Electrophysiological Properties of Pluripotent Human and Mouse Embryonic Stem Cells
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《干细胞学杂志》
a Department of Medicine, University of Hong Kong, Hong Kong, China;
b Departments of Medicine and
c Cellular and
d Molecular Medicine and Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, USA
Key Words. Embryonic stem cells ? Electrophysiology ? Pluripotency ? Ion channels
Correspondence: Ronald Li, Ph.D., Johns Hopkins University, 720 Rutland Avenue, Ross 1165, Baltimore, Maryland 21205, USA. Telephone: 410-614-0035; Fax: 410-502-2802; e-mail: ronaldli@jhmi.edu; and Hung-Fat Tse, M.D., University of Hong Kong, Cardiology Division, Queen Mary Hospital, Pokfulam, Hong Kong. e-mail: hftse@hkucc.hku.hk
ABSTRACT
Embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts. Because ESCs can propagate indefinitely in culture while maintaining their pluripotency to differentiate into all cell types, they may therefore provide an unlimited supply of specialized cells such as cardiomyocytes and neurons for cell-based therapies. For instance, direct injection of pluripotent ESCs after myocardial infarction has been suggested as a means to repair the damaged heart . However, transplantation of cells with undesirable electrical properties into the heart can predispose patients to lethal electrical disorders (arrhythmias) . Therefore, it is critical to understand the electrophysiological profile of undifferentiated ESCs, which has not been characterized. In this study, we hypothesize that ion channels are functionally expressed in mouse (m) and human (h) ESCs, although the specific encoding genes (and/or their isoforms) and their expression levels might differ. We discovered that several specialized ion channels are differentially expressed in pluripotent mESCs and hESCs. Collectively, our experiments reveal further similarities and differences between the two species. We discuss these results in relation to the physiological function of ion channels in hESC biology as well as practical considerations for potential therapeutic applications of hESCs.
MATERIALS AND METHODS
Ionic Currents in Pluripotent mESCs
Figure 1A shows that undifferentiated mESC colonies were homogenously immunostained for the pluripotency markers Oct-4 and SSEA-1 . 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 (IKDR) and could be dose-dependently inhibited by the known K+ channel blocker TEA+ (IC50 = 1.2 ± 0.3 mM, n = 13; Figs. 1B, 1D). IKDR in mESCs was also sensitive to 4-AP (IC50 = 0.5 ± 0.1 mM, n = 17), a more potent K channel blocker than TEA+ , and the Ca2+-activated large-conductance K+ current (IKCa) 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 IKDR (30 mM 4-AP, 30 mM TEA, and 30 mM TEA + 30 mM 4-AP reduced the IKDR 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.
Figure 1. (A): Images of pluripotent mESCs immunostained for SSEA-1 and Oct-4. (B): Representative current tracings recorded from undifferentiated mESCs before (left panels) and after (right panels) blockade by TEA, 4-AP, and IBTX, as indicated. The electrophysiological protocol used for eliciting currents is also given. (C): Current-voltage relationship of IKDR of mESCs. Dose–response relationships for (D) TEA and (E) 4-AP block of IKDR of mESCs. Abbreviations: 4-AP, 4-aminopyridine; IBTX, iberiotoxin; mESC, mouse embryonic stem cell; TEA, tetraethylammonium.
Although voltage-gated Na+ (Nav) and Ca2+ (Cav) currents were completely absent in all pluripotent mESCs tested (n > 200), whether IKDR was present (Fig. 1B) or not (Fig. 2A), a modest yet detectable hyperpolarization-activated inward current (Ih, encoded by the hyperpolarization-activated cyclic nucleotide-modulated nonselective or HCN ion channel family ; –2.2 ± 0.4 pA/pF at –120 mV) was detected in 79 of 270 cells (29.3%; Fig. 2B). Ih in mESCs was reversibly blocked by the HCN inhibitor Cs+ . Application of 1 μM isoproterenol altered neither the kinetics nor amplitude of Ih recorded in mESCs. Inwardly rectifying K currents (IK1) responsible for stabilizing the resting membrane potential were also not present.
Figure 2. (A): Stimulation protocol and representative current traces demonstrating the absence of Nav or Cav currents in an mESC that lacks IKDR. (B): CsCl2-sensitive hyperpolarization-activated currents could be recorded from pluripotent mESCs but not hESCs. (C): Steady-state current-voltage relationships of hyperpolarization-activated currents of mESCs and hESCs. Abbreviations: hESC, human embryonic stem cell; mESC, mouse embryonic stem cell.
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 Ih, 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 . However, neither TEA-subtracted nor 4-AP–subtracted current traces from IKDR 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.
Figure 3. (A): Expression of ion channel transcripts in mESCs probed by semiquantitative reverse transcription–polymerase chain reaction. Dose–response relationships of (B) TEA, (C) 4-AP, and (D) IBTX for inhibition of mESC proliferation assessed by BrdU incorporation (open squares) and cytotoxic effects by MTT (solid squares). Abbreviations: 4-AP, 4-aminopyridine; BrdU, bromodeoxyurindine; IBTX, iberiotoxin; mESC, mouse embryonic stem cell; TEA, tetra-ethylammonium.
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 . 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 IKDR 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 IKDR (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 . 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 . Similar to mESCs, TEA+-sensitive IKDR (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 Ih in all hESCs tested (n = 30; Fig. 2B). Same as mESCs, neither Nav nor Cav currents could be detected in hESCs.
Figure 4. (A): Pluripotent hESCs were positive for alkaline phosphatase, SSEA-4, and TRA-1-60. (B): Representative current tracings recorded from undifferentiated hESCs. The same electrophysiological from Figure 1 was used. (C): Steady-state current-voltage relationship of IKDR in hESCs. (D): IC50 for blockade of IKDR and EC50 for proliferation inhibition by tetraethylammonium in hESCs. Abbreviation: hESC, human embryonic stem cell.
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 Cav, Nav, or Kv 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. . Our data indicate that among the total 104 voltage-gated ion channel genes mentioned above, only the transcripts of 3, 1, and 5 Cav, Nav, and Kv genes were significantly expressed, as defined by Affymetrix. The corresponding gene products were CACNA1A (Cav2.1), CACNA2D2 (Cav 2/ subunit 2), CACNB3 (Cav ?3 subunit), SCN11A (Nav1.9), KCNB1 (Kv2.1), KCND2 (Kv4.2), KCNQ2 (Kv7.2), KCNS3 (Kv9.3), and KCNH2 (Kv11.1) (note, however, that ICa, INa, 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 and the rapid component of the cardiac delayed rectifier (IKr) , were relatively highly expressed. Similarly, KCNB1 and KCNS3, which encode for the delayed rectifier Kv2.1 channels and the silent modulatory -subunit Kv9.3 that heteromerizes with Kv2.1 subunits , respectively, were also expressed. Collectively, these ion channel genes could underlie the KDR 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).
Figure 5. (A): Microarray analysis of pluripotent hESCs for the transcript expression of all genes tested using Affymetrix U133A microarrays (see Materials and Methods). (B): Left, 104 voltage-gated Cav, Nav, and Kv channel genes are clustered. The same expression scale bar shown in (A) was used. Right, same as the left panel, except only transcripts that were defined to be expressed, as defined by Affymetrix, are shown. (C): Bar graph of normalized transcript levels of the expressed ion channel genes. Data were normalized to the expression level of the 50th percentile of the entire microarray. (D): Expression of ion channel transcripts in hESCs probed by semiquantitative reverse transcription–polymerase chain reaction. Abbreviation: hESC, human embryonic stem cell.
DISCUSSION
K.W. and T.X. contributed equally to this study. This work was supported by grants from the National Institutes of Health (R01 HL-52768 and R01 HL-72857 to R.A.L.), the Blaustein Pain Research Centre (to R.A.L.), and the Hong Kong Research Grant Council (HKU 7459/04M to H.F.T., R.A.L., G.R.L., and C.P.L.). S.Y.T. was supported by a postdoctoral fellowship award from the Croucher Foundation. We would also like to thank Dr. Guibin Chen at Johns Hopkins for helpful discussion in microarray analysis.
DISCLOSURES
The authors indicate no potential conflicts of interest.
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b Departments of Medicine and
c Cellular and
d Molecular Medicine and Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, USA
Key Words. Embryonic stem cells ? Electrophysiology ? Pluripotency ? Ion channels
Correspondence: Ronald Li, Ph.D., Johns Hopkins University, 720 Rutland Avenue, Ross 1165, Baltimore, Maryland 21205, USA. Telephone: 410-614-0035; Fax: 410-502-2802; e-mail: ronaldli@jhmi.edu; and Hung-Fat Tse, M.D., University of Hong Kong, Cardiology Division, Queen Mary Hospital, Pokfulam, Hong Kong. e-mail: hftse@hkucc.hku.hk
ABSTRACT
Embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts. Because ESCs can propagate indefinitely in culture while maintaining their pluripotency to differentiate into all cell types, they may therefore provide an unlimited supply of specialized cells such as cardiomyocytes and neurons for cell-based therapies. For instance, direct injection of pluripotent ESCs after myocardial infarction has been suggested as a means to repair the damaged heart . However, transplantation of cells with undesirable electrical properties into the heart can predispose patients to lethal electrical disorders (arrhythmias) . Therefore, it is critical to understand the electrophysiological profile of undifferentiated ESCs, which has not been characterized. In this study, we hypothesize that ion channels are functionally expressed in mouse (m) and human (h) ESCs, although the specific encoding genes (and/or their isoforms) and their expression levels might differ. We discovered that several specialized ion channels are differentially expressed in pluripotent mESCs and hESCs. Collectively, our experiments reveal further similarities and differences between the two species. We discuss these results in relation to the physiological function of ion channels in hESC biology as well as practical considerations for potential therapeutic applications of hESCs.
MATERIALS AND METHODS
Ionic Currents in Pluripotent mESCs
Figure 1A shows that undifferentiated mESC colonies were homogenously immunostained for the pluripotency markers Oct-4 and SSEA-1 . 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 (IKDR) and could be dose-dependently inhibited by the known K+ channel blocker TEA+ (IC50 = 1.2 ± 0.3 mM, n = 13; Figs. 1B, 1D). IKDR in mESCs was also sensitive to 4-AP (IC50 = 0.5 ± 0.1 mM, n = 17), a more potent K channel blocker than TEA+ , and the Ca2+-activated large-conductance K+ current (IKCa) 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 IKDR (30 mM 4-AP, 30 mM TEA, and 30 mM TEA + 30 mM 4-AP reduced the IKDR 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.
Figure 1. (A): Images of pluripotent mESCs immunostained for SSEA-1 and Oct-4. (B): Representative current tracings recorded from undifferentiated mESCs before (left panels) and after (right panels) blockade by TEA, 4-AP, and IBTX, as indicated. The electrophysiological protocol used for eliciting currents is also given. (C): Current-voltage relationship of IKDR of mESCs. Dose–response relationships for (D) TEA and (E) 4-AP block of IKDR of mESCs. Abbreviations: 4-AP, 4-aminopyridine; IBTX, iberiotoxin; mESC, mouse embryonic stem cell; TEA, tetraethylammonium.
Although voltage-gated Na+ (Nav) and Ca2+ (Cav) currents were completely absent in all pluripotent mESCs tested (n > 200), whether IKDR was present (Fig. 1B) or not (Fig. 2A), a modest yet detectable hyperpolarization-activated inward current (Ih, encoded by the hyperpolarization-activated cyclic nucleotide-modulated nonselective or HCN ion channel family ; –2.2 ± 0.4 pA/pF at –120 mV) was detected in 79 of 270 cells (29.3%; Fig. 2B). Ih in mESCs was reversibly blocked by the HCN inhibitor Cs+ . Application of 1 μM isoproterenol altered neither the kinetics nor amplitude of Ih recorded in mESCs. Inwardly rectifying K currents (IK1) responsible for stabilizing the resting membrane potential were also not present.
Figure 2. (A): Stimulation protocol and representative current traces demonstrating the absence of Nav or Cav currents in an mESC that lacks IKDR. (B): CsCl2-sensitive hyperpolarization-activated currents could be recorded from pluripotent mESCs but not hESCs. (C): Steady-state current-voltage relationships of hyperpolarization-activated currents of mESCs and hESCs. Abbreviations: hESC, human embryonic stem cell; mESC, mouse embryonic stem cell.
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 Ih, 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 . However, neither TEA-subtracted nor 4-AP–subtracted current traces from IKDR 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.
Figure 3. (A): Expression of ion channel transcripts in mESCs probed by semiquantitative reverse transcription–polymerase chain reaction. Dose–response relationships of (B) TEA, (C) 4-AP, and (D) IBTX for inhibition of mESC proliferation assessed by BrdU incorporation (open squares) and cytotoxic effects by MTT (solid squares). Abbreviations: 4-AP, 4-aminopyridine; BrdU, bromodeoxyurindine; IBTX, iberiotoxin; mESC, mouse embryonic stem cell; TEA, tetra-ethylammonium.
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 . 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 IKDR 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 IKDR (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 . 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 . Similar to mESCs, TEA+-sensitive IKDR (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 Ih in all hESCs tested (n = 30; Fig. 2B). Same as mESCs, neither Nav nor Cav currents could be detected in hESCs.
Figure 4. (A): Pluripotent hESCs were positive for alkaline phosphatase, SSEA-4, and TRA-1-60. (B): Representative current tracings recorded from undifferentiated hESCs. The same electrophysiological from Figure 1 was used. (C): Steady-state current-voltage relationship of IKDR in hESCs. (D): IC50 for blockade of IKDR and EC50 for proliferation inhibition by tetraethylammonium in hESCs. Abbreviation: hESC, human embryonic stem cell.
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 Cav, Nav, or Kv 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. . Our data indicate that among the total 104 voltage-gated ion channel genes mentioned above, only the transcripts of 3, 1, and 5 Cav, Nav, and Kv genes were significantly expressed, as defined by Affymetrix. The corresponding gene products were CACNA1A (Cav2.1), CACNA2D2 (Cav 2/ subunit 2), CACNB3 (Cav ?3 subunit), SCN11A (Nav1.9), KCNB1 (Kv2.1), KCND2 (Kv4.2), KCNQ2 (Kv7.2), KCNS3 (Kv9.3), and KCNH2 (Kv11.1) (note, however, that ICa, INa, 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 and the rapid component of the cardiac delayed rectifier (IKr) , were relatively highly expressed. Similarly, KCNB1 and KCNS3, which encode for the delayed rectifier Kv2.1 channels and the silent modulatory -subunit Kv9.3 that heteromerizes with Kv2.1 subunits , respectively, were also expressed. Collectively, these ion channel genes could underlie the KDR 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).
Figure 5. (A): Microarray analysis of pluripotent hESCs for the transcript expression of all genes tested using Affymetrix U133A microarrays (see Materials and Methods). (B): Left, 104 voltage-gated Cav, Nav, and Kv channel genes are clustered. The same expression scale bar shown in (A) was used. Right, same as the left panel, except only transcripts that were defined to be expressed, as defined by Affymetrix, are shown. (C): Bar graph of normalized transcript levels of the expressed ion channel genes. Data were normalized to the expression level of the 50th percentile of the entire microarray. (D): Expression of ion channel transcripts in hESCs probed by semiquantitative reverse transcription–polymerase chain reaction. Abbreviation: hESC, human embryonic stem cell.
DISCUSSION
K.W. and T.X. contributed equally to this study. This work was supported by grants from the National Institutes of Health (R01 HL-52768 and R01 HL-72857 to R.A.L.), the Blaustein Pain Research Centre (to R.A.L.), and the Hong Kong Research Grant Council (HKU 7459/04M to H.F.T., R.A.L., G.R.L., and C.P.L.). S.Y.T. was supported by a postdoctoral fellowship award from the Croucher Foundation. We would also like to thank Dr. Guibin Chen at Johns Hopkins for helpful discussion in microarray analysis.
DISCLOSURES
The authors indicate no potential conflicts of interest.
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