technique, RT-PCR, and Western immunoblotting analysis. We found that three types of ionic currents were present in mouse MSCs, including a Ca2+-activated K+ current (IKCa), an inwardly rectifying K+ current (IKir), and a chloride current (ICl). IKir was inhibited by Ba2+, and IKCa was activated by the Ca2+ ionophore A-23187 and inhibited by the intermediate-conductance IKCa channel blocker clotrimazole. ICl was activated by hyposmotic (0.8 T) conditions and inhibited by the chloride channel blockers DIDS and NPPB. The corresponding ion channel genes and proteins, KCa3.1 for IKCa, Kir2.1 for IKir, and Clcn3 for ICl, were confirmed by RT-PCR and Western immunoblotting analysis in mouse MSCs. These results demonstrate that three types of functional ion channel currents (i.e., IKir, IKCa, and ICl) are present in mouse bone marrow MSCs.
- inward rectifier potassium current
- intermediate-conductance calcium-activated potassium current
- volume-sensitive chloride current
bone marrow mesenchymal stem cells (MSCs) are pluripotent adult stem cells that can differentiate into osteoblasts, chondrocytes, adipocytes (25), and excitable cells such as neurons (36), skeletal muscle cells (7), and cardiomyocytes (29). MSCs can be isolated and expanded in vitro (27). In addition, it is believed that MSCs lack the B7 costimulatory molecules CD80 and CD86 and are nonimmunogenic upon allogeneic transplantation (12). These properties make MSCs an ideal cell source for regenerative medicine. It has been demonstrated that the implantation of MSCs to infarcted myocardium induces myocardial regeneration (10) or angiogenesis and improves cardiac function (31). Although the therapeutic effects are encouraging, the potential proarrhythmic effect of transplanted MSCs was observed in both in vitro and in vivo studies (3, 26). These reports pointed out an important concern that the implantation of MSCs might lead to cardiac electrical remodeling and initiate cardiac arrhythmia. These potential adverse effects could hinder the application of MSCs in clinical practice in cardiac diseases. Therefore, it is essential to understand the electrophysiological properties of MSCs before differentiation induction. Recent studies described that multiple ion channel currents were present in human, rabbit, and rat MSCs (5, 9, 15, 16). Differences in both current types and encoding genes have been observed between these species. The present study was designed to investigate the electrophysiological properties of mouse MSCs from bone marrow. We found three functional ionic currents in mouse MSCs different from those observed in human and rat MSCs.
MATERIALS AND METHODS
Mouse bone marrow MSCs (passage 5) from C57Bl/6 mice are kindly provided by Dr. Darwin J. Prockop (Center for Gene Therapy, Tulane University, New Orleans, LA; http://www.som.tulane.edu/gene_therapy), are positive for Sca-1 and negative for CD34, CD45, and CD11B-C surface markers, and can be differentiated into adipocytes and osteocytes. The cells were cultured in Iscove's modified Dulbecco's medium (Sigma-Aldrich, St. Louis, MO) with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% fetal bovine serum (Invitrogen), and 10% horse serum (Invitrogen) and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. When cells grew to ∼70–80% confluence, they were detached for subculture or electrophysiological study by using 0.125% trypsin and 1 mM EDTA in phosphate-buffered saline solution. Cells used in electrophysiological and molecular biological studies were from the early passages 6–8 to limit the possible variations in functional ion channel currents, genes, and proteins induced by cell senescence at later passages, because it has been reported that cell senescence occurs with many passages of in vitro MSC culture (2).
Membrane ionic currents were recorded with the whole cell patch-clamp technique as described previously (16). Borosilicate glass electrodes (1.2-mm outer diameter) were pulled with a Brown-Flaming puller (model P-97; Sutter Instrument, Novato, CA) and had tip resistances of ∼2–3 MΩ when filled with pipette solution. The tip potentials were compensated before the pipette touched the cell. After a gigaohm seal was obtained by negative suction, the cell membrane was ruptured by gentle suction to establish whole cell configuration. Data were acquired with an EPC10 amplifier (Heka, Lambrecht, Germany). Membrane currents were low-pass filtered at 5 kHz and stored on the hard disk of an IBM-compatible computer. Tyrode solution contained (mM) 136 NaCl, 5.4 KCl, 1.0 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES; pH was adjusted to 7.3 with NaOH. The pipette solution contained (mM) 20 KCl, 110 K-aspartate, 1.0 MgCl2, 10 HEPES, 0.05 EGTA, 0.1 GTP, 5.0 Na2-phosphocreatine, and 5.0 Mg2-ATP; pH was adjusted to 7.2 with KOH. The experiments were conducted at room temperature (21–22°C).
Drugs and reagents.
The small-conductance Ca2+-activated K+ channel (SKCa) blocker UCL 1684 was purchased from Tocris (Bristol, UK). Rabbit polyclonal anti-Kir2.1, anti-IKCa, goat polyclonal anti-Clcn3, and goat anti-rabbit and donkey anti-goat IgG-horseradish peroxidase antibodies were products of Santa Cruz Biotechnology (Santa Cruz, CA). Other reagents were obtained from Sigma-Aldrich.
Reverse transcription-polymerase chain reaction.
Total RNA of mouse MSCs from passages 6–8 was extracted using Trizol reagent (Invitrogen) following its enclosed protocol. RNA was treated with DNase I (GE Healthcare) to remove genomic DNA. Reverse transcription (RT) was performed with the RT system (Promega, Madison, WI) protocol in a 20-μl reaction mixture using oligo(dT)15 primers. After the RT procedure, the reaction mixture (cDNA) was used for polymerase chain reaction (PCR).
PCR primers were designed with Primer Premier 5 software (Premier Biosoft International, Palo Alto, CA) and synthesized at the Genome Research Center at the University of Hong Kong. PCR was performed with the Promega PCR Core System I. The cDNA in 2-μl aliquots was amplified using a DNA thermal cycler (Mycycler; Bio-Rad Laboratories, Hercules, CA) in a 25-μ1 reaction volume containing recommended concentrations of PCR components. The amplification was performed under the following conditions: the mixture was initially denatured at 95°C for 2 min, followed by ∼28–30 cycles amplification (denaturation, 95°C for 45 s; annealing, 58°C for 45 s; extension, 72°C for 1 min). This was followed by a final extension at 72°C (5 min) to ensure complete product extension. The PCR products were resolved through 1.5% agarose gel electrophoresis, and the amplified cDNA bands were visualized using ethidium bromide staining and imaged using a Chemi-Genius Bio Imaging System (Syngene, Cambridge, UK).
Cells at ∼70–80% confluence were lysed with a modified RIPA buffer containing (mM) 50 Tris·HCl, 150 NaCl, 1 EDTA, 1 PMSF, 1 sodium orthovanadate, 1 NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 0.1% SDS. Protein concentration was determined using a Bio-Rad protein assay. Cell lysates (50 μg) were mixed with sample buffer and denatured by heating to 95°C for 5 min. Samples were resolved via SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween (TTBS) and then probed with primary antibodies at 4°C overnight with agitation. After being washed with TTBS, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or donkey anti-goat IgG antibody at 1:5,000 dilution in TTBS at room temperature for 1 h. Membranes were washed again with TTBS and then processed to develop X-ray film using an enhanced chemiluminescence detection system (GE Healthcare).
Results are means ± SE. Paired and/or unpaired Student's t-tests were used as appropriate to evaluate the statistical significance of differences between two group means, and analysis of variance was used for multiple groups. Values of P < 0.05 were considered to indicate statistical significance.
Families of membrane ion currents.
Figure 1 illustrates families of membrane currents recorded in undifferentiated mouse MSCs using a standard pipette solution. Three types of membrane currents were observed in mouse MSCs (in a total of 87 cells). One current was activated by depolarization voltages to between −70 and +60 from −80 mV, showing a rapid activation at potentials −70 and +10 mV and a weak inward rectification at +20 to +60 mV (Fig. 1A). These features suggest this current is likely an intermediate-conductance Ca2+-activated K+ channel current (IKCa). The current was observed in 57% (50 of 87) of cells. Another current was activated by voltage steps to between −120 and 0 mV from −40 mV, showing a property typical of inwardly rectifying K+ current (IKir) (Fig. 1B). IKir was observed in 16% (14 of 87) of cells. A third current was elicited by voltage steps to between −100 and +60 from −40 mV, showing a small inward current and a large outward current with outward rectification (Fig. 1C). This type of current was were observed in 34% (30 of 87) of mouse MSCs. IKir and the third type of current were copresent in a small population (8%, 7 of 87) of mouse MSCs.
Ca2+-activated K+ currents.
Figure 2A shows that the SKCa blocker UCL 1684 (1 μM) and the big-conductance IKCa (BKCa) blocker paxilline (1 μM) did not inhibit the outward current, whereas the intermediate-conductance IKCa blocker clotrimazole (1 μM) dramatically inhibited the current amplitude. Similar results were obtained in a total of six cells. These results suggest that only intermediate-conductance IKCa, not small- or large-conductance IKCa, is present in mouse MSCs. In another set of experiments, the membrane current was recorded using a pipette solution containing 800 nM free Ca2+. We found that significant IKCa was detected in 95% (20 of 21) of cells. The percentage of cells with IKCa recorded using the high concentration of free Ca2+ in pipette solution was much higher than that with a standard low-EGTA pipette solution (95% vs. 57%, P < 0.05).
A previous study by our group (15) showed that the Ca2+ ionophore ionomycin increased IKCa in rat MSCs, and the effect was inhibited by clotrimazole. In mouse MSCs with small membrane current, a similar effect was observed. The Ca2+ ionophore A-23187 (1 μM) remarkably increased membrane conductance, and the effect was significantly inhibited by the application of 1 μM clotrimazole (Fig. 2B). Figure 2C shows the current-voltage (I-V) relationships of membrane current recorded using a ramp protocol in a typical experiment. The current evoked by 1 μM A-23187 showed a weak inward rectification and was significantly inhibited by 1 μM clotrimazole. These results demonstrate that intermediate conductance IKCa is present in mouse MSCs.
It is generally believed that inwardly rectifying K+ channels are sensitive to inhibition by Ba2+ (14). We therefore determined the effect of Ba2+ on IKir in mouse MSCs. Figure 3A shows original traces recorded in a representative cell using the voltage protocol shown in the inset in the absence (control) and presence of Ba2+. Ba2+ at 0.5 mM substantially reduced IKir. Ba2+-sensitive current was obtained by digitally subtracting currents before and after application of Ba2+ (Fig. 3A, right). Figure 3B displays the I-V relationships of IKir in the absence and presence of 0.5 mM Ba2+ and Ba2+-sensitive current. Ba2+-sensitive current exhibits an I-V relationship typical of an inwardly rectifying K+ current.
Cl− current in mouse MSCs.
The current with outward rectification shown in Fig. 1C was insensitive to inhibition of K+ channel blockers, including 5 mM tetraethylammonium (TEA), 5 mM 4-aminopyridine (4-AP), and 0.5 mM Ba2+ (n = 5 each), suggesting that the outward rectifying current is not carried by K+ ion. We then employed Cl− channel inhibitors to determine whether the current would be carried by Cl− ions. We found that the Cl− channel blockers 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) inhibited this current. Figure 4A illustrates current traces recorded in a representative cell with the protocol shown in the inset. DIDS at 150 μM substantially suppressed the current, and similar results were obtained in a total of six cells with DIDS and nine cells with 50 μM NPPB, suggesting that the current activation is based on Cl− channels.
To study whether the Cl− channels are volume sensitive in mouse MSCs, we employed a 0.8 T tonic solution and recorded the membrane current using a K+-free pipette solution as described previously (6). Figure 4B illustrates the time course of swelling-induced changes in membrane current at +60 mV in a mouse MSC when bath solution osmolarity (T) was switched from isosmotic (1T) to hyposmotic (0.8T) solution and then to 0.8T with low external Cl− (10 mM). Membrane current in 0.8T gradually increased to a relatively steady-state level and remarkably decreased after exposure to low external Cl−. Similar results were obtained in 87.5% (7 of 8) of cells. The swelling-induced current is volume-sensitive ICl, as described previously in human atrial myocytes (6). Figure 4C displays the currents elicited by voltage steps to between −100 and +70 mV from −40 mV in 1T, 0.8T, and 0.8T with 50 μM NPPB. Similar results were observed in 85.7% (12 of 14) of cells. Figure 4D shows the I-V relationships of the swelling-induced current before and after exposure to NPPB (n = 5). The swelling-induced current outwardly rectified and reversed at −27 mV (−37 mV after correction for the liquid junction potential), near the predicted Cl− equilibrium potential, −35 mV. NPPB remarkably inhibited both inward and the outward currents. These results suggest that volume-sensitive ICl is present in mouse MSCs.
Messenger RNAs of functional ion channels and Western blotting analysis.
To explore the molecular identities of the functional ionic currents, we examined gene expression of various ionic channels in mouse MSCs with RT-PCR, using the specific primers for KCa, Kir, Clcn, and Clca ion channel families as shown in Table 1. The primers of Kv channels were employed as described previously (34). Figure 5A displays the images of RT-PCR products corresponding to gene expression of KCa3.1 (IKCa), Kir2.1 (IKir), and Clcn3 (ICl) in mouse MSCs. Weak expression of Clcn2 and no expression of Kv families were found in mouse MSCs.
Western immunoblot analysis confirmed the protein expressions of KCa3.1, Kir2.1, and Clcn3 as unveiled by gene expression. Figure 5B shows images of KCa3.1, Kir2.1, and ClCn3 proteins (n = 3).
In the present study, we demonstrated that three types of ionic currents (IKCa, IKir, and ICl) were present in undifferentiated mouse bone marrow MSCs. IKCa was inhibited by the intermediate-conductance inhibitor clotrimazole, IKir was blocked by Ba2+, and ICl was inhibited by DIDS or NPPB. The currents' corresponding channels (KCa3.1 for IKCa, Kir2.1 for IKir, and Clcn3 for ICl) were confirmed by RT-PCR for gene expression and Western immunoblotting in protein levels.
IKCa, IKir, and ICl were identified in 57, 16, and 34% of mouse MSCs, respectively, with the use of a standard pipette solution (Fig. 1). However, a high percentage (95%) of cells with IKCa was identified when free Ca2+ was increased in the pipette solution, suggesting that intermediate IKCa is the dominant current in mouse MSCs in maintaining membrane potential and desirable intracellular ion concentrations. On the other hand, the percentage of cells with ICl was increased to 88% when 0.8T external solution was employed, indicating that activation of ICl is dependent on cell volume and/or size of the cells related to cell cycling (18).
The electrophysiological properties of MSCs were initially studied by Kawano et al. (11). They demonstrated that the dominant BKCa current was present in most human MSCs, and L-type Ca2+ current was present in a small population of cells. The activity of BKCa (i.e., KCa1.1) channel was regulated by the spontaneous Ca2+ oscillation, resulting in fluctuations of membrane currents and potentials. Our group (16) and others (9) provided additional information that multiple ion channels were expressed in human MSCs, including nifedipine-sensitive L-type Ca2+ current (ICa.L), transient outward K+ current (Ito), tetrodotoxin-sensitive Na+ current (INa.TTX), and a delayed rectifier K+ current (IKdr) (9, 16). The present and previous studies by our group demonstrated the heterogeneity of electrophysiological properties of MSCs present in different species. We found that human and rat MSCs expressed functional ICa.L, Ito, and INa.TTX, albeit in a small portion of cells. These currents were not present in MSCs from rabbit and mouse. IKir current was observed in rabbit and mouse MSCs but not in human and rat MSCs. In addition, ICl was observed in mouse MSCs (Fig. 4) but not in rat, rabbit, and human MSCs (5, 14, 15). Although functional ICl was detected by its corresponding Clcn3 gene expression in mouse MSCs, Clcn3 in other species needs to be further clarified.
Heterogeneous expression of ion channels also was observed within the same species. For instance, the present study demonstrated that IKCa, IKir, and ICl were heterogeneously expressed in mouse MSCs. This could result from a heterogeneous cell population of the MSCs (30, 32). Consequentially, a subpopulation of MSCs might display a different pattern of ion channel expression. This heterogeneity also may be explained by the fact that cultured MSCs are not synchronously at the same stage of the cell cycle. It should be noted that cells at different stages of the cell cycle might express different patterns of ion channels (20, 21, 37).
Ion channels play a role in cell cycling of proliferative cells (18, 22, 35). Kv1.3 channels and intermediate KCa channels are well known for T lymphocyte activation and its consequent proliferation upon antigen stimulation (13). Human ether-a-go-go K+ channels have been reported to have an oncogenic effect when transplanted in immunodeficient mice (23), and the channel protein could interact with p38 MAPK and evoke p38 MAPK signaling, resulting in the promotion of cell proliferation (8). Moreover, IKir has been found to participate in the proliferation of astrocytes (17) and hematopoietic progenitor cells (28). Although the underlying mechanisms for K+ channels in regulation of cell proliferation remain elusive, the involvement of K+ channels in cell proliferation is well established. In the present study we demonstrated the presence of intermediate IKCa and IKir in mouse MSCs. Future studies are required to find out whether these two types of K+ channels contribute to MSC proliferation.
Clcn3 channel is regarded as one of the candidate channels for volume-regulated anion channels and has been shown to play an important role in cell proliferation and apoptosis (18, 19). Blockade or disruption of Clcn3 channel resulted in arrest of cell cycle and prevention of cell proliferation in several cell types (4, 33, 37). Functional Cl− current encoded by Clcn3, sensitive to cell volume, was observed in mouse MSCs (Fig. 4). Whether this ICl contributes to mouse MSCs proliferation remains to be studied in the future.
Significant beneficial effects were observed in regenerating myocardium and improving cardiac function by implanting bone marrow MSCs into the damaged myocardium (1, 10, 31). However, potential proarrhythmia with the implantation of MSCs has been reported in clinical patients with cell implantation (24), in swine with cardiac infarction treated with MSC implantation (26), and in human MSCs cocultured with neonatal rat ventricular myocytes (3). An understanding of ion channel expression in undifferentiated MSCs in different species will help and/or facilitate possible biological solutions for these concerns and medical challenges.
In summary, the present study demonstrates that three types of functional ion channels are present in mouse bone marrow MSCs, including intermediate-conductance IKCa, IKir, and volume-sensitive ICl. RT-PCR and Western immunoblotting confirm gene expression and proteins of the corresponding functional ion channels. The information obtained from the present study provides a basis for future investigation of how these functional ion channels regulate biological and physiological activity of MSCs.
This study was supported by the Research Grant Council of Hong Kong (HKU 7347/03M). R. Tao was supported by a postgraduate studentship of the University of Hong Kong. We thank Dr. Darwin J. Prockop at the Center for Gene Therapy (supported by National Center for Research Resources Grant P40 RR017447), Tulane University, for providing the mouse MSCs.
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