Gap Junctions and Connexon Hemichannels in Human Embryonic Stem Cells - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:04 |只看该作者 |倒序浏览 |打印

作者：James E. Huettnera, Aiwu Lua,b,c,d, Yun Qub,c,d, Yingji Wub,c,d, Mijeong Kima, John W. McDonaldb,c,d,e $ i" O6 D5 }! S

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      【摘要】
   Correspondence: James E. Huettner, Ph.D., Dept. of Cell Biology & Physiology, Washington University Medical School, 660 South Euclid Avenue (Campus Box 8228), St. Louis, Missouri 63110, USA. Telephone: 314-362-6624; Fax: 314-362-7463; e-mail: huettner@cellbio.wustl.edu or John W. McDonald III, M.D., Ph.D., Kennedy Krieger Institute, International Center for Spinal Cord Injury, 707 North Broadway, Room 518, Baltimore, Maryland 21205, USA. Telephone: 443-923-9210; Fax: 443-923-9215; e-mail: mcdonaldj@kennedykrieger.org
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Intercellular communication via gap junctions is thought to play an important role in embryonic cell survival and differentiation. Classical studies demonstrated both dye and electrical coupling of cells in the inner cell mass of mouse embryos, as well as the development of restrictions against coupling between cells of the inner cell mass and surrounding trophectoderm. Here we demonstrate extensive gap junctional communication between human embryonic stem (ES) cells, the pluripotent cells isolated from the inner cell mass of preimplantation blastocysts. Human ES cells maintained in vitro expressed RNA for 18 of the 20 known connexins; only connexin 40.1 (Cx40.1) and Cx50 were not detected by reverse transcription-polymerase chain reaction. Cx40, Cx43, and Cx45 were visualized by immunofluorescence at points of contact between adjacent cells. Electron microscopy confirmed that neighboring cells formed zones of tight membrane apposition characteristic of gap junctions. Fluorescent dye injections demonstrated extensive coupling within human ES cell colonies growing on mouse embryonic fibroblast (MEF) feeder cells, whereas dye coupling between human ES cells and adjacent MEFs was extremely rare. Physiological recordings demonstrated electrical and dye coupling between human ES cells in feeder-free monolayers and between isolated human ES cell pairs. Octanol, 18--glycyrrhetinic acid, and arylaminobenzoates inhibited transjunctional currents. Dye uptake studies on human ES cell monolayers and recordings from solitary human ES cells gave evidence for the surface expression of connexon hemichannels. Human ES cells provide a unique system for the study of human connexin proteins and their potential functions in cellular differentiation and the maintenance of pluripotency.
      【关键词】 Connexin Dye coupling Electron microscopy Patch clamp Reverse transcription-polymerase chain reaction2 p, X: {* I( S6 ~6 b( o: A
   INTRODUCTION

Gap junction channels mediate electrical and biochemical communication in a wide variety of somatic cells and tissues .$ y+ r  F( y) {0 g, m# d+ e

More than 20 different connexin (Cx) subunits that contribute to vertebrate gap junction channels have been cloned and characterized .

In rodents, expression of zygotic connexin genes, including cx31, cx40, and cx43, begins as early as the two- to four-cell stage . These patterns of communication are thought to be important in the regulation of embryonic development, but identifying the salient molecules exchanged through gap junctions early in development has proven difficult." _9 g# P3 p5 n6 L7 X8 A

Pluripotent mammalian embryonic stem (ES) cells, which are derived from the inner cell mass of preimplantation blastocysts, have the capacity to differentiate into cells of all three germ layers  ES cells have appeared, the physiological properties of human ES cell gap junctions have not been characterized. Moreover, the roles that gap junctions play in the process of differentiation remain poorly understood.

In the present study, we demonstrate the formation of gap junction channels between human ES cells maintained in vitro. Our results show that human ES cells express RNA encoding most of the known human connexin genes. Electrical and dye coupling is prominent in human ES cell colonies and monolayers and between individual pairs of cells. In addition, human ES cells exhibit currents and dye loading from calcium-free solutions that are characteristic of connexon hemichannels present on the surface membrane. Thus, human ES cells provide a useful system for studying the role of human gap junctional communication during development and cellular differentiation." i6 O0 C2 f0 P

MATERIALS AND METHODS
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Mouse Embryonic Fibroblast Feeder Layer' D2 l. q' P' n

Mouse embryonic fibroblast (MEF) feeder layers were prepared in a modification of previously published protocols . Briefly, pregnant CD1 strain mice were sacrificed by CO2 asphyxiation. Male and female embryos were dissected and eviscerated under sterile conditions. The tissue was minced and triturated in 0.25% trypsin/EDTA (Gibco, Grand Island, NY, http://www.invitrogen.com) to a single-cell suspension and plated at a density of 0.5¨C1.0 embryo equivalents per 75-cm2 flask containing Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) (HyClone, Logan, UT, http://www.hyclone.com), supplemented with 2 mM glutamine, 0.1 mM nonessential amino acids (Gibco), and 10% fetal bovine serum (FBS) (HyClone). Medium was changed every other day. MEFs were harvested on day 3 by trypsinization and frozen as stocks using dimethyl sulfoxide (DMSO). Frozen stocks were thawed and plated in 75-cm2 flasks in complete medium at a density of 1.0 x 106 ml¨C1. Cell division was inhibited by the addition of 10 µg ml¨C1 mitomycin C (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 2.5 hours on day 3. Cells were washed, retrypsinized to a single-cell suspension, and replated at a density of 1.0 x 106 cells ml¨C1 in 35-mm dishes or 24-well plates. Human ES cells were plated onto MEFs during the succeeding 2 days to 2 weeks.  T8 p, X3 v% U+ e5 o7 G( D3 A

Human ES Cell Culture1 y) v7 W; _* e: |* U/ V

Two lines of human ES cells were used in completing these studies: one donated by BresaGen (NIH code, BG01; provider¡¯s code, human ESBGN.01; passage 42; BresaGen, Athens, GA) and the other purchased from WiCell Research Institute (NIH code, WA01; provider¡¯s code, H1; passage 24; WiCell Research Institute, Madison, WI, http://www.wicell.org) and maintained according to the providers¡¯ protocols .( R# X' b. h4 k, F. }* P1 [
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Chromosome Counts
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Chromosome counts of cell line BG01 were performed between passage 50 and passage 72. The cells were grown to 70% confluence in a 25-cm flask and fed with medium containing colcemide (final concentration, 0.02 µg ml¨C1) 1 hour prior to analysis. Cells were washed with PBS, trypsinized for 5 minutes, and collected by centrifugation (70g, 3 minutes) in a conical tube. Cells were incubated for 6 minutes at room temperature in 5 ml of hypotonic (0.56%) KCl and then fixed by five changes of methanol/acetic acid (3:1; total volume, 1 ml). To create cell spreads, the cell suspension was dropped from at least 1 foot above the surface of the slide and allowed to air dry. Slides were stained with Giemsa for at least 15 minutes, washed with running water for 1 minute, and air-dried. Chromosome spreads were photographed, and representative photos were used to provide chromosomal counts.
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Reverse Transcription-Polymerase Chain Reaction8 K& Y( }( N9 m$ M3 k2 r) ]

Total RNA was prepared from human ES cells and from MEF cultures using the RNeasy mini kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Small amounts of contaminating DNA were removed by treatment of the RNA samples with RNase-free DNase I (Promega, Madison, WI, http://www.promega.com) according to the manufacturer¡¯s instructions. Two micrograms of total RNA was used to prepare single-strand cDNA. Omniscript reverse transcriptase (Qiagen) was used for reverse transcription following the manufacturer¡¯s instructions. Polymerase chain reactions (PCRs) were performed according to Di et al. , with some modification using HotStarTaq DNA polymerase (Qiagen). Primers for PCR, which are listed in Table 1, were chosen using the PrimerSelect module of Lasergene (DNAStar, Madison, WI, http://www.dnastar.com) based on their specificity among homologous human connexins and their selectivity for human versus mouse connexin sequences. The PCR program was 94¡ãC for 15 minutes to activate the dormant polymerase; followed by 32¨C36 cycles of 94¡ãC for 1 minute, 60¡ãC for 1 minute (55¡ãC for Cx59 and Cx62), and 72¡ãC for 1 minute; and completed with a final extension of 72¡ãC for 10 minutes. As all paired primers lie within a single exon (most connexins are encoded in a single exon), a negative control for each reverse transcription-polymerase chain reaction (RT-PCR) was run using the same conditions but without reverse transcriptase to eliminate the possibility of genomic DNA contamination. In a 50-µl reaction mixture, 2 µl of 1 ng/µl human placenta DNA (Sigma-Aldrich) was used as a positive control. All primer pairs were also tested in PCRs with mouse genomic DNA (not shown) and MEF cell cDNA to rule out spurious amplification of mouse connexin sequences. RT-PCR products were separated on 1.5% agarose gels. The gels were stained with ethidium bromide and visualized by UV transillumination (Spectroline, Westbury, NY, http://www.spectroline.com). PCR products amplified from human ES cell cDNA were cut from the gel and partially sequenced to verify their identity.

Table 1. Primers for reverse transcription-polymerase chain reaction; q! ?+ L$ q! N: [0 v( M5 `3 j1 G
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Immunofluorescence8 j8 ~' M+ W& ]  U/ Y" R' e4 y* t" K

To visualize connexins, human ES cell cultures were washed and fixed with 4% paraformaldehyde plus 0.025% glutaraldehyde in 0.12 M sodium phosphate buffer, pH 7.4. Cultures were rinsed with PBS, permeabilized in 0.1% Triton X-100, and blocked in 10% normal goat serum in PBS. Cultures were incubated for 1 hour at room temperature or overnight at 4¡ãC with primary antibodies (Table 2) anti-Cx40 and Cx43 at 1:100 dilution and anti-Cx45  at 1:1,000 dilution. Cultures were then washed in PBS and incubated for 1 hour at room temperature with Cy3-conjugated goat anti-rabbit immunoglobulin at 1:100 dilution. Cell nuclei were counterstained with Hoechst 33342. Specimens were imaged using a Nikon microscope equipped with differential interference contrast microscopy optics, fluorescence epi-illumination, a cooled charge-coupled device camera (Cooke Corp. Ltd., Romulus, MI, http://www.cookecorp.com), and Metamorph acquisition software.! K7 c* e0 G7 f. y3 R

Table 2. Antibody antigen characteristics2 @7 J& E) W  V) x) f5 C

For surface antigen immunofluorescence, cells were fixed with 4% paraformaldehyde in 0.12 M sodium phosphate buffer, pH 7.4, blocked with 10% normal goat serum in PBS and incubated at room temperature for 1 hour in primary antibodies at 1:50 dilution (Table 2). Cultures were washed with PBS and incubated at room temperature for 1 hour with 1:100 dilutions of fluorescein isothiocyanate- or Cy3-conjugated goat anti-mouse IgG or IgM, as appropriate (Table 2).

For double immunofluorescence of rabbit anti-Oct3/4 and mouse anti-human nuclear antigen (Table 2), cells were fixed with 4% paraformaldehyde in 0.12 M sodium phosphate, pH 7.4, rinsed with PBS, permeabilized with 0.1% Triton X-100, and blocked with 10% normal goat serum. Cultures were incubated simultaneously with anti-Oct3/4 (1:200) and anti-human nuclear antigen (1:100) for 1 hour at room temperature. Cells were then rinsed and incubated for 1 hour at room temperature in a mixture of Cy2-conjugated goat anti-mouse IgG and Cy3-conjugated goat anti-rabbit immunoglobulin, both at 1:200 final dilution. Preliminary single immunofluorescence control experiments confirmed the specificity of the secondary antibodies and the adequacy of our filter set to prevent spillover between the Cy2 and Cy3 channels (data not shown).6 P+ G$ B6 b: H2 ]6 B9 A

Electron Microscopy% S6 R9 @0 M7 |  L" k
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Feeder-free human ES cells, prepared as described above, were fixed at 5 days in vitro with 2.5% glutaraldehyde in 0.1 M PBS, postfixed in 1% OsO4, block-stained with 1% uranyl acetate, dehydrated in ethanol, and then embedded in polybed 812. Thin sections were cut and stained with uranyl acetate and lead citrate and then examined using a JEOL 100CX transmission electron microscope.

Electrophysiology! \7 [' V" F$ V) {# \3 G4 C

Whole-cell recordings were obtained with borosilicate glass pipettes. Passive properties and resting membrane potentials were evaluated using electrodes filled with (in mM) 140 potassium glucuronate, 5 KCl, 10 EGTA, 5 magnesium-ATP, 1 Tris-GTP, and 10 HEPES, pH adjusted to 7.4 with KOH. The open-tip resistance ranged from 1 to 3 MOhm. The bath was perfused at 1¨C2 ml/minutes with Tyrode¡¯s solution (in mM) 150 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 D-glucose, and 10 HEPES adjusted to pH 7.4 with NaOH. After seal formation in the Tyrode¡¯s bath, a wide-bore (300 µm) multichannel delivery pipette was positioned to provide local perfusion of various external solutions. Currents were recorded with Axoclamp 200B and/or 200A amplifiers (Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com), filtered at 1¨C5 KHz with the internal Bessell filter of the clamp, and digitized at 10¨C50 KHz. In-house software controlled the voltage commands and data acquisition to disc. Recordings were accepted for analysis if the seal resistance before breakthrough exceeded 1 GOhm. Immediately after breaking through to the whole-cell configuration the input resistance, series resistance and membrane capacitance were determined from the average of currents recorded during six hyperpolarizing voltage steps. In addition to the potassium glucuronate intracellular and Tyrode¡¯s extracellular solutions described above, permeation through surface hemichannels was evaluated using internal and external solutions of the following compositions.$ q8 s7 Q, H! U3 }9 ~
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Internal Solutions. Cesium glucuronate: 140 mM cesium glucuronate, 5 mM CsCl, 5 mM magnesium-ATP, 1 mM Tris-GTP, 10 mM EGTA, 10 mM HEPES; pH adjusted to 7.4 with CsOH.

N-Methyl-D-glucamine chloride: 140 mM N-methyl-D-glucamine (NMDG) chloride, 5 mM magnesium-ATP, 1 mM Tris-GTP, 0.1 mM EGTA, 10 mM HEPES; pH adjusted to 7.4 with N-methyl-D-glucamine.
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Tetraethylammonium chloride: 120 mM tetraethylammonium (TEA) chloride, 5 mM MgCl2, 5 mM magnesium-ATP, 1 mM Tris-GTP, 10 mM EGTA, 10 mM HEPES; pH adjusted to 7.4 with TEA hydroxide.

External Solutions. NaCl: 160 mM NaCl, 1 mM calcium glucuronate, 10 mM HEPES; pH adjusted to 7.4 with NaOH.. `) S" s# |1 C4 X

Sodium glucuronate: 160 mM sodium glucuronate, 1 mM calcium glucuronate, 10 mM HEPES; pH adjusted to 7.4 with NaOH.3 T( I* W- s% I* j
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NMDG-chloride: 160 mM N-methyl-D-glucamine chloride, 1 mM Ca-glucuronate, 10 mM HEPES; pH adjusted to 7.4 with N-methyl-D-glucamine.

NMDG-glucuronate: 160 mM N-methyl-D-glucamine d-glucuronate, 1 mM Ca-glucuronate, 10 mM HEPES; pH adjusted to 7.4 with N-methyl-D-glucamine.

Calcium-free: 160 mM NaCl, 0.5 mM EGTA, 10 mM HEPES; pH adjusted to 7.4 with NaOH.
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In these solutions, small inorganic cations (sodium and cesium) or anions (chloride) are replaced by larger organic cations (TEA, NMDG) or anions (glucuronate), which should pass through the large diameter connexon pore but be impermeable through most conventional voltage-gated ion channels . Mean ¡À SEM values are reported for all measured parameters. Statistical significance was assigned for p 6 y6 }" k* G: h8 H

Dye Injections

Whole-cell electrodes were used to inject individual cells with fluorescent dyes dissolved in the same internal solution used for electrophysiology experiments. Initially sulforhodamine B (555 Da; Sigma-Aldrich) or Lucifer Yellow CH (443 Da; Sigma-Aldrich) were injected at 0.4%; however, most experiments used aldehyde-fixable AlexaFluor hydrazides. AlexaFluor 488 (570 Da) and 568 (731 Da) hydrazides were purchased as 10 mM solutions in 200 mM KCl (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) and were diluted 1:100 into internal solution. For some experiments, AlexaFluor 568 hydrazide was co-injected with Lucifer Yellow coupled to 10 kDa anionic dextran, which is too large to pass through gap junction channels and therefore remains within the injected cell. Cells injected with fluorescent hydrazides were imaged live and in some cases were also fixed for subsequent immunofluorescence. Cultures were fixed in 0.12 M sodium phosphate buffer, pH 7.4, containing 4% paraformaldehyde alone to preserve antigenicity for Oct3/4 and the human nuclear antigen, although our preliminary experiments revealed superior retention of fluorescent AlexaFluor hydrazides by cells that were fixed with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde.
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Pharmacological Agents

Compounds previously demonstrated to inhibit electrical or dye coupling between cells , were purchased from Sigma-Aldrich and prepared as 50 mM stocks in DMSO. Addition of 0.2% DMSO alone was found to have no effect on currents through gap junctions or hemichannels.; `) a: u8 p8 R* [4 a* A. M$ A
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RESULTS8 p' Y; N8 \+ u, M4 h* e( K

Undifferentiated human ES cells were maintained on MEFs (Fig. 1A) and were also grown independent of feeder cells using a modification of previously described protocols , which allowed for unambiguous discrimination between human ES cells and any contaminating mouse cells. As illustrated in Figure 1G¨C1J, strong nuclear expression of Oct3/4 was maintained in human ES cells but was not observed in the rare MEFs that survived through initial passages to feeder-free dishes.

Figure 1. Human embryonic stem (ES) cells in culture. (A): Differential interference contrast (DIC) microscopy image of human ES cells clustered on a mouse embryonic fibroblast (MEF) feeder layer. (B): Phase contrast image of a feeder-free human ES cell monolayer. (C): Interphase chromosome spreads from feeder-free human ES cells after 56 total passages, the final four passages under feeder-free growth conditions. (D¨CF): Immunofluorescent visualization of stage-specific embryonic antigen-4 (green) (D), TRA-1-81 (red) (E), and TRA-1-60 (red) (F) antigens in feeder-free human ES cells monolayers. (G¨CJ): Double immunofluorescence for Oct3/4 (red) (G) and human nuclear antigen (green) (H) together with Hoechst 33342 nuclear counterstaining (blue) (I), which reveals an MEF nucleus (arrow). (J): Composite of (G¨CI) with the DIC image of the same field. Scale bars = (A), 30 µm; (B), 10 µm; (C), 3 µm; (D), 10 µm; and (E, F, G¨CJ), 30 µm./ ^1 z; i6 |# {& `) |' I
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Connexin Expression in Human ES Cells: C1 |; a: ~  o/ m
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As an initial screen for the production of gap junction constituents by human ES cells, we evaluated expression of connexin mRNA by RT-PCR (Fig. 2). In replicate experiments from four separate platings, PCR products were observed for nearly all of the known human connexins (Table 1). Only Cx40.1 and Cx50 were not detected in any samples from undifferentiated human ES cell. Strong PCR products were observed for Cx25, Cx26, Cx30.3, Cx31, Cx31.1, Cx31.9, Cx37, Cx40, Cx43, Cx45, and Cx46 for all of the undifferentiated human ES cultures that were sampled. The remaining connexins (Cx30, Cx30.2, Cx32, Cx36, Cx47, Cx59, and Cx62) were also reliably detected in undifferentiated human ES cells but yielded weaker PCR product bands in some of the replicate experiments.& |- J0 e9 u" b( V% M
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Figure 2. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis of connexin expression in undifferentiated human embryonic stem (ES) cells. Experiments were performed as outlined in Materials and Methods using PCR primers in Table 1. For each connexin, four lanes were run in the following order: lane 1, PCR using reverse-transcribed mouse embryonic fibroblast (MEF) cDNA; lane 2, PCR of the RT-negative control in which DNase-treated total RNA from human ES cells was used without reverse transcriptase to rule out the possibility of genomic DNA contamination; lane 3, PCR using reverse-transcribed human ES cell cDNA; lane 4, PCR using human placenta genomic DNA as a positive control. PCR products were detected for all 20 of the positive controls but not for any of the MEF cDNA or RT-negative control lanes. Abbreviations: bp, base pairs; L, 100-base pair DNA size ladder.

To test for production of connexin proteins and to visualize their subcellular distribution, we performed immunofluorescence on human ES cell monolayers (Fig. 3A¨C3C). Antibodies to Cx40, Cx43, and Cx45 , were confirmed by transmission electron microscopy (Fig. 3D¨C3F). The density of tight membrane appositions was greater in samples from near-confluent high-density cultures than in similar samples from low-density cultures.

Figure 3. Gap junction distribution and function in human embryonic stem (ES) cells. Distribution of Cx40 (A), Cx43 (B), and Cx45 (C) in human ES cells visualized by immunofluorescence (red). Nuclei counterstained with Hoechst dye (blue). (D¨CF): Transmission electron micrographs of human ES cells. Arrows indicate regions of close membrane apposition characteristic of gap junctions. (E): Higher power view of the region boxed in (D). (G): Dye coupling between human ES cells revealed by spread from the cell indicated by the asterisk, which was injected with sulforhodamine B through a whole-cell electrode. Scale bars = (A¨CC), 20 µm; (D, F), 200 nm; (E), 100 nm; and (G), 30 µm.
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Physiology and Dye Coupling of Human ES Cells

To characterize the physiology of human ES cells and to provide an initial test for gap junction-mediated communication between cells, we obtained whole-cell recordings from 33 human ES cells grown in confluent monolayers. Immediately after attaining the whole cell configuration, the average input resistance was 103 ¡À 22 MOhm, and the membrane capacitance was 304 ¡À 102 pF. With internal potassium glucuronate and external Tyrode¡¯s solution, the mean zero current potential was ¨C26 ¡À 1.7 mV. The low input resistance and high membrane capacitance values are consistent with electrical coupling mediated by gap junctions among cells in the monolayer. Because many gap junctions permit the passage of molecules smaller than 1,000 Da, we initially tested for gap junction-mediated dye transfer by including sulforhodamine B (557 Da) within our recording electrodes. As illustrated in Figure 3G, sulforhodamine B readily spread from the recorded cell into neighboring cells of the monolayer. Comparable results were obtained with electrodes that contained Lucifer yellow CH (443 Da; data not shown). To confirm that dye transfer was restricted to low molecular weight compounds, we injected a mixture of low (Alexafluor hydrazide 568; 731 Da) and high (Lucifer Yellow coupled to Dextran; 10 kDa) molecular weight probes into human ES cells grown as monolayers. The low molecular weight Alexafluor hydrazide passed freely to neighboring cells (Fig. 4A), whereas fluorescent dextran was retained within the injected cell (n = 15).

Figure 4. Lack of dye coupling between human embryonic stem (ES) cells and mouse feeder cells. (A): AlexaFluor 568 hydrazide (731 Da; red) delivered into a single human ES cell (asterisk) quickly spread to neighboring cells, whereas coinjected 10-kDa Lucifer Yellow-dextran (yellow) remained within the injected cell. (B): In the same field of view, AlexaFluor 488 hydrazide delivered subsequently into a single mouse embryonic fibroblast (MEF) (arrow) quickly spread to other MEFs but not to the adjacent human ES cells. (C): Composite of (A) and (B) with the differential interference contrast (DIC) microscopy image of the same field. (D, E): Double immunofluorescence for Oct3/4 (D) and human nuclear antigen (E) in the same field as (A¨CC). (F): Composite of (D) and (E) with the DIC image. (G): AlexaFluor 568 hydrazide (red) was injected into a single human ES cell (asterisk); AlexaFluor 488 hydrazide (green) was injected into a nearby MEF (arrow). (H): Composite of (G) and the DIC image of the same field. (I¨CL): Subsequent double immunofluorescence for Oct3/4 and human nuclear antigen. Composite views show the same field as (G) and (H) with (I) or without (L) the DIC image. (M): AlexaFluor 488 hydrazide (green) was injected into a single MEF (arrow), and AlexaFluor 568 hydrazide (red) was injected into a single human ES cell within a colony. This is the only case that displayed convincing dye spread between the human ES cells and adjacent MEF cells (arrowhead). (N): Composite of (M) with the DIC image of the same field. Scale bars = 30 um.  |* L+ B4 c: a& r5 i: ~

The observation of strong dye coupling between human ES cells under feeder-free conditions led us to test for coupling between human ES cells and adjacent MEF feeder cells. To perform these experiments, individual human ES cells were injected with Alexafluor hydrazide 568 (Fig. 4A, 4C), and single nearby MEFs were injected with Alexafluor hydrazide 488 (570 Da; Fig. 4B, 4C). For some experiments, cultures were fixed after dye injection, and cell identity was subsequently verified by immunofluorescence for Oct3/4 and human nuclear antigen (Fig. 4D¨C4F, 4I¨C4L). In all cases (n = 16), human-to-human and mouse-to-mouse dye transfer was observed; however, only one clear example was obtained of dye spread from human ES cells to adjacent mouse cells (Fig. 4M, 4N). These experiments confirm strong gap junction-mediated coupling between undifferentiated human ES cells. In contrast, human ES cell coupling to mouse fibroblast feeder cells is extremely rare and is unlikely to be essential for maintenance of human ES cell pluripotency.
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Recordings from Coupled Cell Pairs' P( r9 ~3 j# \: I' L9 V( W

To evaluate cell-to-cell coupling directly , we passaged human ES cells at lower density and used two electrodes to record simultaneously from both cells in isolated pairs that were in contact (Fig. 5). Both of the cells were initially clamped to 0 mV. Currents through gap junctions connecting the two cells were recorded by stepping the potential in one cell to values positive and negative to 0 mV, while maintaining the second cell clamped at 0 mV. This procedure was then repeated with the first cell held at 0 mV and the second cells stepped to different potentials. Such experiments yielded transjunctional conductance values that ranged from 2.7 to 79 nS, with a mean of 20 ¡À 5 nS (n = 15 cell pairs). In all cases, the junctional current reversed polarity at zero transjunctional potential, and there was good agreement in the junctional conductance as determined for steps applied to either of the two cells (Fig. 5E). Linear regression yielded a correlation coefficient of r = .996. Current voltage relations for whole cell current and for transjunctional current were relatively linear for transjunctional voltages that ranged from ¨C30 to  30 MV.
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Figure 5. Transjunctional currents recorded from human embryonic stem (ES) cell pairs. (A): Whole-cell currents recorded simultaneously from two adjacent human ES cells. Currents were elicited in both cells by voltage steps from ¨C30 to  30 mV delivered either to cell 1 (traces on the left) or to cell 2 (traces on the right). (B): Plots of current recorded in cell 1 (circles) or cell 2 (squares) as a function of test potential. Filled symbols indicate that the cell was clamped to the test potential, and open symbols indicate that the cell was clamped to 0 mV. (C): Transjunctional current as a function of transjunctional potential. (D): Differential interference contrast and fluorescence images of an isolated cell pair that was studied by simultaneous whole-cell recording. Sulforhodamine B was included in the electrode used for the cell on the left. Scale bar = 30 µm. (E): Transjunctional conductance as determined by voltage steps applied alternately to both cells of 15 human ES cell pairs. Abbreviation: GJ, gap junction.

Pharmacology of Transjunctional Currents& L0 @/ [& Q5 T: N9 t

A number of compounds, including 18--glycyrrhetinic acid, long-chain alcohols (e.g., octanol and heptanol), and arylaminobenzoates (e.g., flufenamic acid and niflumic acid), have been shown to reduce cell-to-cell coupling mediated by gap junctions. Although some of these drugs are not entirely specific for gap junction channels . Inhibition of current flow by these compounds, together with the selective passage of low molecular weight dyes described above (Fig. 4A), provides the strongest evidence that the low resistance connections between adjacent cells are formed by gap junctions, rather than by incomplete mitosis or some other type of cytoplasmic continuity.: M  S+ t& Z4 O! Y

Figure 6. Pharmacology of transjunctional currents recorded from human embryonic stem (ES) cell pairs. (A): Transjunctional conductance plotted as a function of time. Period of exposure to 100 µM AGA is indicated by the bar. Conductance was determined from currents evoked when one cell was stepped to  40 mV while the other cell was held at 0 mV. (B): Time course of inhibition of transjunctional conductance by 1 mM octanol. (C): Mean ¡À SEM percent inhibition of transjunctional conductance produced by 1 mM OCT, 100 µM FFA, 100 µM AGA, 100 µM NFA, 100 µM La, or 2 mM Co. (D): Time course of inhibition of transjunctional conductance by 100 µM FFA. Note: Relative to (A), the time scales are expanded threefold and fivefold in (B) and (D), respectively. Abbreviations: AGA, 18 -glycyrrhetinic acid; Co, cobalt chloride; FFA, flufenamic acid; La, lanthanum chloride; NFA, niflumic acid; OCT, octanol.# g9 w- H& K2 y$ M* E; z' t

Connexon-Mediated Currents in Isolated Human ES Cells/ A# r6 C! |5 o

In a number of reports, individual isolated cells that are capable of forming gap junctions have been shown to display voltage-dependent hemichannel currents mediated by connexons that have not paired up with a partner on an adjacent cell. Examples include horizontal cells isolated from catfish .
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To test for the presence of functional hemichannels, we recorded from isolated human ES cells. In contrast to human ES cells growing in monolayers, which display low input resistance owing to the strong coupling to neighboring cells, the isolated human ES cells (n = 31) had significantly higher input resistance (717 ¡À 111 MOhm, p 5 K+ V4 |: _2 \
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Figure 7. Connexon hemichannel-mediated currents in isolated human embryonic stem (ES) cells. (A): Whole-cell currents elicited in an isolated cell by voltage steps from a holding potential of ¨C80 mV to test potentials of ¨C30 to  80 mV. (B): Plots of instantaneous (open symbols) and steady-state (filled symbols) currents as a function of test potential. (C): Plot of inward tail current recorded at ¨C80 mV (inverted) as a function of the preceding test potential. Smooth curve: best fit of the Boltzmann equation in the form: I = B/(1 exp (¨CA x (Vm ¨CVo))) C, where I is the recorded current, Vm is the membrane test potential, and Vo, A, B, and C are fitted parameters. Vo is the potential at half maximal conductance ( 55 mV), B and C are the maximal and minimal conductance, respectively, and A can be expressed as zq/kT, where z (best fit = 2.5) is the valence of charge q, k is Boltzmann¡¯s constant, and T is the temperature in kelvin.; g: C* R/ k! J

In a few cells, the currents activated and decayed along a single exponential time course; however, in most cases, two or more exponentials were required for an adequate fit to activation and deactivation. Moreover, in a number of cells, the activation and deactivation rates exhibited dramatic dependence on the duration that the cell was held at activating or deactivating voltages, which suggests the existence of multiple slowly equilibrating closed and open states. Similar dependence of kinetic parameters on the frequency of stimulation has been reported for recombinant Cx46 hemichannels expressed in Xenopus oocytes .

Brief voltage steps from holding potentials of ¨C80 or  60 mV (Fig. 8A) were used to evaluate the instantaneous current-voltage (I-V) relation for hemichannel-mediated whole-cell currents. As shown in Figure 8B, the instantaneous I-V of open hemichannels was linear and reversed near 0 mV. With external Tyrode¡¯s solution, the whole-cell conductance through surface hemichannels in isolated human ES cells ranged from 3.6 to 90.5 nS, with a mean of 40.2 ¡À 5.6 nS (n = 20 cells). To evaluate the ionic selectivity of channels underlying these currents we substituted large organic cations (NMDG and TEA) or anions (glucuronate) for the small diameter inorganic ions (potassium, cesium, sodium, and chloride) present in our standard intracellular and extracellular solutions. Consistent with the nonselective permeation properties of connexons, we observed relatively linear I-V relations, which reversed between ¨C10 and  10 mV, using a variety of different internal and external solutions (Fig. 8D). These included internal solutions containing potassium glucuronate, cesium glucuronate, NMDG-chloride, or TEA-chloride as the major intracellular salts, and paired with NaCl, NMDG-chloride, sodium glucuronate, or NMDG-glucuronate as the major extracellular salts (Materials and Methods). The linear instantaneous I-V relations, and the lack of any dramatic change in potential at which current reversed polarity, indicate that the large organic ions pass through the channels as readily as the smaller inorganic ions. This stands in contrast to most conventional voltage-gated channels, which typically are selective for smaller inorganic ions .( L1 m8 K* x! A; b5 n% a0 N

Figure 8. Hemichannel current-voltage relations. (A): Whole-cell currents recorded in Tyrode¡¯s solution at holding potentials of ¨C80 and  60 mV. The cell was initially held at ¨C80 mV and briefly (15 ms) stepped six times through a series of test potentials ranging from  100 to ¨C110 mV. The holding potential was jumped to  60 mV for 9 seconds, and the series of test potential steps was repeated six times. Finally, the holding potential was returned to ¨C80 mV, and the test potential steps were applied six more times. The solid line plots the holding current. Currents recorded at the end of each test potential step are superimposed as points. Internal solution: 140 mM NMDG, 10 mM HEPES, 0.1 mM EGTA, pH adjusted to 7.4 with HCl. (B): Current recorded in (A) as a function of test potential for holding potentials of ¨C80 mV (open circles, average of the first six steps to each test potential) and  60 mV (filled circles, average of the last two steps from  60 mV to each test potential). (C): Mean ¡À SEM percent inhibition of hemichannel conductance produced by 1 mM OCT, 100 µM FFA, 100 µM AGA, 100 µM CARB, 100 µM La, or 2 mM Co. (D): Current-voltage relations in extracellular NaCl (open circles), N-methyl-D-glucamine (NMDG) glucuronate (filled circles), NMDG-chloride (filled squares), and sodium-glucuronate (filled triangles). Internal solution: 140 mM NMDG, 10 mM HEPES, 0.1 mM EGTA, pH adjusted to 7.4 with HCl. Abbreviations: AGA, 18 -glycyrrhetinic acid; CARB, carbenoxolone; Co, cobalt chloride; FFA, flufenamic acid; La, lanthanum chloride; NFA, niflumic acid; OCT, octanol.* F: T/ t" r5 c# C: A

Consistent with previous work  have demonstrated that connexons are not all equally sensitive to block by organic gap junction antagonists, which suggests that human ES cell hemichannels may include individual connexins, or connexin combinations, that are relatively resistant to inhibition by the organic blockers.! R3 u* d/ o, f! Y8 e7 L

Evaluation of Junctional Versus Nonjunctional Connexin Distribution in Cell Pairs

It is of interest to ask whether the hemichannel-mediated currents recorded in isolated cells simply reflect the absence of a suitable partner for cell-to-cell junction formation or whether cells that have formed junctions with their neighbors might retain a significant proportion of unpaired connexons on their surface. To evaluate this question, we again recorded from pairs of cells, but both cells were stepped simultaneously to the same positive potential, relative to the bath. This protocol will maintain the transjunctional voltage at 0 mV and hence eliminate junctional current. Out of 22 cells (11 pairs) tested in this way, six cells displayed slowly activating outward currents characteristic of connexon hemichannels, and all six of these cells were paired with a cell that lacked hemichannel-mediated current. In these six cells, the ratio of surface hemichannel conductance to transjunctional conductance ranged from 0.36 to 2.1, with a mean of 1.14 ¡À 0.32 (n = 6 cells). Linear regression for hemichannel conductance and transjunctional conductance yielded a correlation coefficient of r = .742. These results suggest that connexons preferentially combine to form gap junctions, but hemichannels will be present in cells that express an excess of connexons, outnumbering the available partners on neighboring cells., H* X$ [* I9 B- e' o
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Dye Loading Through Surface Membrane Hemichannels
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In addition to depolarizing voltage steps, opening of cell surface hemichannels is favored by removal of extracellular calcium . To determine whether hemichannels were present on the surface of human ES cells in high-density monolayers, we exposed cells to sulforhodamine B or propidium iodide in calcium-free Tyrode¡¯s solution. As demonstrated in Figure 9C¨C9F, a subset of cells contained the dye after a brief incubation in calcium-free, but not in calcium-containing, solutions. As mentioned above, our recordings from cell pairs suggest that hemichannels are present on cells that express a higher number of connexons than their nearest neighbors. Thus, the uneven distribution of dye uptake is likely to reflect the location of cells with a higher relative level of connexin expression than surrounding cells.9 b+ d6 h1 r: ~" t8 H; N
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Figure 9. Conductance and dye loading through surface membrane hemichannels in calcium-free medium. (A): Whole-cell current evoked by calcium-free external solution in an isolated human embryonic stem (ES) cell. The cell was held at ¨C20 mV in NaCl external solution and briefly (40 ms) stepped six times through a series of test potentials from  60 to ¨C100 mV. Calcium-free external solution was applied by local perfusion and the series of test potential steps was repeated, followed by recovery. (B): Current recorded in (A) as a function of test potential in control NaCl (open circles, average of the first six steps to each test potential) and calcium-free (filled circles, average of the last two steps to each test potential) external solution. Undifferentiated human ES cell cultures were exposed to propidium iodide in Tyrode¡¯s solution containing 2 mM calcium (C, E) or 0 mM calcium (D, F) for 15 minutes in the dark, rinsed, and observed immediately. Photomicrographs were taken after brief fixation and incubation with Hoechst 33258. Scale bars = (C, D), 100 µm; (E, F), 30 µm.4 H, Q( ?; j6 _  C8 X

DISCUSSION, \4 R# D/ Q3 `, c' v, U2 Z' ]
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ES cells are derived from the inner cell mass, a cluster of cells that display extensive gap junction-mediated coupling in early human embryos . Our results show that connexin expression and gap junction coupling are maintained in human ES cells propagated in vitro. Recordings from isolated cell pairs provide direct evidence for the formation of functional gap junction communication pathways between human ES cells in culture. Brief steps of transjunctional voltage elicited linear currents between the cells. Conductance across the junctions varied from approximately 3 to approximately 80 ns among the cell pairs sampled. In theory, low resistance coupling might arise as an artifact of incomplete cell division or some other form of cytoplasmic continuity. However, strong confirmation that gap junctions underlie the conductance between adjacent cells comes from the reversible blockade by pharmacological agents known to inhibit gap junction channels and from the failure of high molecular weight fluorescent dextran to spread from injected cells.( P9 N( ?1 Z. q9 i6 G. K

Mammalian embryos express a large number of different connexins. Studies in rodents have reported zygotic expression of transcripts for Cx30, Cx31, Cx31.1, Cx36, Cx40, Cx43, Cx45, and Cx57 , antibodies to Cx40, Cx43, and Cx45 labeled points of contact between adjacent human ES cells. Thus, undifferentiated human ES cells express most of the known connexins at the level of RNA, and at least some of these are translated into protein and delivered to the surface membrane at points of intercellular contact.' K% X, h! L4 c% N2 U
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Cell-to-cell coupling via gap junctions is a common feature of embryonic development, although the role of embryonic cell junctions remains elusive. Direct evidence for electrical coupling among embryonic cells was first described in squid embryos , but there is clearly a change in communication as tissues differentiate in vivo. In human ES cell monolayer cultures, dye injected into individual cells spreads quickly and uniformly to all surrounding cells. Therefore, cultured human ES cells remain extensively coupled through repeated passages, preserving the communication pathways that exist within the inner cell mass. In mixed cultures that included both human ES cells and mouse embryonic fibroblast feeder cells, we observed dye coupling within each population; however, coupling between human ES and MEF cells was extremely rare. These results suggest that preservation of human ES cell pluripotency does not require direct transmission of signals from MEF cells to human ES cells via gap junction channels, but they leave open the possibility that coupling between adjacent human ES cells may play a role in coordinating cellular responses to external signals that maintain pluripotency or that stimulate differentiation.& k3 Z9 M5 e+ z" k/ G& V2 n
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Indeed, the function of embryonic cell-to-cell communication has not been determined . Clearly, the functional implications of connexin expression on human ES cell growth and differentiation merit further investigation.

Previous studies in both native and recombinant systems have documented a number of differences in the properties of gap junctions or hemichannels formed by different connexins, including differences in kinetics, unitary conductance, and permeability for specific dyes . The large number of connexin RNAs expressed in embryos and human ES cells suggests that, in both cases, the cells may produce heteromeric connexons containing two or more distinct connexin subunits. Further work will be needed to quantify the relative connexin protein levels and to determine the stoichiometry of connexins that combine to form connexon hexamers in these cells.
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In general, strong depolarization or reduction in extracellular calcium promotes the opening of surface membrane hemichannels . In medium that contained 1¨C2 mM calcium, currents through human ES cell hemichannels were minimal when cells were maintained at their normal zero current potential between ¨C15 and ¨C30 mV, suggesting that most hemichannels remain closed in cells at rest.

Investigation of human ES cells is still at an early stage. Identifying characteristics of human ES cells are currently limited to the expression of a few relatively specific markers and the ability to differentiate into cells from all three germ layers. The gold standard for proof of an undifferentiated nonhuman embryonic stem cell phenotype is the ability to generate a chimeric animal following injection into the blastocyst inner cell mass. This of course is not a testable option for human ES cells. Our data provide additional ultrastructural, immunocytochemical, and physiological properties that may aid in proper confirmation of undifferentiated human ES cells. In addition to expression of the Oct3/4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 antigens, the expression profile for connexins (positive for Cx25, Cx26, Cx30.3, Cx31, Cx31.1, Cx31.9, Cx37, Cx40, Cx43, Cx45, and Cx46; negative for Cx40.1 and Cx50) and physiological dye coupling can be added to the growing list of human ES cell characteristics in the undifferentiated state. Collectively, our results establish human ES cells as a good model system for the investigation of direct intercellular communication channels during early stages of differentiation.! `* `. B! u' v6 P9 E5 R( x
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DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS! R- Y- g' T4 a) V0 W1 m
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We are grateful to Tim Wilding for technical assistance and Dr. Tom Steinberg for generously providing anti-Cx45 antibodies. This work was supported by grants from the NIH (NS045023 to J.E.H.; NS39577 and NS40520 to J.W.M.), the Sam Schmidt Race for a Cure Foundation, and the Jack Orchard ALS Foundation.
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