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Stem Cells 2004;22:883-889 www.StemCells.com
© 2004 AlphaMed Press

RAPID COMMUNICATION

Presence of Functional Gap Junctions in Human Embryonic Stem Cells

Raymond C.B. Wong, Alice Pébay, Linh T.V. Nguyen, Karen L.L. Koh, Martin F. Pera

Monash Institute of Reproduction and Development, Monash University, Clayton,Australia

Key Words. Human embryonic stem cells • Gap junction • Connexin 43 • Connexin 45

Correspondence: Dr. A. Pébay, Ph.D., Monash Institute of Reproduction and Development, Monash University, 246 Clayton Road, Clayton, VIC 3168,Australia. Telephone: 0061-395947302; Fax: 0061-95947311, e-mail: alice.pebay{at}med.monash.edu.au


   ABSTRACT
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gap junctions are intercellular channels that allow both chemical and electrical signaling between two adjacent cells. Gap junction intercellular communication has been implicated in the regulation of various cellular processes, including cell migration, cell proliferation, cell differentiation, and cell apoptosis. This study aimed to determine the presence and functionality of gap junctions in human embryonic stem cells (hESCs). Using reverse transcription—polymerase chain reaction and immunocytochemistry, we demonstrate that human ES cells express two gap junction proteins, connexin 43 and connexin 45. Western blot analysis revealed the presence of three phosphorylated forms (nonphosphorylated [NP], P1, and P2) of connexin 43, NP being prominent. Moreover, scrape loading/dye transfer assay indicates that human ES cells are coupled through functional gap junctions that are inhibited by protein kinase C activation and extracellular signal-regulated kinase inhibition.


   INTRODUCTION
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Gap junctions are intercellular channels that consist of two hemi-channels, termed connexons, each localized in the membrane of adjacent cells. Each connexon consists of six integral membrane proteins, termed connexins [1]. Gap junction intercellular communication (GJIC) allows cell–cell exchange of inorganic salts and small metabolites of less than ~1 kDa with a maximum diameter of ~1.5 nm [2]. Such intercellular coupling has been implicated in the control of various cellular processes, including intercellular buffering of cytoplasmic ions, electrical synchronization, control of cell migration, cell proliferation, cell differentiation, metabolism, and apoptosis [2–4].

Gap junctions have long been implicated in cellular growth control. Although the molecular mechanisms remain largely unknown, it is generally believed that upregulation of GJIC is associated with inhibition of cellular growth, whereas downregulation of GJIC correlates with stimulation of growth [5]. Most, if not all, cancer cells lack functional GJIC [6]. A recent hypothesis suggests that GJIC deficiency is also a characteristic of stem cells [6–9]. This hypothesis is based on the fact that two types of epithelial adult stem cell, keratinocyte stem cells [10] and corneal epithelial stem cells [11], do not express connexin and are GJIC deficient. Studies on several presumptive adult progenitor cells (including immortalized and transformed cells) also support this hypothesis, because these different cell types also lack functional gap junctions [12–16]. However, gap junctions have not been studied extensively in embryonic stem (ES) cells. It has been reported that connexin 43 and connexin 45 mRNAs are expressed in undifferentiated mouse ES cells [17].

hESCs are pluripotent cells derived from the inner cell mass of in vitro fertilized human blastocysts [18,19]. ES cells can be grown in vitro indefinitely while maintaining a normal karyotype, and throughout long periods of cultivation in vitro they remain pluripotent, processing the ability to develop into multiple cell types representative of all three embryonic germ layers and extra-embryonic tissues [20]. This work aimed to study the expression of connexins in hESCs and to investigate the presence of functional gap junctions within hESC colonies.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All experiments were performed at least 3 times.

Cell Culture
HES-3 and HES-4 were cultured as previously described [21]. Briefly, hESCs were grown in the presence of a mitotically inactivated mouse embryonic fibroblast feeder layer (MEF) in Dulbecco’s modified Eagle medium supplemented with 0.1 mM ß-mercaptoethanol, 1% (vol/vol) nonessential amino acid solution, 1% (vol/vol) insulin/transferring/selenium, 2 mM L-glutamine, 0.25 U/ml penicillin, 0.25 µg/ml streptomycin (all from Invitrogen, Mount Waverley, Australia), and 20% (vol/vol) fetal calf serum (Hyclone, Logan UT). hESCs were kept in a 5% CO2 incubator at 37°C. Medium was changed every 2 days, and cells were passaged by mechanical dissection of colonies under microscopic control followed by dispase treatment every 7 days.

ReverseTranscription–Polymerase Chain Reaction
mRNA was extracted from HES-3 and HES-4 cells and reverse transcribed as previously described [19]. The cDNA samples were amplified by polymerase chain reaction (PCR) with sense and anti-sense primers (Sigma, Castle Hill, Australia) designed for the specific detection of human DNA target sequences using 0.25 U Taq DNA polymerase (Biotech International Ltd., Perth,Australia) and 2 mM of each primer in a buffer including 67 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 16.6 mM [NH4]2SO4, 0.45% Triton X-100, and 0.25 mM deoxynucleotide triphosphate. The primer sequences were as follows: connexin 43, sense 5'-ATGAGCAGTCT-GCCTTTCGT-3' and anti-sense 5'-TCTGCTTCAAGTG-CATGTCC-3'; connexin 45, sense 5'-GGAAGATGGGCT-CATGAAAA-3' and anti-sense 5'-GCAAAGGCCTGTAAC ACCAT-3'. PCR was performed under the following conditions: one cycle of initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 2 minutes, extension at 74°C for 2 minutes, ending with a final incubation at 74°C for 7 minutes. The amplified DNA fragments were sized by electrophoresis on 2% (wt/vol) agarose gel and stained with ethidium bromide. Molecular sizes (bp) were calculated using 1 kb plus DNA ladder markers. The products were purified and sequenced.

Immunocytochemistry
Cells were fixed in 100% ethanol for 5 minutes, air dried, and incubated in antibodies and reagents in the following sequences: rabbit polyclonal antibody against connexin 43 or connexin 45 (both from Chemicon, Temecula, CA), anti-rabbit secondary antibody conjugated with fluorescein isothiocyanate (Dako, Glostrup, Denmark), rabbit immunoglobulin G–blocking antibody (Dako), Germ Cell Tumor Monoclonal-2 (GCTM-2, this laboratory), and biotinylated anti-mouse immunoglobulin M and streptavidin Texas red (Amersham, Piscataway, NJ). Nuclei were counterstained with Hoechst-33342 (Chemicon). Specificity was verified by the absence of any staining in the negative controls (no addition of the primary antibody).

Western Blot Analysis
HES-3 cells were incubated with dispase (10 mg/ml, Roche, Mannheim, Germany) for 10 minutes to release the colonies intact from the culture dish with minimal feeder cell contamination and were lysed by addition of a reducing loading buffer in Laemmli sample buffer containing ß-mercaptoethanol. Protein separation, transfer, and immunoblotting were carried out as previously described [22] using a rabbit polyclonal antibody against connexin 43. Peroxidase-coupled secondary antibodies (Dako) were detected by exposure of autoradiographic films in the presence of a chemiluminescent detection reagent (ECL,Amersham).

Scrape Loading/Dye Transfer Assay
GJIC was determined by the scrape loading/dye transfer assay as described previously [8]. Briefly, the cells were washed three times in a prewarmed Ca2+Mg2+ phosphate-buffered saline (PBS) buffer (140 mM NaCl, 5.5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES [Invitrogen], pH 7.35). In Ca2+Mg2+-free experiments, the colonies were then washed with a prewarmed Ca2+Mg2+-free buffer (140 mM NaCl, 5.5 mM KCl, 10 mM glucose, 10 mM HEPES, and 2 mM EGTA, pH 7.35). In some experiments, cells were treated with phorbol 12-myristate 13-acetate (PMA; 1 µM, 60 minutes) or U0126 (60 µM, 60 minutes). The colonies were scraped with a scalpel blade and incubated for 1 minute with Lucifer yellow (1 mg/ml, Sigma) and rhodamine-dextran (1 mg/ml, Molecular Probes, Leiden, The Netherlands) diluted either in the Ca2+Mg2+-PBS buffer or in the Ca2+Mg2+-free buffer. The dye diffusion was observed by fluorescence. Before using this assay, we verified that the loading of Lucifer yellow by scraping a part of the colonies did not result in a loading of Lucifer yellow into all cells of the colony (data not shown).


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Both HES-3 and HES-4 cells expressed mRNA transcripts for connexin 43 and connexin 45 (Fig. 1AGo). Expression of the connexin proteins in stem cells was confirmed by costaining cultures with the stem cell surface antigen GCTM-2 and antibodies against the two connexins (Figs. 2Go, 3Go). Although con-nexin 43 was mainly localized at the cell surface (Fig. 2Go), connexin 45 was mostly cytoplasmic (Fig. 3Go). Because the antibodies used are capable of recognizing the mouse con-nexins 43 and 45, we can conclude that MEF cells do not express detectable levels of these proteins under these conditions (Figs. 2Go, 3Go). Because there are three phosphorylated forms of connexin 43 in other cell types, we checked its phosphorylated state in hESCs. Immunoblot analysis with an antibody reactive with all of the phosphorylated forms of connexin 43 revealed the presence of a triplet of bands representing the nonphosphorylated (NP) and the phosphorylated (P1, P2) forms of connexin 43 (Fig. 1BGo). These results also indicate that connexin 43 is mostly NP in hESCs (Fig. 1BGo).



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  		Figure 1. (A): Messenger RNA expression of connexin 43 and connexin 45 in HES-3 and HES-4 cells. Control reactions omitting the addition of reverse transcriptase [Cx43 (-ve) and Cx45 (-ve)] or primers (-ve control) were all negative. Sequencing of polymerase chain reaction products confirmed identity of human transcript without mutation. (B): Western blot analysis of connexin 43 in HES-3 cells. Abbreviations: Cx, connexin; NP, nonphosphorylated; P1 and P2, phosphorylated.

 


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  		Figure 2. Expression of connexin 43 in human embryonic stem cells. Immunostaining of HES-3 cells (A) with Hoechst 33342 (B), connexin 43 (C), and GCTM2 (D). Higher magnification of HES cells stained with connexin 43 (E). Merging image of HES-3 cells and MEF dually immunostained with connexin 43 and GCTM2 (F). Scale bars = 50 µm. Abbreviation: MEF, mouse embryonic fibroblast.

 


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  		Figure 3. Expression of connexin 45 in human embryonic stem cells. Immunostaining of HES-3 cells (A) with Hoechst 33342 (B), connexin 45 (C), and GCTM2 (D). Merging image of HES-3 cells and MEF dually immunostained with connexin 45 and GCTM2 (E). Scale bars = 50 µm. Abbreviation: MEF, mouse embryonic fibroblast.

 
We next examined the presence or absence of functional gap junctions in hESCs using the scrape loading/dye transfer assay with Lucifer yellow and rhodamine-dextran. Because of its low molecular weight (522 Da), Lucifer yellow diffuses from cell to cell through functional gap junctions. On the other hand, rhodamine-dextran (10,000 Da) is too large to diffuse through gap junctions and thus serves as a negative control to confirm that the Lucifer yellow transfer is solely due to gap junction coupling and not due to membrane fusions or formation of cytoplasmic bridges. When hESCs were scraped and incubated in presence of these fluorescent dyes, we observed extensive Lucifer yellow diffusion through hESC colonies, whereas rhodamine-dextran remained at the site of the scrape injury (Figs. 4A–CGo). Control colonies incubated with either marker in the absence of scraping demonstrated no uptake or dye transfer of Lucifer yellow or rhodamine-dextran (data not shown). Moreover, we did not observe any Lucifer yellow diffusion between hESCs and the supportive feeder cells (Fig. 4Go). The Lucifer yellow diffusion in hESCs was not affected by the absence of Ca2+Mg2+ (Figs. 4D–FGo) but was inhibited by PMA, a protein kinase C (PKC) activator, and by the extracellular signal-regulated kinase (ERK) kinase inhibitor U0126 (Figs. 4G–LGo). Altogether, these data demonstrate that hESCs are coupled through functional gap junctions and suggest that these gap junctions are not regulated by Ca2+Mg2+. These results also suggest that activation of PKC, as well as inhibition of ERK phosphorylation, inhibit GJIC in hESCs.



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  		Figure 4. Gap junctional intercellular communication in human embryonic stem cells. (A, D, G, J): Light and fluorescence micrographs with Lucifer yellow (B, E, H, K) and rhodamine-dextran (C, F, I, L) in HES-3 cells. Rhodamine-dextran was used as a negative control, showing no dye transfer across to the neighboring cell. Cells were incubated in the presence (A–C) or absence (D–F) of Ca2+Mg2+ or in the presence of phorbol 12-myristate 13-acetate (G–I) or U0126 (J–L). Scale bars = 100 µm.

 

   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that hESCs expressed connexin 43 and connexin 45. Earlier studies by Oyamada et al. [17] showed that transcripts of both connexin 43 and connexin 45 were present in undifferentiated mouse ES cells (J1 cell line). Moreover, Nishi et al. [23] detected connexin 43 mRNA transcripts in another mouse ES cell line (CCE), in undifferentiated F9 embryonic carcinoma (EC) cells, and in the inner cell mass of the mouse embryo. Other studies also demonstrated the presence of connexin 43 protein in undifferentiated P19 cells [24] and NT2/D1 cells [25], pluripotent mouse and human EC cells, respectively. In addition, connexin 45 mRNA transcripts were found in F9 EC cells [26]. During the preparation of this manuscript, Carpenter et al. [27] demonstrated the presence of connexin 43 and connexin 45 mRNA and proteins in hESC lines H1, H7, and H9 (grown on a Matrigel) with a localization of connexin 43 at the border of the hESC colonies. In our study, connexin 43, as well as connexin 45, was localized in the entire colonies and not only at the edges. This difference in the pattern of expression might relate to differences between ES cell lines, the different cell culture conditions (culture on feeder cell layers versus culture on Matrigel), or both.

Post-translational phosphorylation of gap junction proteins is considered to be a major regulatory mechanism for a wide spectrum of connexin processes, including trafficking, assembly/disassembly, and gap junctional communication [28]. In this study, we detected NP and phosphorylated (P1 and P2) forms of connexin 43 in hESCs, and we show that connexin 43 is mostly present under its NP form. Phosphory-lation of connexin 43 seems to influence GJIC both positively and negatively [28] depending on the cell types. Thus, the significance of phosphorylated and NP forms of connexin 43 in hESCs requires additional investigation.

This study also demonstrates transfer of Lucifer yellow in hESCs, indicating that hESCs are coupled through functional gap junctions, which are inhibited by PKC activation and ERK inhibition. Our results are consistent with the finding that undifferentiated mouse ES cells are also capable of GJIC [17] and the results of Carpenter et al. [27], who also described functional gap junctional communication in hESCs grown on Matrigel. Collectively, these results with human and mouse ES cells can be contrasted with results on several adult stem cell populations. Further studies are required to assess the role of GJIC in hESC survival or differentiation. Because dissociation of hESCs to single cells causes considerable cell death, it is possible that gap junctional communication is important to the survival of these cells.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was supported by Monash University, ES Cell International, and the National Institutes of Health (NIGMS GM68417).

Raymond Wong and Alice Pébay contributed equally to this work


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  14. Holland MS, Tai MH, Trosko JE et al. Isolation and differentiation of bovine mammary gland progenitor cell populations. Am J Vet Res 2003;64:396–403.[CrossRef][Medline]

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  18. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.[Abstract/Free Full Text]

  19. Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.[CrossRef][Medline]

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  22. Wolvetang EJ, Wilson TJ, Sanij E et al. ETS2 overexpression in transgenic models and in Down syndrome predisposes to apoptosis via the p53 pathway. Hum Mol Genet 2003;12:247–255.[Abstract/Free Full Text]

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  25. Bani-Yaghoub M, Bechberger JF, Naus CC. Reduction of connexin43 expression and dye-coupling during neuronal differentiation of human NTera2/clone D1 cells. J Neurosci Res 1997;49:19–31.[CrossRef][Medline]

  26. Hennemann H, Schwarz HJ, Willecke K, Characterization of gap junction genes expressed in F9 embryonic carcinoma cells: molecular cloning of mouse connexin31 and -45 cDNAs. Eur J Cell Biol 1992;57:51–58.[Medline]

  27. Carpenter MK, Rosler ES, Fisk GJ et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 2004;229:243–258.[CrossRef][Medline]

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Received October 23, 2003; accepted for publication July 8, 2004.



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