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RAPID REPORT |
1 Department of Physiology and Biophysics, Institute of Molecular Cardiology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA2 Department of Pharmacology, Center for Molecular Therapeutics, Columbia University, New York, NY 10032, USA
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Abstract |
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j) of 30–80pS. The macroscopic Ij obtained from hMSC–cardiac myocyte cell pairs exhibited asymmetrical Vj dependence, while single channel events revealed j of the size range 40–100pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.
(Received 25 November 2003;
accepted after revision 2 February 2004;
first published online 6 February 2004)
Corresponding author I. S. Cohen: Department of Physiology and Biophysics, 8661 SUNY, Stony Brook, NY 11794-8661, USA. Email: icohen{at}physiology.pnb.sunysb.edu
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Introduction |
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Methods |
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Human mesenchymal stem cells (hMSCs; mesenchymal stem cells, human bone marrow; PoieticsTM) were purchased from Clonetics/BioWhittaker (Walkersville, MD, USA) and cultured in mesenchymal stem cell (MCS) growth medium and used from passages 2–4. Isolated and purified hMSCs can be cultured for many passages (12) without losing their unique properties, i.e. normal karyotype and telomerase activity (van den Bos et al. 1997; Pittenger et al. 1999). HeLa cells that were transfected with rat Cx40, rat Cx43 or mouse Cx45 were cocultured with hMSCs. Production, characterization and culture conditions of transfected HeLa cells have been previously described (Valiunas et al. 2000, 2002). Adult dogs of either sex were killed by an approved protocol at SUNY Stony Brook by an injection of sodium pentobarbital (80 mg kg-1 body weight). Cardiomyocytes were isolated from the canine ventricle as previously described (Yu et al. 2000). We have adopted the method of primary culture of canine cardiomyocytes following the procedure described for mouse cardiomyocytes (Zhou et al. 2000). The cardiomyocytes were plated at 0.5–1 (104 cells cm-2 in minimal essential medium (MEM) containing 2.5% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) onto mouse laminin (10µgml-1) precoated coverslips. After 1h of culture in a 5% CO2 incubator at 37°C, the medium was changed to FBS-free MEM. Stem cells were added after 24h and coculture was maintained in Dulbecco's modified Eagle's medium (DMEM) with 5% FBS. Cell Tracker Green (Molecular Probes, Eugene, OR, USA) was used to distinguish hMSCs from HeLa cells in coculture in all experiments (Valiunas et al. 2000).
Anti-connexin antibodies, immunofluorescent labelling and immunoblot analysis of the cells
Commercially available mouse anticonnexin monoclonal and polyclonal antibodies (Chemicon International, Temecula, CA, USA) of Cx40, Cx43 and Cx45 were used for immunostaining and immunoblots as described earlier (Laing & Beyer, 1995). Fluorescein-conjugated goat antimouse or antirabbit IgG (ICN Biomedicals, Inc.) was used as secondary antibody.
Electrophysiological measurements
Glass coverslips with adherent cells were transferred to an experimental chamber perfused at room temperature (
22°C) with bath solution containing (mM): NaCl, 150; KCl, 10; CaCl2, 2; Hepes, 5 (pH 7.4); glucose, 5. The patch pipettes were filled with solution containing (mM): potassium aspartate, 120; NaCl, 10; MgATP, 3; Hepes, 5 (pH 7.2); EGTA, 10 (pCa 8); filtered through 0.22µm pores. When filled, the resistance of the pipettes measured 1–2M
. Experiments were carried out on cell pairs using a double voltage-clamp. This method permitted us to control the membrane potential (Vm) and measure the associated junctional currents (Ij).
Dye flux studies
Dye transfer through gap junction channels was investigated using cell pairs. Lucifer Yellow (LY) (Molecular Probes) was dissolved in the pipette solution to reach a concentration of 2 mM. Fluorescent dye cell-to-cell spread was imaged using a 16 bit 64 000 pixel grey scale digital CCD-camera (LYNXX 2000T, Spectra Source Instruments) (Valiunas et al. 2002). In experiments with heterologous pairs LY was always injected into the cells which were tagged with Cell Tracker Green. The injected cell fluorescence intensity derived from LY is 10–15 times higher than the initial fluorescence from Cell Tracker Green.
Signal recording and analysis
Voltage and current signals were recorded using patch clamp amplifiers (Axopatch 200). The current signals were digitized with a 16 bit A/D-converter (Digidata 1322A, Axon Instruments, Union City, CA, USA) and stored with a personal computer. Data acquisition and analysis were performed with pCLAMP 8 software (Axon Instruments). Curve fitting and statistical analyses were performed using SigmaPlot and SigmaStat, respectively (SPSS, Chicago, IL, USA). The results are presented as means ±S.E.M.
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Results |
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) and steady state conductances (gj,ss, •) (determined at the beginning and at the end of each Vj step, respectively) were plotted versusVj. The left panel shows a quasi-symmetrical relationship from five hMSC pairs. The continuous curves represent the best fit of data to the Boltzmann equation with the following parameters: half-deactivation voltage, Vj,0=-70/65mV; minimum gj, gj,min= 0.29/0.34; maximum gj, gj,max= 0.99/1.00; gating charge, z= 2.2/2.3 for negative/positive Vj, respectively. Summarized plots from six asymmetrical cases are shown in the right panel. The gj,ss declined in sigmoidal fashion at negative Vj and showed a reduced voltage sensitivity to positive Vj. Boltzman fitting for negative Vj revealed the following values: Vj,0=-72mV, gj,min= 0.25, gj,max= 0.99, z= 1.5.
Figure 2D and E illustrates typical multichannel recordings from a hMSC pair. Using 120 mM potassium aspartate as a pipette solution we have observed channels with unitary conductances of 28–80pS range. Operation of channels with 50pS conductance (see Fig. 2D) is consistent with previously published values (Valiunas et al. 1997, 2002) for Cx43 homotypic channels. This does not preclude the presence of other channel types; it merely suggests that Cx43 forms functional channels in hMSCs.
To further define the nature of the coupling we cocultured hMSCs with human HeLa cells stably transfected with Cx43, Cx40 and Cx45 (Elfgang et al. 1995) and found that hMSCs were able to couple to all these transfectants. Figure 3A illustrates an example of junctional currents recorded between hMSC and HeLaCx43 cell pairs, one that manifested symmetrical and one that exhibited asymmetrical voltage-dependent currents in response to a series (from ±10mV to ±110mV) of symmetrical transjunctional voltage steps (Vj). The quasi-symmetrical record suggests that the dominant functional channel is homotypic Cx43 while the asymmetrical record suggests the activity of another connexin in the hMSC (presumably Cx40 as shown by immunohistochemistry, see Fig. 1) that could be either a heterotypic or heteromeric form or both. These records are similar to those previously published for transfected cells: heterotypic and mixed/heteromeric forms of Cx40 and Cx43 (Valiunas et al. 2000, 2001). Co-culture of hMSCs with HeLa cells transfected with Cx40 (Fig. 3B) also revealed symmetrical and asymmetrical voltage-dependent junctional currents consistent with the coexpression of Cx43 and Cx40 in the hMSCs similar to the data for HeLaCx43–hMSC pairs. HeLa cells transfected with Cx45 that coupled to hMSCs always produced asymmetrical junctional currents with pronounced voltage gating when the Cx45 (HeLa) side was negative (Fig. 3C). This is consistent with the dominant channel forms in the hMSC being Cx43 and Cx40 as both produce asymmetrical currents when they form heterotypic channels with Cx45 (Valiunas et al. 2000, 2001). This does not exclude Cx45 as a functioning channel in hMSCs but it does indicate that Cx45 is a minor contributor to cell to cell coupling in hMSCs. The lack of visualized plaques in the immunostaining for Cx45 (Fig. 1) further supports this interpretation.
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, three preparations), the Boltzmann fit (dashed line) at negative Vj values revealed the following parameter values: Vj,0=-70mV, gj,min= 0.31, gj,max= 1.00, z= 2.2. The middle panel shows data from hMSC–HeLaCx40 pairs including three symmetrical (•) and two asymmetrical () gj,ss–Vj relationships. The continuous lines correspond to a Boltzmann fit to symmetrical data (Vj,0=-57/76mV, gj,min= 0.22/0.29, gj,max= 1.1/1.0, z= 1.4/2.3; negative/positive Vj) and the dashed line is a fit to the asymmetrical data (Vj,0=-57/85mV, gj,min= 0.22/0.65, gj,max= 1.1/1.0, z= 1.3/2.2; negative/positive Vj). The data from the six complete experiments from hMSC–HeLaCx45 cell pairs are shown on the right panel. The gj,ss plot versusVj was strongly asymmetrical and the best fit of the data to the Boltzmann equation at positive Vj values revealed following parameter values: Vj,0= 31mV, gj,min= 0.07, gj,max= 1.2, z= 1.8.
Figure 3E shows Lucifer Yellow transfer from an hMSC to an hMSC (upper panel), from a HeLaCx43 to an hMSC (middle panel) and from an hMSC to a HeLaCx43 (bottom panel). The junctional conductance of the cell pairs was simultaneously measured by methods described earlier (Valiunas et al. 2002) and revealed conductances of 13, 16 and 18nS, respectively. The transfer of Lucifer Yellow was similar to that previously reported for homotypic Cx43 or coexpressed Cx43 and Cx40 in HeLa cells (Valiunas et al. 2002). Cell Tracker Green was always used in one of the two populations of cells to allow heterologous pairs to be identified (Valiunas et al. 2000). Lucifer Yellow was always delivered to the cell containing cell tracker. The fluorescence intensity generated by the Cell Tracker Green was 10–15 times less than fluorescence intensity produced by the concentration of Lucifer Yellow delivered to the source cell.
We also cocultured hMSCs with adult canine ventricular myocytes as shown in Fig. 4. Immunostaining for Cx43 was detected between the rod-shaped ventricular myocytes and hMSCs as shown in Fig. 4A. The hMSCs also couple electrically with cardiac myocytes. Both macroscopic (Fig. 4B) and multichannel (Fig. 4C) records were obtained. Junctional currents in Fig. 4B are asymmetrical while those in Fig. 4C show unitary events of the size range typically resulting from the operation of homotypic Cx43 or heterotypic Cx43–Cx40 or homotypic Cx40 channels (Valiunas et al. 2000, 2001). Heteromeric forms are also possible whose conductances are the same or similar to homotypic or heterotypic forms.
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Discussion |
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What are the implications of junctional current asymmetry recorded from various cell pairs including an hMSC? A number of studies have shown that heterotypic gap junction channels typically generate asymmetrical currents while heteromeric mixtures give rise to symmetrical-like (weakly asymmetrical) currents. Co-expression of two connexins is expected to display variability in terms of symmetry and asymmetry based on the population size of homotypic, heterotypic and potential heteromeric channel forms. The size of the homotypic, heterotypic and heteromeric populations would be dependent on connexin–connexin interactions during assembly and connexon–connexon interactions in the plasma membranes of two closely apposed cells (Brink et al. 1997; Valiunas et al. 2001; Beyer & Berthoud, 2002). In the case of the stem cell pairs, assuming only Cx43 and Cx40 are coexpressed, only two types of heterotypic channels are possible, each with equal but oppositely signed voltage dependence. Two equally distributed populations will result in symmetrical behaviour while unequal distribution (dominance of one form over the other) will result in asymmetry. This is consistent with the data and interpretation of Valiunas et al. (2000, 2001). Any number of possible heteromeric forms are also potential candidates for generating asymmetrical junctional currents. The problem is that no one single form (of 192 possible for two coexpressing connexins) can be studied in isolation from others. Thus attributing asymmetry to heteromeric forms is equivocal. If all the connexins had equal affinity for each other and themselves then coexpression of two connexins would be dominated by heteromeric forms. If on the other hand self-recognition and affinity are preferred over heterologous interactions then homotypic forms would dominate.
Lucifer Yellow passage between an hMSC and HeLaCx43 cell (see Fig. 3F) is yet another indicator of robust gap junction-mediated coupling. The transfer of Lucifer Yellow between hMSCs and HeLa cells transfected with Cx43 is similar to that of homotypic Cx43 or coexpressed Cx43 and Cx40. It excludes homotypic Cx40 as a dominating channel type as Cx40 is some 5 times less permeable to Lucifer Yellow than Cx43 (Valiunas et al. 2002).
We have defined the lower limit or minimum number of functional connexins present in stem cells (Cx43, Cx40 and possibly Cx45). The literature has already provided us with examples of hMSC transformation into heart cells (Toma et al. 2002). Does the functional expression of connexins change under such transformations and as a consequence affect coupling, or are hMSCs fixed with regard to the pool of potentially expressible connexins? This is yet to be determined. Our results show that hMSCs couple to one another via Cx43 and Cx40. We have also established that hMSCs can form heterologous junctions with cardiac myocytes and cultured HeLa cells transfected with specific connexins (Cx40, Cx43, Cx45).
To date, there are more than 20 connexins identified in humans encoded by a multigene family (Beyer & Berthoud, 2002). We recognize that there is the potential for the expression of other connexins in hMSCs. However, our present study focused on cardiac-specific connexins. Our data strongly suggest that Cx43 and Cx40 are dominant functional forms in stem cells. We have also probed for Cx26 and Cx32 and found no expression (data not shown). A thorough demonstration of which connexins are expressed of the many human connexins awaits further investigation.
The hMSC–cardiomyocyte coupling conductance was notably lower than those values obtained from hMSC–HeLa cell pairs. The gap junction conductance between hMSC–cardiomyocyte pairs ranged from 0.1 to 5.5nS, which reflects an approximate range of 2–110 channels and is considerably lower compared to conductance values (40–200nS) reported earlier for adult ventricular cell pairs (Maurer & Weingart, 1987; Polontchouk et al. 2002). The lower value might, in fact, reflect a reduced ability of hMSCs and cardiomyocytes to form gap junctions, which could be influenced by several experimental conditions and factors. The first is the amount of time given for the hMSC to form junctions with the cardiomyocytes, which typically was less than 12h. After that time the hMSCs become flattened and spread out, making contacts with many other hMSCs. To find a single hMSC–cardiomyocyte pair then is rather difficult. Second, in the freshly isolated cardiomyocytes, Cx43 immunolocalizes mostly at intercalated discs at the cell ends (Huang et al. 1996; Eppenberger & Zuppinger, 1999) (see also Fig. 4A). Therefore, the lower junctional conductance between hMSCs and myocytes is to some degree configuration dependent where the hMSC must contact the end of a myocyte. This latter conformation, illustrated in Fig. 4A, was not common. Finally, the reduction in gap junction expression over the first 48h after isolation and subsequent culturing (Huang et al. 1996) plays a significant role. It is entirely possible that prolonged coculturing of hMSCs with cardiomyocytes would reveal heterogeneous cell pairs with larger junctional conductances than reported here but the technical reasons sited above limit our ability to define the meaning of the low junctional conductance measured between hMSCs and myocytes. Moreover, it has been found that the formation of only 15 channels and possibly even less is quite sufficient to ensure action potential propagation in neonatal rat heart cells (Rook et al. 1988, 1990). In a preliminary report we have shown that these same hMSCs transfected with a pacemaker gene (HCN2) are able to effect alterations in pacing in situ and in vitro, which strongly suggests that the level of coupling between hMSCs and myocytes is physiologically sufficient (Plotnikov et al. 2003).
Related studies have shown that skeletal muscle myotubes can effect some measure of cardiac repair but a recent study by Leobon et al. (2003) has shown in vitro that myotubes do not form gap junctions with cardiac myocytes. While the data of that study exclude gap junction-mediated communication (in vitro) it does not eliminate other forms of interaction between cells. One possibility is ephaptic-like transmission similar to the work of Arvantitaki and collaborators that is referred to by Eccles (1964). Other characterizations of electrical transmission without gap junctions are summarized by Bennett (1977). The experimental procedures used by Leobon et al. (2003) would not define or illustrate a ‘restricted space' electrical synapse like those examples given by Bennett (1977). A further complication in interpreting the data of Leobon et al. (2003) is the fact that their experiments are done in vitro with cardiac myocytes and the cited data illustrating myotubes enhanced performance is in situ. The myotubes in situ might well have differentiated and possibly formed junctions with cardiac myocytes.
These data support the possibility of using hMSCs as a therapeutic substrate for repair of cardiac tissue. Other syncytia such as vascular smooth muscle or endothelial cells should also be able to couple to the hMSCs because of the ubiquity of Cx43 and Cx40 (Wang et al. 2001). Thus they might also be amenable to hMSC-based therapeutics, as follows: hMSCs can be transfected to express ion channels which then can influence the surrounding syncytial tissue. Alternatively, the hMSCs can be transfected to express genes that produce small therapeutic molecules capable of permeating gap junctions and influencing recipient cells. Further, for short-term therapy, small molecules can be directly loaded into hMSCs for delivery to recipient cells. The success of such an approach is critically dependent on gap junction channels as the final conduit for delivery of the therapeutic agent to the recipient cells. We have demonstrated the feasibility of one such approach by transfecting hMSCs with mHCN2, a gene encoding the cardiac pacemaker channel, and delivering them to the canine heart where they generate a spontaneous rhythm (Plotnikov et al. 2003).
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K. Wang, T. Xue, S.-Y. Tsang, R. Van Huizen, C. W. Wong, K. W. Lai, Z. Ye, L. Cheng, K. W. Au, J. Zhang, et al. Electrophysiological Properties of Pluripotent Human and Mouse Embryonic Stem Cells Stem Cells, October 1, 2005; 23(10): 1526 - 1534. [Abstract] [Full Text] [PDF]
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