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In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST-BODIPY-Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X-Rhod-1. Immunofluorescence experiments showed the presence of connexin-43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage-dependent calcium signaling in cocultured MSCs. MSCs showed a time-dependent increase of labeling of ST-BODIPY-Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non-fluorescent DHP, Nifedipine, completely abolished ST-BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization-induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage-dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte-like phenotype. J. Cell. Biochem. 100: 86–99, 2007. © 2006 Wiley-Liss, Inc.
Bone marrow mesenchymal stem cells (MSCs) can be easily isolated and expanded ex vivo and are relatively safe in terms of rejection reaction, thus providing a promising model for development of stem cell therapeutics. In particular they have been considered of potential clinical use for the repair of infarcted myocardium [Pittenger and Martin, 2004].
Early clinical trials on small cohorts of patients with acute myocardial infarction have generally shown that implantation of autologous unfractionated bone marrow mononuclear cells or of MSCs significantly improve cardiac function [Laflamme and Murry, 2005]. However, a recent clinical trials update from the American Heart Association [Cleland et al., 2006] reports no benefit of stem cells implantation in the ASTAMI study and a small but significant benefit in REPAIR-AMI.
Several studies have reported that animals injected after an experimental infarction with autologous or etherologous MSCs have an improved recovery [Leri et al., 2005].
However, several technical problems on the detection of successfully implanted and differentiated cells are still open [Laflamme and Murry, 2005]. Due to the diverging results obtained by various investigators, the possibility of a fruitful use of MSCs has remained a matter of controversy. In particular Wang et al.  found that the transplantation of undifferentiated bone marrow MSCs in the rat infarcted myocardium originated a limited amount of cardiomyocytes together with individual cells or clusters expressing fibroblastic phenotype. A further issue is the finding that a small number of donor cells might fuse with the host cells giving rise to chimeric phenotypes [Zhang et al., 2004].
On another side a full characterization of MSCs biology in vitro is still not available and several doubts remain on the phenotypes they can develop and on the conditions required [Javazon et al., 2004]. At this regard recent results from Bayes-Genis et al.  report that human MSCs spontaneously express markers of cardiac phenotype in vitro.
A vast literature reports about MSCs differentiation under defined in vitro conditions into the cells of several tissues, like osteocytes, chondrocytes, adipocytes [Pittenger et al., 1999], skeletal muscle fibers [Bhagavati and Xu, 2004; Gang et al., 2004], hepatocytes [Lee et al., 2004]. In particular, regarding heart repair, they have been shown to potentially originate both endothelial vascular cells and cardiomyocytes. Among the different strategies proposed to promote in vitro cardiac differentiation of MSCs, chronic exposure to 5-azacytidine has been amply tested [reviewed by Leri et al., 2005], as well as stimulation by exogenous cytokines and growth factors [Xaymardan et al., 2004, Shim et al., 2004], and coculture with neonatal cardiac cell [Lagostena et al., 2005] or immortalized cardiac cell lines [Rangappa et al., 2003]. Cocultures between MSCs and adult cardiomyocytes were performed mainly to show functional connection between the two cell types [Valiunas et al., 2004]. Other results with adult cardiomyocytes and MSCs were controversial [Wang et al., 2005; Yoon et al., 2005] and failed to clearly show striated and functional cardiac cells during the coculture time. It is interesting that bone marrow-derived endothelial progenitor cells taken from peripheral blood have been seen to differentiate into cardiomyocytes when cocultured with neonatal rat cardiomyocytes [Badorff et al., 2003].
Recent results suggest, moreover, that the differentiation of MSCs may depend on stochastic events and the cells actually fail to acquire a truly functional status [Belema Bedada et al., 2005].
Thus, many issues remain to be clarified on the processes following MSCs culture [Javazon et al., 2004] and transplantation in the myocardium. In particular, the cellular mechanism involved in the interaction between MSCs and recipient's adult cardiomyocytes has not yet been fully described. On the basis of the above uncertainty, the present investigation aims to reproduce and study in vitro the changes induced by host's myocardial microenvironment on donor's MSCs.
As an experimental model we cocultivate adult rat MSCs and adult cardiomyocytes, while characterizing the appearance of contractile proteins, calcium channels, and calcium control changes occurring in MSCs as well as their time-course during coculture.
Our experiments showed the presence of gap-junctions both in cardiomyocytes/MSCs and in MSCs/MSCs contacts, the appearance of voltage-dependent calcium signals in cocultured MSCs but no myofibrillar structures development at any steps of coculture and therefore the absence of contractile activity. We propose that MSCs grown in vitro in a cardiac microenvironment integrate with host cells but show a limited plasticity in the differentiation process towards a functional contractile phenotype.
- Top of page
- MATERIALS AND METHODS
- Supporting Information
A vast literature has presented the potential of MSCs to differentiate into cardiomyocytes in both in vitro and in vivo models [Haider and Ashraf, 2005]. Even with the amount of experiments performed, many details on the mechanisms and limits of this process are still not clear. Controversial results have emerged even in comparable conditions, stimulating speculations on basic physiological and developmental issues, such as the potential of bone marrow derived stem cells to fuse with resident cells [Zhang et al., 2004] or which pathways may lead to cell dedifferentiation and redifferentiation. A further problem is the lack of a clear definition of which markers are evidence for differentiation [Belema Bedada et al., 2005]. The in vivo experiments are faced with the difficult issue to find the relatively few injected cells in the organ and to properly identify them among the vast amount of non-myocyte cells that form the heart. Moreover, recent results introduced the hypothesis that adult stem cells may represent loose ends of progenitor cells that stopped serving an important physiological role and failed to acquire a fully functional phenotype [Belema Bedada et al., 2005].
A basic issue is also related to the puzzling question posed by the discovery that some gastrointestinal tumors may be due to mobilized MSCs that migrate to the inflamed tissue [Houghton et al., 2004]. There is, nevertheless, a substantial agreement that some benefits upon MSCs transplantation may be achieved, even if it is questioned whether it arises directly from the regeneration of cardiac tissue or from more general improvements of the healing process, derived by increase of vascularization or decrease in fibrotic scar [Pittenger and Martin, 2004].
The in vitro assessment of the differentiation potential of MSCs has been mainly monitored in cultures containing either MSCs alone treated with 5-azacytidine [Xu et al., 2004] or with a cocktail of defined growth factors [Shim et al., 2004; Xaymardan et al., 2004]. Cell treatment with 5-azacytidine has a long scientific history, as it has been at first proposed to induce differentiation in several cell lines, mainly in an attempt to reduce the aggressivity of transformed cells. It apparently acts by modifying methylation [Momparler, 2005]. Other experiments studied coculture model of MSCs with neonatal cardiomyocytes [Lagostena et al., 2005] or cardiomyocyte-cell lines [Rangappa et al., 2003; Beeres et al., 2005], but not extensively with primary adult cardiomyocytes. We are presenting a study in controlled conditions of the effect of adult cardiomyocytes on the expression of markers of differentiation in MSCs. Previous data on MSCs/adult cardiomyocytes coculture [Wang et al., 2005; Yoon et al., 2005] have been presented but apparently did not clearly show the presence of cardiomyocytes that retain a normal functional morphology along the culture period.
After establishing a culture protocol, we have then examined and found connexin-43 expression both between MSCs and between cardiomyocytes and MSCs. The formation of gap junctions is a marker of cell–cell interaction and is required to allow electrical signal propagation, a prerequisite for any functional grafting in the myocardium. While even in MSCs alone expression of Cx43 is present in coculture conditions we observed a clear evidence of this signal only in the MSCs/cardiomyocytes contacts but not in the MSCs/BAE-1 contacts (Fig. s1).
Some authors have showed evidence of a complete establishment of muscular protein networks in MSCs upon clonal selection in the presence of 5-azacytidine [Xu et al., 2004] and the expression of markers typical of cardiac myocytes, but not functional differentiation in MSCs cocultured with neonatal cardiomyocytes [Lagostena et al., 2005].
In our results even after 1 week of coculture, we have not seen any ordered sarcomeric structure that can indeed be easily identified in cardiac myocytes by a ordered pattern in the Fourier transform of the confocal images. In particular, sarcomeric-α-actinin immunostaining revealed the same pattern in control and cocultured MSCs, with an evident staining of stress fibers and focal adhesion. In agreement with our immunofluorescence results Bayes-Genis et al.  recently shown that in human MSCs sarcomeric-α-actinin, but not sarcomeric myosin, was spontaneously expressed.
Furthermore, the appearance of such structures could not be considered by itself a specific marker of muscular differentiation, as many different cell types in culture as well as in situ present stress fibers with a periodic sarcomere-like organization [Peterson et al., 2004] and myofibroblast cell lines of mesenchymal origin express sarcomeric proteins [Mayer and Leinwand, 1997].
Our coculture procedure is affected by clear limitations, such as a restriction to 1 week of the observation and the presence of an excitation-contraction uncoupler, that allows a longer survival period of the cardiac myocytes. One might speculate that even longer time might lead to a new destiny of the MSCs or at least of a subpopulation. Still, most in vitro experiments that have described differentiations have been performed for even shorter times. The excitation-contraction uncoupling drug does not by itself in our hands induce persistent changes in the cardiac myocyte as cells resume contraction rapidly upon washout of the drug even after several days in culture, as demonstrated by the presence of contractions upon potassium stimulation. In control experiments (data not shown) cardiac myocytes have been field stimulated during rapid washout of BDM and recovery of contraction occurred within minutes.
We acknowledge that our approach is still rudimentary, as we do not maintain the coculture under continuous electrical stimulation. Therefore, during coculture the cardiomyocytes may have lost their properties which provide the “cardiac niche.” To avoid this possibility we carefully controlled during potassium depolarization experiments that all cardiomyocytes present in the observed field showed a clear contractility (supplementary data, Fig. s3), and this test was used to validate both the depolarization and the cardiomyocytes functionality.
A sound hypothesis could nevertheless be that differentiation of MSCs into cardiac myocytes could require at least a stage of periodic contraction, in a way resembling the embryonic to adult development in the heart.
ST-BODIPY-Dihydropyridine specific labeling of live cocultured MSCs indicates an increase in voltage-gated calcium channels (VGCC) expression. Moreover the absence of labeling in the MSCs/BAE-1 coculture suggests that cardiac microenvironment could be necessary in this process.
However this behavior is not necessarily a marker of differentiation toward an excitable cell, as many different cell types express VGCC for many different functions, including growth factor induced fibroblast cell migration [Yang and Huang, 2005].
Our observation of depolarization-induced calcium transients in MSCs in proximity with myocytes is in agreement with ST-BODIPY-Dihydropyridine specific labeling of cocultured MSCs. Moreover, in a similar experiment [Beeres et al., 2005], it has been observed the electrotonic propagation of action potentials across MSCs implanted in an in vitro lesion of a layer of beating myocytes. In both cases the result could be in perspective clearly beneficial, even if the MSCs themselves are incapable of action potentials and contraction. We have been unable to observe any clear-cut contraction in MSCs upon depolarization and in the presence of calcium increases. The only movements we have detected were clearly related to close-by myocytes that were pulling the MSCs.
As a summary our results agree with the hypothesis that it is difficult to obtain in vitro a complete differentiation of bone marrow derived stem cells, at least in the absence of a complete reprogram as can be achieved with 5-azacytidine. The limited plasticity could anyway explain the improvement observed in in vivo experiments and in patients, as the formation of gap-junctions could restore conduction that is blocked instead by fibroblasts, and the presence of MSCs could reduce the formation of scar tissue.