Maintenance of mESCs and hESCs

The mESC line R1 [4] (kind gift from Dr. Andras Nagy, University of Toronto, Toronto), which has been genetically engineered to constitutively express the green fluorescent protein (GFP), was used in this study to assist their identification from mouse embryonic fibroblast (MEF) cells. mESCs were maintained in their undifferentiated stage by growing on mitomycin-treated MEF feeder layer [5] in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Carlsbad, CA, http://www.lifetech.com) supplemented with 20% fetal bovine serum (GIBCO), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol, 0.1 mM nonessential amino acids, and 1,000 U/ml leukemia inhibitory factor (Chemicon, Temecula, CA; http://www.chemicon.com).

For hESCs, the H1 line (WiCell Research Institute, Madison, WI, http://www.wicell.org) [6] that has been stably transduced by the recombinant lentivirus LV-CAG-GFP (see below for further description of the lentiviral vector used), as we have recently described, was used [7, 8]. hESCs were maintained on irradiated MEF feeder layer and propagated as previously described. The culture medium consisted of DMEM supplemented with 20% fetal bovine serum (HyClone, Logan, UT, http://www.hyclone.com), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% nonessential amino acids. MEF cells were obtained from 13.5-day-old embryos of CF-1 mice.

Lentivirus-Mediated Stable Genetic Modification of hESCs

For stable genetic modification, we used the self-inactivating HIV1-based lentiviral vector (LV) [9]. The plasmid pLV-CAG-GFP was created from pRRL-hPGK-GFP SIN-18 (generously provided by Dr. Didier Trono, University of Geneva, Geneva, Switzerland) by replacing the human phosphoglycerate kinase 1 (hPGK) promoter with the CAG promoter, an internal composite constitutive promoter containing the cytomegalovirus enhancer and the β-actin promoter. Recombinant lentiviruses were generated using the three-plasmid system [10] by cotransfecting HEK293T cells with pLenti-CAG-GFP, pMD.G, and pCMV R8.91. The latter plasmids encode the vesicular stomatitis virus G envelope protein and the HIV-1 gag/pol, tat, and rev genes required for efficient virus production, respectively. Lenti-viral particles were harvested by collecting the culture medium at 48 hours after transfection and were stored at −80°C before use.

hESCs were transduced by adding purified lentiviruses to cells at a final concentration of 10,000 TU ml⁻¹ with 8 μg/ml polybrene to facilitate transduction. The multiplicity of infection was approximately 5 for each round of transduction. After 4–6 hours of incubation with LV-CAG-GFP, 2 ml fresh medium per 60 mm dish was added. Transduction was allowed to proceed for at least 12–16 hours. Cells were washed with phosphate-buffered saline (PBS) twice to remove residual viral particles. For generating stably LV-CAG-GFP–transduced hESCs, green portions of hESC colonies were microsurgically segregated from the nongreen cells, followed by culturing under undifferentiating conditions for expansion. This process was repeated until a homogenous population of green hESCs, as confirmed by fluorescence-activated cell sorter, was obtained. Further details are also provided in another recent publication [11].

Immunostaining

mESCs or hESCs were fixed in 4% paraformaldehyde for 15 minutes at 21°C, washed with PBS, and permeabilized with 0.1% Trition X-100/PBS. The cells were then blocked with 10% bovine serum albumin with 0.075% saponin or 4% goat serum in PBS for 2 hours at 21°C. Fixed cells were incubated with the primary antibodies at a dilution of 1:25 (for SSEA-4 and TRA-1-60 in hESC staining) (Chemicon) overnight at 4°C, followed by incubation with fluorescent-labeled secondary antibodies for 50 minutes at 21°C and visualization by laser-scanning confocal microscopy.

Cell Proliferation Assay

Cell proliferation was determined in 96-well plates using a non-radioactive chemiluminescent bromodeoxyurindine (BrdU) kit (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) according to the manufacturer's protocols. mESCs or hESCs were treated with specified concentrations of tetraethylammonium (TEA), 4-aminopyridine (4-AP), or iberiotoxin (IBTX) for 24 hours. BrdU labeling solution was then added to give a final concentration of 10 μM of BrdU. Medium was removed after 2 hours, and cells were fixed with the addition of FixDenat solution (Roche Dianostics) for 30 minutes at room temperature. After removing FixDenat, 100 μl freshly diluted 1:100 anti-BrdU peroxidase solution was added to the wells for 30 minutes, followed by washing three times. Finally, 100 μl of substrate solution was added, and luminescence was read by a multiwell scanning spectrophotometer automatic luminometer.

Cell viability was determined in 96-well plates using a colorimetric MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] kit (Roche Diagnostics). Briefly, specified concentrations of TEA, 4-AP, or IBTX were added for 24 hours. Then 10 μl of MTT labeling reagent (5 mg/ml in PBS) was added to each well, followed by incubation at 37°C for 4 hours. Solubilization solution (100 μl) was added to dissolve the formazan crystals formed. Absorbances at 540 nm were read by a spectrophotometer. Untreated cells were used as control (i.e., 100% survival).

Electrophysiology

Only GFP-expressing mESCs and hESCs (∼15 pF) were selected for experiments. Electrophysiological recordings were performed at room temperature using whole-cell patch clamp. Pipette electrodes (TW120F-6; World Precision Instruments, Sarasota, FL, http://www.wpiinc.com) were fabricated using a Sutter P-87 horizontal puller and fire-polished and had final tip resistances of 2–4 Mμ. All recordings were performed at room temperature in a bath solution containing (in mM) NaCl 110, KCl 30, CaCl₂ 1.8, MgCl₂ 0.5, HEPES 5, and glucose 10, pH adjusted to 7.4 with NaOH. The internal solution for patch recordings contained (in mM) NaCl 10, KCl 130, MgCl₂ 0.5, HEPES 5, EGTA 1, and MgATP 5, pH adjusted to 7.3 with KOH. For recording Ca²⁺-activated large-conductance K⁺ current (IK_Ca), 2.5 mM Ca²⁺ was added to the pipette solution containing (in mM) K-aspartate 110, NaCl 10, KCl 20, MgCl₂•6H₂O 1, Na₂-phosphocreatine 5, HEPES 10, K₂-EGTA 5, Mg₂ATP 5, and GTP 0.1, pH adjusted to 7.2 with KOH. Blockers were diluted to the final concentrations in the bath solution as indicated and administrated via superfusion (at least 10 ml) using a fast-exchange perfusion system. The current amplitude at +50 mV was monitored every 30 seconds after 5 minutes of incubation until steady-state current blockage was achieved.

Half-blocking concentrations (IC₅₀) were determined from the following binding isotherm: I = I_blocker-ins + (1 − I_blocker-ins) / (1 + [blocker]/IC₅₀)ⁿ), in which IC₅₀ is the half-blocking concentration, I_blocker-ins is blocker-insensitive component, n is the Hill coefficient, and I₀ and I are the peak currents measured at the voltage indicated before and after application of the blocker, respectively. All IC₅₀ and half-effective concentration (EC₅₀) values reported were calculated from individual determinations (i.e., curve fitting to data from individual experiments). The curves presented in the Figures were fitted to averaged data points pooled from all experiments.

Reverse Transcription–Polymerase Chain Reaction

Total RNA was prepared from mESCs or mouse brain using ToTALLY RNA Kit (Ambion Inc., Austin, TX, http://www.ambion.com). Single-stranded cDNA was synthesized from approximately 1 μg of total RNA using random hexamers and SuperScript reverse transcription (RT) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's protocols, followed by polymerase chain reaction (PCR) amplification with gene-specific primers for ion channel genes. Primers, annealing temperatures, product sizes, and the corresponding references are given in Table 1. 18S ribosomal RNA (498 bp) was used as an internal control. The reaction was conducted using the following protocol: initial denaturing of the template for 5 minutes at 94°C followed by 32 repeating cycles of denaturing for 1 minute at 94°C, annealing for 1 minute, extension for 1 minute at 72°C, and a final elongation at 72°C for 7 minutes. The PCR products were size-fractionated by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. All the primers were tested with preparations from mouse brain and water as positive and negative controls, respectively. To analyze RNA expression in hESCs by RT-PCR, total RNA was extracted using a NucleoSpin RNAII kit (Clontech, Palo Alto, CA, http://www.clontech.com), treated with DNase I, and used for RT-PCR with SuperScript One-Step RT-PCR with the Platinum Taq system (Invitrogen). The primers for gene-specific RT-PCR are given in Table 2. Equal aliquots of the PCR products were electrophoresed through 2% agarose gels and visualized by ethidium bromide staining.

Microarray Analysis

Microarray analysis was performed using Affymetrix human genome U133A array (Affymetrix, Santa Clara, CA http://www.affymetrix.com), which represents 18,400 transcript and variants, including 14,500 well-characterized human genes. Total RNA was extracted from pluripotent hESCs (H1) and hybridized to microarrays according to the protocols provided by the manufacturer. The software Genespring 6.0 (Silicon Genetics, Redwood City, CA, http://www.silicongenetics.com) was used for microarray data analysis. Data were normalized to the expression level of the 50th percentile of the entire chip and filtered to show genes that are labeled as expressed (i.e., present flags) as defined by Affymetrix analysis.

Statistics

All data reported are means ± SEM. Statistical significance was determined for all individual data points and fitting parameters using one-way analysis of variance and Tukey's Honestly Significantly Different post-hoc test at the 5% level.

Ionic Currents in Pluripotent mESCs

Figure 1A shows that undifferentiated mESC colonies were homogenously immunostained for the pluripotency markers Oct-4 and SSEA-1 [12, 13]. In 159 of 304 (52.3%) undifferentiated mESCs, depolarization-activated time-dependent noninactivating outward currents that increased progressively with positive voltages could be recorded (8.6 ± 0.9 pA/pF at +40 mV; Figs. 1B, 1C). These outwardly rectifying currents resemble the delayed-rectifier K⁺ currents (IK_DR) and could be dose-dependently inhibited by the known K⁺ channel blocker TEA⁺ [14, 15] (IC₅₀ = 1.2 ± 0.3 mM, n = 13; Figs. 1B, 1D). IK_DR in mESCs was also sensitive to 4-AP (IC₅₀ = 0.5 ± 0.1 mM, n = 17), a more potent K channel blocker than TEA⁺ [16, 17], and the Ca²⁺-activated large-conductance K⁺ current (IK_Ca) blocker IBTX (100nM) (current inhibition = 33.2% ± 12.7%, n = 3) (Figs. 1B, 1E). As shown in Figures 1D and 1E, increasing TEA or 4-AP to 30 mM could not lead to complete current inhibition. Indeed, even combined application of 30 mM TEA and 30 mM 4-AP also could not completely eliminate the IK_DR (30 mM 4-AP, 30 mM TEA, and 30 mM TEA + 30 mM 4-AP reduced the IK_DR to 45.8% ± 2.1%, 39.3% ± 7.6%, and 45.1% ± 8.1%, respectively; p > .05). The currents remaining after combined TEA/4-AP blockade were also not sensitive to 1 mM BaCl₂, implicating the presence of some background current.

Although voltage-gated Na⁺ (Na_v) and Ca²⁺ (Ca_v) currents were completely absent in all pluripotent mESCs tested (n > 200), whether IK_DR was present (Fig. 1B) or not (Fig. 2A), a modest yet detectable hyperpolarization-activated inward current (I_h, encoded by the hyperpolarization-activated cyclic nucleotide-modulated nonselective or HCN ion channel family [18]; −2.2 ± 0.4 pA/pF at −120 mV) was detected in 79 of 270 cells (29.3%; Fig. 2B). I_h in mESCs was reversibly blocked by the HCN inhibitor Cs⁺ [19, 20]. Application of 1 μM isoproterenol altered neither the kinetics nor amplitude of I_h recorded in mESCs. Inwardly rectifying K currents (I_K1) responsible for stabilizing the resting membrane potential were also not present.

To obtain insights into the molecular identities of the ionic currents identified, total RNA was isolated from pluripotent mESCs for RT-PCR. Figure 3A shows that Kv1.1, 1.2, 1.3, 1.4, 1.6, 4.2, and BK (or Maxi-K) transcripts but not Kv1.5, 2.1, 3.1, 3.2, and 4.3 were detected. Consistent with the presence of I_h, HCN2 and HCN3 transcripts were also expressed. Inhibition by 4-AP and insensitivity to extracellular TEA ions is a pharmacological hallmark of A-type currents [21]. However, neither TEA-subtracted nor 4-AP–subtracted current traces from IK_DR blockade revealed any transient outward current with the typical rapid inactivation feature (data not shown). Therefore, although Kv1.1, 1.2, 1.6, and BK channels might underline the delayed rectifier current recorded, we conclude that Kv1.4- and Kv4.2-encoded transient outward K⁺ currents were not functionally expressed.

Effects of Ion Channel Blockers

To investigate possible physiological roles of the ionic currents identified, we next studied the functional consequences of their pharmacological blockade by assessing the effects of extracellular application of K⁺ channel blockers on cell proliferation. Specifically, we measured DNA synthesis as an index for replication by quantifying BrdU incorporation into genomic DNA during the S phase of the cell cycle, which is proportional to the rate of cell division [22]. Application of TEA⁺ significantly inhibited the proliferation of mESCs in a dose-dependent manner (Fig. 3B, open squares). The EC₅₀ was 20.1 ± 3.7 mM (n = 3), approximately 20-fold higher than the IC₅₀ for IK_DR inhibition. Similarly, 4-AP (EC₅₀ = 2.7 ± 0.2 mM; Fig. 3C, open squares) and IBTX (EC₅₀ = 133.9 ± 25.9 nM; Fig. 3D, open squares) also dose-dependently reduced cell proliferation. Of note, the rank orders of these agents to inhibit proliferation follow the trend of their potencies to block IK_DR (i.e., IBTX > 4-AP > TEA). To assess the cytotoxic effect of K⁺ channel blockers, a colorimetric MTT kit was used to examine for changes in metabolism. Notably, the EC₅₀ values for inhibition of metabolic activity by TEA (62.7 ± 9.0 mM; Fig. 3B, solid squares), 4-AP (4.6 ± 0.5 mM; Fig. 3C, solid squares), and IBTX (333.9 ± 64.6 nM; Fig. 3D, solid squares) were significantly higher than those for inhibiting cell proliferation (p < .05). These results implicate that the metabolic influences of these blockers were relatively insignificant at concentrations at which they effectively exert their inhibitory effects on ESC proliferation, presumably by K⁺ channel blockade.

Electrophysiological Properties of hESCs: Similarities and Differences

Although hESCs and mESCs share several similarities, significant differences are known to exist between the two species [23]. Therefore, we also examined the previously unexplored electrophysiological properties of hESCs. Pluripotent hESCs were positive for markers such as alkaline phosphatase, Oct4, SSEA4, and TRA-60 (Fig. 4A), consistent with previous reports [6]. Similar to mESCs, TEA+-sensitive IK_DR (IC₅₀ = 2.1 ± 0.2 mM; Fig. 4D) was also detected in hESCs (∼100%), but the current density was approximately sixfold higher (47.5 ± 7.9 pA/pF at +40 mV, n = 12, p < .05) (Figs. 4B, 4C). Similar to mESCs, application of TEA⁺ dose-dependently inhibited hESC proliferation as assayed by BrdU incorporation, with an EC₅₀ (11.6 ± 2.0 mM) (Fig. 4D). Unlike mESCs, however, there was no measurable I_h in all hESCs tested (n = 30; Fig. 2B). Same as mESCs, neither Na_v nor Ca_v currents could be detected in hESCs.

Microarray Analysis of Ion Channel Genes in hESCs

Using Affymetrix U133A chips, we performed microarray analysis of pluripotent hESCs to examine the expression of ion channels at the transcriptomic level. Figure 5A shows the expression profile of all genes tested in undifferentiated hESCs. For voltage-gated ion channels, a total of 36, 19, and 49 genes on the U133A chips were identified as Ca_v, Na_v, or K_v channel genes, respectively. For inspection, their expression profile is extracted in Figure 5A and further summarized in Figures 5B and 5C by normalizing signals to the average expression level of the entire microarray in a manner similar to that of cytokines and their receptors in hESCs, as recently reported by Dvash et al. [24]. Our data indicate that among the total 104 voltage-gated ion channel genes mentioned above, only the transcripts of 3, 1, and 5 Ca_v, Na_v, and K_v genes were significantly expressed, as defined by Affymetrix. The corresponding gene products were CACNA1A (Ca_v2.1), CACNA2D2 (Ca_v α2/δ subunit 2), CACNB3 (Ca_v β3 subunit), SCN11A (Na_v1.9), KCNB1 (K_v2.1), KCND2 (K_v4.2), KCNQ2 (K_v7.2), KCNS3 (K_v9.3), and KCNH2 (K_v11.1) (note, however, that I_Ca, I_Na, and Kv4.2-encoded transient outward K⁺ currents could not be electrophysiologically recorded, like mESCs). Of note, KCNQ2 and KCNH2, which underlie the noninactivating, slowly deactivating M-current [25] and the rapid component of the cardiac delayed rectifier (IK_r) [26], were relatively highly expressed. Similarly, KCNB1 and KCNS3, which encode for the delayed rectifier K_v2.1 channels and the silent modulatory α-subunit K_v9.3 that heteromerizes with K_v2.1 subunits [27–29], respectively, were also expressed. Collectively, these ion channel genes could underlie the K_DR current identified, although further experiments will be needed to confirm and dissect their molecular identities. By contrast, no HCN transcript was expressed in pluripotent hESCs. RT-PCR confirmed the array results for five of nine channels (Fig. 5D).