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Am J Physiol Cell Physiol 293: C1539-C1550, 2007. First published August 8, 2007; doi:10.1152/ajpcell.00089.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Electrophysiological properties of human adipose tissue-derived stem cells

Xiaowen Bai,1,* Junyi Ma,2,* Zhizhong Pan,2 Yao-Hua Song,1 Susanne Freyberg,1 Yasheng Yan,1 Daynene Vykoukal,1 and Eckhard Alt1

1Department of Molecular Pathology, University of Texas, MD Anderson Cancer Center, Houston, Texas; and 2Department of Anesthesiology, University of Texas, MD Anderson Cancer Center, Houston, Texas

Submitted 5 March 2007 ; accepted in final form 3 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human adipose tissue-derived stem cells (hASCs) represent a potentially valuable cell source for clinical therapeutic applications. The present study was designed to investigate properties of ionic channel currents present in undifferentiated hASCs and their impact on hASCs proliferation. The functional ion channels in hASCs were analyzed by whole-cell patch-clamp recording and their mRNA expression levels detected by RT-PCR. Four types of ion channels were found to be present in hASCs: most of the hASCs (73%) showed a delayed rectifier-like K+ current (IKDR); Ca2+-activated K+ current (IKCa) was detected in examined cells; a transient outward K+ current (Ito) was recorded in 19% of the cells; a small percentage of cells (8%) displayed a TTX-sensitive transient inward sodium current (INa.TTX). RT-PCR results confirmed the presence of ion channels at the mRNA level: Kv1.1, Kv2.1, Kv1.5, Kv7.3, Kv11.1, and hEAG1, possibly encoding IKDR; MaxiK, KCNN3, and KCNN4 for IKCa; Kv1.4, Kv4.1, Kv4.2, and Kv4.3 for Ito and hNE-Na for INa.TTX. The IKDR was inhibited by tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which significantly reduced the proliferation of hASCs in a dose-dependent manner (P < 0.05), as suggested by bromodeoxyurindine (BrdU) incorporation. Other selective potassium channel blockers, including linopiridine, iberiotoxin, clotrimazole, and apamin also significantly inhibited IKDR. TTX completely abolished INa.TTX. This study demonstrates for the first time that multiple functional ion channel currents such as IKDR, IKCa, Ito, and INa.TTX are present in undifferentiated hASCs and their potential physiological function in these cells as a basic understanding for future in vitro experiments and in vivo clinical investigations.

proliferation; differentiation; delayed rectifier K+ current; voltage-dependent Ca2+ current; Ca2+-activated K+ current; tetrodotoxin-sensitive Na+ current; transient outward K+ current

ADULT STEM CELLS HAVE BEEN identified in different organs such as bone marrow, adipose tissue, skin, skeletal muscle, and liver (8, 11, 40, 43, 49). Until recently, bone marrow has been the major tissue source of adult stem cells for both experimental and clinical studies (5, 40). However, problems associated with bone marrow-derived stem cells (BMSCs) are relatively low cell yield, often requiring culturing for expansion, and extraction-related discomfort (50). Recent studies support the existence of stem cells in adipose tissue. Adipose tissue-derived stem cells (ASCs) are very similar to BMSCs. Several reports showed that ASCs can be relatively easily recovered, expanded, and have the potential to differentiate into various cell types, including chondrocytes, adipocytes, osteoblasts, hepatocytes, epithelial cells, smooth muscle cells, and endothelial cells, when cultured in defined medium in vitro (7, 34, 43, 47, 50). ASCs can also differentiate into excitable cells, such as cardiomyocytes and neurons (25, 41). Direct injection of both cultured and uncultured ASCs into infarcted hearts has shown beneficial effects (1). ASCs transplanted into the ischemic myocardium differentiated into vascular endothelial cells. Left ventricular function was improved in ASCs when compared with a control group at 4 wk (29). Transplantation of human ASCs (hASCs) into the lateral ventricle of the rat improved functional deficits in ischemic brain injured by middle cerebral artery occlusion. Injected hASCs migrated to the injured cortex, and some of the cells differentiated into neurons (24). In addition, ASCs are easy to obtain in large quantities. Therefore, ASCs are a potentially important cell source for autologous cell-based clinical therapy.

The surface antigen profiles and differentiation potential of ASCs have been well documented (50). However, currently, information about the electrophysiological properties of ion channels in undifferentiated ASCs is missing. Ion channels are widely expressed in different types of cells. They play fundamental roles in diverse key functions such as maintenance of physiological homeostasis, cell proliferation, and signal transduction in a variety of cell types and different cell stages (13, 36). Experimental evidence in cell biology and pharmacology demonstrate that cancer cells and other proliferating cells exhibit ion channel expression, ion conductance, and electrical properties very different from that of resting cells (14). The present study was therefore designed to investigate ion channels present in undifferentiated hASCs and their potential physiological function in these cells, as a basic understanding for future in vitro experiments and in vivo clinical investigations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and culture of hASCs. Subcutaneous adipose tissue was obtained from three patients undergoing elective operations (not liposuctions) under an IRB-approved protocol. Cells were isolated from the fat tissue, as described previously (20) with modifications. Fat tissue was minced and incubated for 90 min at 37°C on a shaker with Liberase Blendzyme 3 (Roche) at a concentration of 4 units per gram of fat tissue in PBS. The digested tissue was sequentially filtered through 100-µm and 40-µm filters (Fisher Scientific) and centrifuged at 450 g for 10 min. The supernatant containing adipocytes and debris was discarded, and the pelleted cells were washed twice with Hanks’ balanced salt solution (Cellgro) and finally resuspended in growth media. Growth media contained alpha-modification of Eagle's medium (Cellgro), 20% FBS (Atlanta Biologicals), 2 mM glutamine (Cellgro), 100 U/ml penicillin with 100 µg/ml streptomycin (Cellgro). Plastic adherent cells were designated hASCs and grown in Nunclon culture vials (Nunc) at 37°C in a humidified atmosphere containing 5% CO2 followed by daily washes to remove red blood cells and nonattached cells. After confluence of hASCs (passage 0), cells were seeded at a density of 3,000 cells/cm2 (passage 1).

Flow cytometric analysis of phenotype in hASCs. hASCs of the third passage from three samples were harvested by treatment with 0.05% trypsin-0.53 mM EDTA and then fixed for 10 min in 1% paraformaldehyde. Following fixation, cells were washed twice with PBS. Cell aliquots (1x106 cells/1 ml) were stained with primary antibodies at room temperature for 30 min. The primary antibodies were FITC-conjugated anti-human CD44 (Chemicon, cat. no. CBL154F), CD34, CD90, HLA-DR (U.S. Biological, cat. no. C3111362/C2441-63/CH6100-01), or phycoerythrin-conjugated anti-human CD11b, CD105 (eBioscience, cat. no. 12-0118-73/12-1057), CD14, CD45 (U.S. Biological, cat. no. C5120250/C2399-20N). Isotype-matched normal mouse IgGs were used as controls. The antibodies were purchased from Chemicon (cat. no. CBL601P, CBL601F, CBL600F, and CBL600P). Flow cytometry was performed on a fluorescence-activated cell sorter (FACSCalibur, BD Biosciences, San Jose, CA), and data analysis was performed with Cell Quest software (Becton-Dickinson).

Immunofluorescence staining. hASCs of passage 3 cultured on glass coverslips were washed three times with PBS and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were washed three times with PBS containing 0.3% Triton X-100 (Sigma) and blocked with 10% donkey serum for 30 min at room temperature. Cells were incubated with mouse anti Ki67 (Zymed) for 1 h at 37°C. After three washes, cells were incubated with an Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (Invitrogen) at a dilution of 1:1,000 for 1 h at room temperature. Hoechst 33342 dye was used to stain nuclei. The cells were examined under a fluorescence microscope.

Cell differentiation. The adipogenic differentiation potential for hASCs was analyzed as described previously (40). The cells of passage 3 were harvested using trypsin/EDTA and plated in 24-well plates at 30,000 cells/cm2 for 16 h to allow attachment. At 100% confluence, cells were then switched to adipogenic medium containing low glucose DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Cellgro), 100 µM L-ascorbate acid (Sigma), 1 µM dexamethasone, 0.5 mM IBMX, 100 µM indomethacin, and 10 µg/ml human recombinant insulin (Sigma) for 21 days. Cells of the control group were cultured in low-glucose DMEM plus 10% FBS (control medium). The medium was changed every 3 days. Adipogenesis of ASCs was assessed by incubating cells with Oil Red O solution (Sigma) to stain neutral lipids in the cytoplasm. Osteogenic differentiation (37) was performed by incubating the cells of 100% confluence on coverslips with high-glucose DMEM plus 10% FBS supplemented with 0.1 µM dexamethasone, 200 µM L-ascorbic acid, and 10 mM β-glycerol phosphate (Sigma). Media were changed every 3 days for 3 wk. Cells of the control group were cultured in high-glucose DMEM plus 10% FBS for 3 wk. To assess mineralization, calcium deposits in cultures were stained with Alizarin Red S (Sigma). The cells were then observed under a phase contrast microscope.

Analysis of mRNA expression of ion channels in hASCs using RT-PCR technique. Total RNA was extracted from hASCs of passage 3 using TriZol reagent (Life Technologies). cDNA was synthesized from 1 µg total RNA using SuperScript II reverse transcriptase (Life Technologies ) in 20 µl, according to the manufacturer's instructions. cDNA samples were subjected to PCR amplification using AccuPrime Super Mix I (Invitrogen) with specific primers for human ion channels. The primer sequence and PCR conditions for each ion channel subunit and the housekeeping gene GAPDH are listed in Table 1. Control reactions consisted of the above-mentioned PCR amplification mix with primers but no cDNA template. PCR was performed with an Eppendorf mastercycler gradient (Eppendorf). Cycles were programmed as follows: 94°C for 10 min, 35 cycles of 30 s denaturation at 94°C, 30 s at annealing temperature indicated in Table 1, 45-s extension at 72°C, with a final extension at 72°C for 5 min. Expression of GAPDH mRNA was used as an internal control. The product size was confirmed by running 10 µl of sample on 1.5% agarose gel electrophoresis. All PCR products were sequenced to verify identity (data not shown). Water and commercially available human heart and brain total RNA (Ambion) were used as negative and positive controls, respectively.


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  		Table 1. Primer pairs and conditions used for PCR

 
Western blot analysis. Passage 3 hASCs were lysed in RIPA lysis buffer (Upstate), including protease inhibitor cocktail (Roche Diagnostics) according to the manufacturer's instructions. The proteins were separated on SDS/PAGE gels and then transferred to polyvinylidene difluoride membranes. After being blocked with 5% nonfat milk, the membranes were incubated for 1 h at room temperature with rabbit anti-HCN2, Kir2.2, and {alpha}1C subunit of L-type calcium channel (CaV1.2) (Alomone Laboratories), respectively. Following incubation with goat anti-rabbit horseradish peroxidase secondary antibody (Pierce), labeled proteins were detected using the SuperSignal chemiluminescence detection system (Pierce).

Whole cell patch-clamp recordings. hASCs (passage 3) from three samples were detached from culture flasks using trypsin-EDTA. The cells were transferred to glass coverslips in 24-well plates and cultured for at least 2 h in culture medium. For recordings, a coverslip with hASCs was transferred to a chamber that was perfused with modified Tyrode solution at a rate of 1.5 ml per minute. The perfusing solution was equilibrated to 5% CO2 and 95% O2 and maintained at room temperature (21–22°C). The contents of the perfusing solution were (in mM): 136 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 glucose, 10 HEPES, with a pH adjusted to 7.3 with NaOH and osmolarity of 320–330 mosmol/l. Visualized whole cell voltage-clamp recordings were obtained with a glass pipette (resistance 2-3 M{Omega}) filled with a solution containing (in mM): 140 potassium gluconate, 10 NaCl, 1 MgCl2, 2 EGTA, 10 HEPES, 2 ATP, 0.25 GTP, with pH adjusted to 7.3 with KOH and osmolarity of 300-310 mosmol/l. An AxoPatch-1D amplifier and AxoGraph software (Axon Instruments) were used for data acquisition and for online and off-line data analyses. A seal resistance of 2 G{Omega} or above and an access resistance of 10 M{Omega} or less were considered acceptable. Series resistance was optimally compensated by 80%, and it was decreased from 80% to 60% when current oscillation was detected. The access resistance was monitored throughout the experiment. The cells were held at –80 mV. Voltage steps from –60 mV to +80 mV at a 20-mV interval for 300 ms were used to induce voltage-dependent currents. Data were digitized at 20 kHz and filtered at 2 kHz. The experiments were conducted at room temperature (21°C–22°C). The following ion channel blockers were used: linopirdine, clofilium tosylate, tetraethyl ammonium (TEA), iberiotoxin, 4-aminopyridine (4-AP), apamin, E4031, clotrimazole, and TTX. All drugs were purchased from Sigma.

Cell proliferation. DNA incorporation as an indicator of cell proliferation was studied using a colorimetric bromodeoxyurindine (BrdU) kit (Roche Diagnostics) according to the manufacturer's instructions. 2.5x103 hASCs at passage 3 were grown in 96-well plates for 12 h in 20% FBS and then starved in 2% FBS for 24 h. Cells were then switched to 20% FBS supplemented with a series of concentrations of linopirdine, clofilium tosylate, TEA, iberiotoxin, 4-AP, apamin, E4031, clotrimazole, and TTX for 24 h. BrdU solution was then added into the medium to a final concentration of 10 µM of BrdU. Medium was discarded after 2 h, and cells were fixed with FixDenat solution for 30 min at room temperature. After FixDenat was removed, cells were incubated with 100 µl freshly diluted 1:100 anti-BrdU peroxidase solution for 30 min. The cells were washed three times; 100 µl of substrate solution was added to incubate for 30 min at room temperature, 25 µl 1 M H2SO4 was added to terminate the reaction, and then absorbance at 450 nm was measured in an ELISA plate reader. Cytotoxic effects of different ion channel blockers on hASCs were analyzed by checking cell viability by staining dead cells with propidium iodide (PI). The method was described as follows: 1.0x105 hASCs at passage 3 were grown in 12-well plates for 12 h in 20% FBS culture medium and then starved in 2% FBS-containing medium for 24 h. Cells were then switched to 20% FBS medium supplemented with a series of concentrations of linopirdine, clofilium tosylate, TEA, iberiotoxin, 4-AP, apamin, E4031, clotrimazole, and TTX for 24 h. Then cells were digested with trypsin-EDTA and resuspended with PBS. PI solution was then added to the cell suspension to a final concentration of 2 µg/ml for flow cytometric analysis. PI-positive cells represent dead cells (17).

Data analysis. Data reported were expressed as means ± SD. Statistical significance of differences between groups was tested using Student's t-test or one-way ANOVA. A level of P ≤ 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
hASCs expressed stem cell surface markers and showed proliferative and differentiation potential. ASCs isolated from human adipose tissue exhibited fibroblast or spindle-like morphology (Fig. 1A) and proliferative potential. The self-renewal capacity of ASCs was verified by immunofluorescence staining of Ki67, a marker of proliferative cells (16) (Fig. 1B). The surface markers on ASCs at passage three were analyzed by flow cytometry from three samples (Fig. 2A). The cells are positive for CD44 (95.44 ± 2.66%), CD90 (95.87 ± 3.51%), CD105 (98.54 ± 1.89%), and negative for CD11b (0.58 ± 0.69%), CD14 (0.08 ± 0.02%), CD34 (0.08 ± 0.07%), and CD45 (0.11 ± 0.10%), which preclude contamination by hematopoietic cells. HLA-DR, a class II antigen of HLA, was also negative (1.31 ± 0.70%) in ASCs, suggesting that ASCs are not immunogenic and might eventually be considered for heterologous transplantation. The expression pattern of surface proteins on our preparations of hASCs was in agreement with that of hASCs and hMSCs reported by others (22, 27).


Figure 1
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  		Fig. 1. Phase contrast photomicrographs of human adipose tissue-derived stem cells (hASCs) at passage three. A: cells show spindle or triangle-like morphology. B: Immunofluorescence analysis of Ki67 expression in hASCs (passage 3). Blue color indicates nuclei (B1); green color indicates signal of Ki67 (B2). Overlay of nuclei and Ki67 signals is shown in B3.

 

Figure 2
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  		Fig. 2. A: flow cytometric analysis of surface marker expression in hASCs. Black traces indicate isotype controls; red traces show surface antigen expression level. B: differentiation potential of hASCs. Cells were cultured in adipogenic (b) or osteogenic medium (d) or adipogenic control medium (a) or osteogenic control medium (c) for 3 wk. Adipogenesis and osteogenesis of hASCs were confirmed by Oil Red O staining (a, b) or Alizarin Red S staining (c, d), respectively. Scale bar = 50 µm.

 
To evaluate the multipotent capacity of hASCs, cells were cultured in adipogenic and osteogenic medium for 3 wk. hASCs in adipogenic induction medium displayed characteristic multiple intracellular bright white oil droplets. These oil droplets showed red vesicles when stained with Oil Red O stain (Fig. 2B, b). No lipid droplets were observed in cells cultured in control medium (Fig. 2B, a). Cells cultured in osteogenic medium showed black regions within the monolayer, which indicated calcification deposits from differentiated osteoblasts. Calcification of extracellular matrix assessed by Alizarin Red S stain (Fig. 2B, d) showed red staining of mineralized deposits, which was not observed in cells cultured in control medium (Fig. 2B, c).

Families of ion channel currents in hASCs. Whole cell patch-clamp recordings were made from a total of 144 hASCs of passage 3 (Fig. 3) from three patients. The membrane currents were elicited by 300-ms voltage steps between –60 and +80 mV from a holding potential of –80 mV. All cells recorded in this study demonstrated outward currents. Most hASCs (106, 73%) showed a slowly activating, delayed rectifier-like K+ current, or IKDR (Fig. 4A). In 27 cells, a transient outward K+ current Ito was detected (Fig. 4B). TTX-sensitive transient inward sodium current (INa TTX) was observed in a small population of cells (11 of 144). The inward current generally coexisted with the outward currents (Fig. 4C). We did not find any significant difference of those currents in three patients.


Figure 3
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  		Fig. 3. Original gel showing mRNA expression of ion channel subunits from hASCs by RT-PCR. On the left and right side of each image are DNA size markers (100-bp ladder). Ion channel subtypes are noted on the top of bands. hASCs are positive for {alpha}1C, {alpha}1H, HCN2, Kir2.1, Kir2.2, Kir3.4, Twik1, Kv1.1, Kv1.4, Kv1.5, Kv2.1, Kv4.1, Kv4.2, Kv4.3, Kv7.3, Kv11.1, hEAG1, MaxiK, KCNN3, KCNN4, hNE-Na. Water and commercially available human heart and brain total RNA were used as negative and positive controls, respectively.

 

Figure 4
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  		Fig. 4. Ion channel currents detected in hASCs by whole cell patch-clamp recordings. Membrane currents were elicited by 300-ms voltage steps between –60 and +80 mV from a holding potential of –80 mV. All examined cells demonstrated outward currents. A: slowly activating outward current (IKDR). B: a transient outward current (Ito). C: transient inward current that coexisted with the outward current. Among 144 hASCs examined, most hASCs (106 of 144) showed IKDR.

 
Outward currents in hASCs. A high percentage of the hASCs (106/144) showed only IKDR (Fig. 5A). This current displayed voltage dependence and was activated slowly by 300-ms voltage steps. The threshold for current activation was around –20 mV (Fig. 5B). No significant inactivation was detected at positive potentials during prolonged pulses of 2 s in duration (Fig. 5E). The current-voltage (I-V) relationship of outward currents displayed significant outward rectification (Fig. 5D). The activation variable [G/Gmax, (maximum conductance change)] was determined from the I-V relationship of steady-state current for each cell and fitted with the Boltzmann equation to obtain the voltage for half-activation (V0.5). Mean V0.5 for activation of the outward current was 21.5 ± 1.5 mV (Fig. 5C, n = 5). These activation properties of outward currents indicate the involvement of IKDR.


Figure 5
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  		Fig. 5. Delayed rectifier outward current (IKDR) detected in hASCs. A: voltage-dependent outward currents activated by 300-ms voltage steps. B: outward currents from A were inhibited by 4-AP. C: activation curve (G/Gmax). Mean V0.5 for activation was 21.5 ± 1.5 mV (n = 5). D: current-voltage (I-V) relationship of the outward currents. 4-AP significantly inhibited the membrane currents (n = 6, P < 0.05). E: significant inactivation was not detected at positive potentials during prolonged pulses of 2 s in duration. F: effect of different ion channel blockers on IKDR. Outward current amplitudes in the presence of blockers were normalized to predrug amplitudes. Data reported were expressed as means ± SD. Statistical significance of differences of current before and after ion channel blockers treatment was analyzed using paired Student's t-test (*P < 0.05).

 
The nonselective K+ channel blocker 4-AP (3 mM) was used to test the involvement of IKDR. Figure 5, B and D demonstrates that 4-AP significantly inhibited the outward currents at test potentials from 0 to +80 mV (73.5 ± 6.8%, n = 6, P < 0.05). The effects of different channel blockers are shown in Fig. 5F. TEA (15 mM) inhibited the outward currents to 48.8 ± 5.2% of control at test potentials of +80 mV (n = 6, P < 0.05). It is known that IKDR has a slowly activating component (Iks) and a rapidly activating component (Ikr) (56). Linopirdine (10 µM), a Iks blocker (46), significantly inhibited the outward currents at test potential of +80 mV (72.8 ± 8.5% of control, n = 5, P < 0.05). However, E4031 (52) and clofilium tosylate (6, 33) (10 µM), selective Ikr blockers, had no effect on the outward currents in hASCs at any test potential (100 ± 11.0% of control, n = 8, P > 0.05; 95 ± 5.0% at –80 mV, n = 6, P > 0.05, respectively). Because of the relatively low concentration of EGTA (2 mM) in the electrode solution, the outward currents recorded may also include Ca2+-activated potassium currents (IKCa). According to their single-channel conductance, IKCa channels have been classified into three types: large-conductance channels (BKCa), small-conductance channels (SKCa), and intermediate-conductance channels (IKCa). Selective IKCa blockers were applied to test whether IKCa was present in hASCs (Fig. 5F). Iberiotoxin (100 nM), a selective blocker of BKCa (10), substantially inhibited the outward currents at +80 mV in a total of three cells (31.0 ± 3.6%, P < 0.05). The selective blocker of IKCa, clotrimazole (54) (1 µM), reduced the outward currents to 78.7 ± 7.2% at +80 mV (n = 5, P < 0.05). The selective blocker of SKCa, apamin (15) (100 nM), reduced the outward currents to 83.7 ± 3.4 at +80 mV (n = 3, P < 0.05). These results demonstrate the presence of three types of IKCa in hASCs. The outward current was completely blocked by TEA plus 4-AP, which indicates that a majority of the outward current is due to potassium ions. Chloride influx through chloride channels, if expressed, may contribute a minimal linear current to the dominating outward K current during the voltage steps.

Transient outward currents (Ito) were recorded from a typical cell evoked by 300-ms voltage steps from –60 to +80 mV with a prepulse to –80 mV (Fig. 6A, a) or –30 mV (Fig. 6A, b). Ito coexisted with other types of outward currents when holding potential was at –80 mV (Fig. 6A, a). Ito was eliminated when the holding potential was at –30 mV (Fig. 6A, b). Figure 6A, c shows a pure Ito obtained by subtracting (6A, b) from (6A, a). The I-V relationship of Ito showed significant outward rectification (Fig. 6A, d). The half-activation voltage of Ito in a representative cell is shown in Fig. 6A, e. Ito was detected in a small population of hASCs (19%).


Figure 6
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  		Fig. 6. A: Ito in ASCs. Ito was recorded from a typical cell evoked by 300-ms voltage steps from –60 to +80 mV with a prepulse to –80 mV (a) or –30 mV (b). a: Ito coexisted with other types of outward currents. b: Ito was eliminated when holding potential was –30 mV. c: current traces after subtracting b from a, showing pure transient outward currents. d: I-V relationship of Ito showed significant outward rectification. e: activation curve for Ito from a representative cell with a V0.5 of 24.2 mV. B: transient inward currents in hASCs. a: inward currents were elicited by depolarizing voltage steps. b: TTX completely abolished the inward currents. c: Magnified sodium currents from A. d: I-V relationship of INa in control and in TTX (300 nM).

 
Inward current in ASCs. Only a small population of cells exhibited transient inward current, which coexisted with outward current. Transient inward currents were elicited in 11 of 144 cells (Fig. 6B, a). TTX at 300 nM completely abolished the inward current, without affecting the outward current (Fig. 6B, b). Figure 6B, d shows I-V relationship of INa in cells before and after application of 300 nM TTX. Inward currents were activated at around –20 mV and peaked at about 15 mV.

In another set of experiments, we analyzed the inward rectifier current by stimulating the cell with a wide voltage range from 60 mV to –120 mV. However, we did not find current during stimulation from –120 mV to –80 mV after leak subtraction. We also examined whether functional L-type calcium current (ICa.L) was present in hASCs. KCl in the external solution was substituted by the same concentration of TEA. CaCl2 was substituted by BaCl2. Three-hundred nanomoles of TTX was added to block INa. Under this condition, no Ca2+ current was detected in any cells examined (data not shown, n = 10). These results indicate that TTX-sensitive sodium channels, but not calcium channels, contribute to the inward current recorded in hASCs.

RT-PCR analysis of mRNA expression of ion channels. The mRNA expression of ion channel subtypes related to functional outward and inward currents on ASCs (n = 3) was analyzed by RT-PCR (Fig. 3). The 31 types of ion channel subunit mRNA detected were as follows: L-type calcium channel subunit ({alpha}1C, {alpha}1D); T-type calcium channel subunit ({alpha}1G, {alpha}1H); hyperpolarization-activated and cyclic nucleotide-gated channel (HCN): HCN1, HCN2, and HCN3; inward rectifier channel (Kir): Kir2.1, Kir2.2, Kir2.3, Kir3.1, Kir3.4, and Twik1; voltage-gated K+ channel (Kv): Kv1.1, Kv1.4, Kv1.5, Kv2.1, Kv3.1, Kv4.1, Kv4.2, Kv4.3, Kv7.1, Kv7.3, and Kv11.1; ether-à-go-go K+ channel: hEAG1 and hEAG2; human large-conductance, voltage- and calcium-activated K+ channel: MaxiK; human intermediate-small-conductance, voltage- and calcium-activated K+ channel: KCNN3b and KCNN4; cardiac TTX-insensitive voltage-dependent Na+ channel {alpha}-subunit: SCN5A; and TTX-sensitive voltage-activated Na+ channel: hNE-Na (SCN9A). PCR results showed that mRNA expression of {alpha}1C, {alpha}1H, Kir2.1, Kir2.2, Kir3.4, Twik1, Kv1.1, Kv1.4, Kv1.5, Kv2.1, Kv4.1, Kv4.2, Kv4.3, Kv7.3, Kv11.1, hEAG1, MaxiK, KCNN3, KCNN4, and hNE-Na was positive, while mRNA expression of other channel subunits was not detected (Fig. 3).

We used commercial mRNA from brain and heart as positive controls and water as a negative control (Fig. 3). All PCR products showed the expected size, and their identities were confirmed by sequencing. These results provide a molecular basis for the functional ionic currents (IKDR, IKCa, Ito, INa.TTX) observed in hASCs. Positive mRNA expression of Kv1.1, Kv2.1, Kv1.5, Kv7.1, Kv11.1, and hEAG1 indicates that these ion channel subunits might be responsible for IKDR. Significant expression of MaxiK, KCNN3, and KCNN4 mRNAs provided molecular evidence for the presence of three types of IKCa. A population of ASCs (19%) displayed Ito. It is generally believed that Ito is encoded by Kv1.4, Kv4.2, or Kv4.3 (53). High mRNA expression level of Kv1.4, Kv4.2, and Kv4.3 in ASCs suggests that Ito may also be contributed, in part, by Kv1.4, Kv4.2, and Kv4.3. Only a small percentage of cells (8%) demonstrated transient inward current that coexisted with the outward current in ASCs (Fig. 6B). The inward current was completely inhibited by TTX. Positive expression levels of mRNA for TTX-sensitive Na+ channel hNE-Na and absence of mRNA for TTX-insensitive Na+ channel SCN5A (data not shown) further indicate that INa.TTX is present in ASCs.

Western blot analysis protein expression of HCN2, Kir2.2, and CaV1.2. mRNA expression of HCN2, Kir2.2, and CaV1.2 were positive in hASCs; however, we did not find related currents. We further checked the protein expression of HCN2, Kir2.2, and CaV1.2 by Western blot analysis (Fig. 7). hASCs were positive for Cav1.2, Kir2.2, and negative for HCN2.


Figure 7
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  		Fig. 7. Western blot analysis of protein expression of Kir2.2, Cav1.2, and HCN2 in hASCs. Lane 1 indicates Kir2.2 expression, lane2 indicates Cav1.2 expression, and lane 3 indicates HCN2 expression.

 
Effect of ion channel blockers on proliferation and cytotoxin of hASCs. To investigate potential physiological function of the ionic channels on hASCs, we studied the effects of ion channel blockers on proliferation and cytotoxicity of hASCs. Cell proliferation was evaluated indirectly by quantifying BrdU incorporation as a method reflecting and indicating cell proliferation. The IKDR was slowly activated and was inhibited by nonspecific potassium channel blockers TEA and 4-AP, which significantly reduced the proliferation of hASCs in a dose-dependent manner (P < 0.05). To preclude the effect of cytotoxicity of ion channel blockers on proliferation of hASCs, we examined the cytotoxic effect of K+ channel blockers by staining dead cells with PI. Notably, TEA significantly inhibited proliferation of hASCs (Fig. 8A, light gray line) at lower concentrations (30 mM) without exerting toxic effects. However, higher concentrations of TEA (120 mM) had a significantly toxic effect on hASCs (Fig. 8A: dark gray line). In a similar manner, low concentrations of 4-AP inhibited the proliferation of hASCs, and high concentrations of 4-AP exerted a cytotoxic effect on hASCs (Fig. 8B). Other selective potassium channel blockers, including linopiridine, iberiotoxin, clotrimazole, and apamin also significantly inhibited IKDR. However, they did not affect proliferation of hASCs (Fig. 8, C, E–G). Clofilium tosylate, a blocker of Ikr, did not affect IKDR; however, it inhibited proliferation of hASCs (Fig. 8I). TTX completely abolished INa.TTX but had no effect on proliferation on hASCs (Fig. 8H). E4031 affected neither IKDR nor cell proliferation (Fig. 8D).


Figure 8
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  		Fig. 8. Effects of different doses of ion channel inhibitors on proliferation (light gray line) and cytotoxin (dark gray line) of hASCs. Proliferative effects were assessed by bromodeoxyuridine (BrdU) incorporation, and cytotoxic effects were assessed by analyzing PI-positive cells using flow cytometry. The abscissa of each figure represents concentration of ion channel blockers. The left ordinate of each figure indicates percentage of PI-positive dead cells reflecting cytotoxic effect on hASCs, and the right ordinate indicates absorbance reflecting BrdU incorporation. Control culture was not treated with ion channel inhibitors. Data were reported as means ± SD; n = 3. Statistical significance of differences between groups was tested using ANOVA (*P < 0.05 vs. control; **P < 0.01 vs. control).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this report, we demonstrate for the first time the presence of three types of K+ currents in human adipose tissue-derived stem cells: IKDR, IKCa, Ito, and one type of Na+ current INa.TTX. RT-PCR analysis of mRNA expression of ion channel subunits further confirmed the presence of these channels. However, there are some discrepancies between mRNA expression levels and the presence of detectable functional ion channels. We detected high expression levels of mRNA for the HCN2 in all samples of hASCs, but the corresponding current If (35) was not detected. Similarly, mRNA for Kir2.1, Kir3.4, Kir2.2, and Twik1, which are associated with the inward rectifier current (IK1), were expressed in our hASCs. However, we did not detect IK1 in hASCs. High expression levels of mRNA for {alpha}1C and {alpha}1H subunits, which are associated with L-type calcium channel and T-type calcium channel, respectively, were positive in hASCs; however, we could not detect the corresponding functional ion channels. Similar observations have been reported in human BMSCs (hBMSCs) and human embryonic stem cells (hESs), a type of totipotent stem cell, by other research groups (21, 51). HERG (Kv11.1) mRNA expression is detected in hASCs; however, in our preliminary study, we didn't see detectable HERG currents when the holding voltage was stepped from 20 mV to –50 mV. The results of E4031 further confirm that there is no functional HERG current in hASCs. HERG present in cardiomyocytes constitute the channel responsible for the rapidly activating component of delayed rectifier K+ current (IKr), which contributes to the final repolarization of the ventricular action potential (44). Suppression of HERG channel causes action potential and QT interval prolongation, which is related with the induction of fatal arrhythmias (42). The expression of HERG mRNA might indicate the potential of hASCs to differentiate into cardimyocytes. Western blot analysis showed the protein expression of Kir2.2 and Cav1.2 channel proteins was positive in hASCs, but HCNN2 was absent. There are several possible reasons why the related current was not detected: 1) HCNN2 expression in hASCs may be only maintained at the mRNA level; 2) protein subunits of Kir2.2 and Cav1.2 in some hASCs may not assemble into functional channels; and 3) only a low percentage of hASCs had functional L-type Ca2+ channels. Our group has previously demonstrated the existence of functional L-type calcium channel in genetically selected cardiac progenitor cells isolated from hASCs. Only 0.045–0.4% of all hASCs showed functional L-type calcium channels (3). Therefore, it is most likely that cells with functional calcium channels were by chance not included among the 144 hASCs that we patched.

The presence of multiple ion channels indicates that hASCs may be heterogeneous. It is possible that the CD markers that we analyzed for this study could not distinguish subpopulations of hASCs. Different types of committed progenitor cells, such as cardiac progenitor cells, are present in hASCs (3, 48), which may affect the expression pattern of ion channels. In addition, the heterogeneity of ion channels detected in hASCs may be also due to the fact that the cells that we examined were at different phases of the cell cycle. Ion channel expression may change with cell cycle progression (38) but can also vary with different progenitor lineages and stages of our cell population.

Recently, the electrophysiological characteristics of hBMSCs and hESs have been described. Expression patterns of ion channels in hASCs showed more similarity to hBMSCs (30), except 15% of hBMSCs had functional L-type Ca2+ channels compared with hESs. Only IKDR was detected in hESs. Neither Na+ nor Ca2+ currents were detected in hESs (51).

hASCs and hBMSCs are both adult multipotent stem cells derived from mesenchyme. A comparative analysis of stem cells isolated from bone marrow and adipose tissue clearly showed that ASCs and BMSCs were not different regarding the success rate of isolating stem cells, morphology, surface markers, ability to form colonies, and differentiation potential. They grow as plastic fibroblast-like adherent cells. They are positive for CD29, CD44, CD90, and CD105 and negative for CD11b, CD31, CD34, and HLA-DR. They can differentiate into multiple tissue-specific cells, including adipocytes, osteoblasts, neurons, and hepatocytes (2, 22, 26, 27, 31, 40). An alternative explanation for their similarities is that ASCs and BMSCs may be derived from the same vascular associated early stem cells. The slightly different expression pattern of ion channels between ASCs and BMSCs also reflects that these two types of tissue-derived stem cells may be made up of different percentages of committed progenitor cells. However, whether the differences between them are due to inherent differences or differing culture conditions requires further analysis.

Expression of multiple ion channels in ASCs indicates possible functions of these different channels in the cellular physiological activity of hASCs. For example, K+ channels are key players in controlling membrane potentials. They also establish the electrochemical gradient that determines the movement of other ions across the plasma membrane. In addition, potassium channels participate in cell volume regulation (9, 12). Specific inhibitors are required to assess the contribution of these channels to proliferation of hASCs. Our current results showed that low concentrations of the nonselective potassium channel inhibitors TEA and 4-AP significantly inhibited proliferation of hASCs without affecting cell viability (Fig. 8, A and B). This result is consistent with reports from other groups that K+ channels play an important role in the cell proliferation processes (4, 23). If K+ channels are inhibited, proliferation is impaired. In general, initiation of G1 progression requires K+ channel activity. Blockade of K+ channels can induce G1 arrest in the OP cell cycle (19). K+ channels are involved in the EGF-mediated mitogenic signal transduction process, required for voltage-gated K+ channels participating in G1/S-phase transition of the cell cycle (32). K+ channels also physically interact with Ca2+-binding protein calmodulin to regulate cell growth (18, 45). However, contribution of BKCa, IKCa, SKCa, Iks, and INaTTX to proliferation of hASCs could be excluded as their selective blockers significantly suppressed outward K+ current but had no effect on proliferation. E-4031 affected neither IKDR nor cell proliferation. Ten micromoles of clofilium tosylate, a selective blocker of Ikr, did not affect outward K+ but inhibited proliferation of hASCs. This is because the pharmacological action of clofilium tosylate is much more complex than previously anticipated. In addition to inhibiting K+ current, clofilium tosylate also inhibits Na+ current and Ca2+ current (28, 55). The inhibitory effect of clofilium tosylate on cell proliferation may due to its effect on these different ion channels. Therefore, the mechanism of action remains a topic for further investigation.

In summary, this is the first systemic characterization of ion channels present in an unselected population of ASCs, as an important step toward their possible clinical use. These novel findings have an impact on the understanding of the mechanisms of stem cell proliferation and differentiation.

One current concern with cell-based therapy is that certain stem cells might exhibit electrical properties that could negatively interfere with the normal conductance of the heart. It was reported that transplantation of skeletal myoblasts for myocardial regeneration caused severe arrhythmias (39). This raises the concern that arrhythmic events might be caused by injected cells' intervention in the natural conductance among cells in the infarcted heart. The characterization of ion channel expression in undifferentiated ASCs provides a basic understanding for further assessment of the safety issue of stem cell-based therapy, not only for treatment of infarcted hearts, but also for other possible biological applications in the future.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Grant 543102 from the Alliance of Cardiovascular Researchers (to E. Alt) and by American Heart Association Southeast Affiliate Award 0555331B (to Y. H. Song).


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Alt, Univ. of Texas, MD Anderson Cancer Center, SCRB2, Unit 951, 7435 Fannin St., Houston, TX 77054 (e-mail: ealtmd{at}aol.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* These authors contributed equally to this work. Back


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