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Right arrow Cardiac - physiology
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Eur J Cardiothorac Surg 2005;28:685-691
© 2005 Elsevier Science NL

Fusion of bone marrow-derived stem cells with cardiomyocytes in a heterologous in vitro model {star}

Jens Garbade a , * , Andreas Schubert a , Ardawan Julian Rastan a , Dominik Lenz b , Thomas Walther a , Jan Fritz Gummert a , Stefan Dhein a , Friedrich-Wilhelm Mohr a

a Department of Cardiac Surgery, Heart Center, University of Leipzig, Struempellstr. 39, 04289 Leipzig, Germany
b Department of Paediatric Cardiology, Heart Center, University of Leipzig, Germany

Received 26 November 2004; received in revised form 17 June 2005; accepted 27 June 2005.

* Corresponding author. Tel.: +49 341 8651420; fax: +49 341 8651452. (Email: jgmed93{at}hotmail.com).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 
OBJECTIVE: Recent studies have demonstrated that transplanted bone marrow-derived stem cells (BMCs) possess a broad differentiation potential and are able to form new cardiomyocytes. However, the identity of BMCs as true cardiomyocytes is still ambiguous. Therefore, we investigated the fate of transplanted fluorescence labeled BMCs and cardiomyocytes in co-culture. Methods: For cell tracking we used two different fluorescent probes, Vybrant/DiO and Vybrant/DiI. BMCs were taken from human sternal marrow, purified using a Ficoll-gradient-centrifugation, treated with 5-azacytidine and stained with Vybrant/DiO. Furthermore, isolated spontaneous beating cardiomyocytes of neonatal rats (CM) were labeled with Vybrant/DiI. Thereafter, the BMCs were transplanted into CM-cultures and investigated on day 1, 4, 7, 14 and 28 using two-color fluorescence phenotyping by laser-scanning-cytometry (LSC). Two-color positive cells were harvested by patch–clamp technique and ß-MHC mRNA expression was analyzed by single-cell PCR. Results: Two different morphological phenotypes were observed by LSC. First, isolated DiO labeled BMCs without contact or with direct cell contact to DiI labeled CMs. Second, some BMCs and CMs were double positive for DiO/DiI spontaneously forming hybrids. This population increased by 18% from day 1 to 4 and decreased only slightly until day 28. Additionally, few two-color positive cell formations expressed both human and rat specific ß-MHC mRNA as well as only human ß-MHC mRNA indicating that cell-fusion and transdifferentiation has occurred. Conclusion: These observations provide in vitro evidence for spontaneous cell fusion and transdifferentiation of BMCs in co-culture, raising the possibility that the observed phenomenons may contribute to development or maintenance of these cell types.

Key Words: Bone marrow stem cells • Cell fusion • Transdifferentiation • Heart failure • Myocardial regeneration


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 
The potential of adult stem cells to function as cellular therapy in chronic cardiac diseases relies on their ability to plasticity, clonogenicity, self-renewal and to replenish multiple cell types within the tissue they reside in. This is different from haematopoietic alignment. Based on different experimental research transdifferentiation of bone marrow-derived cells into cardiomyocytes, supporting angiogenesis in ischemic heart diseases and improvement of cardiac function could be demonstrated [1,2]. Recently, the safety and feasibility of stem cell transplantation has been established in humans by clinical trials on intracoronary infusion of autologous mononuclear bone marrow-derived and progenitor cells. Enhanced contractility, improvement in stroke volume index and increased myocardial viability were demonstrated at 3 and 12 months, respectively [3,16].

However, a great debate has arisen about this phenomenon of plasticity of adult stem cells. Since the independent publications by Murry et al. [4] and Balsam et al. [5] these observations have been challenged by many authors, including the scientific contribution of ongoing human studies. The rarity of cardiogenic differentiation by endogenous stem cells, the modified activation by cytokines, the uncertainty of myocyte origin in transplanted hearts and the confounding possibility of cell fusion after in vitro grafting as well as in vivo observations lead to unsettled and controversial issues in regard to the plasticity for myocardial regeneration. However, more recent publications suggest that cell fusion is considered an alternative mechanism to cell reprogramming by transdifferentiation leading to the generation of hybrid cells with donor cell origin and simultaneous expression of recipient cell markers and characteristics [6–10].

Until now it is not clear whether active or passive mechanisms lead to improved left ventricular function. If active mechanisms were assumed, a transdifferentiation of BMCs to contractile cardiomyocytes is required, whereas secondary factors include the formation of a new vascular network of infarcted myocardium and the modulation of paracrine factors leading to enhanced myocardial regeneration.

Therefore, we evaluated the fate and the ability of fluorescence labeled human bone marrow-derived mononuclear cells to regenerate new cardiomyocytes in a heterologous in vitro model with spontaneous beating neonatal rat cardiomyocytes. Our aim was to elucidate the expression of species- and cardiospecific ß-MHC mRNA to identify the possible mechanism responsible for their developmental cell plasticity.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A
 References
 
2.1 Cell culture and manipulation
All procedures were approved by the animal care committee of the University Leipzig and were performed in accordance with the ‘Guide for the Care and Use of Laboratory Animals’, prepared by the Institute of Laboratory Resources, National research Council, and published by the National Academies Press, revised 1996. BMCs were obtained from human sternal marrow in patients that underwent elective cardiac surgery. This procedure was reviewed and approved by our institutional review board.

Neonatal rat ventricular cardiomyocytes (CM) were isolated from 1 to 2 day old Sprague-Dawley rats as described previously [11] with some modifications. In brief, neonatal rats were decapitated and ventricles were dissociated enzymatically at 37 °C with a repetitive protocol using collagenase type CLS II (Biochrom) with an enzyme activity of 270 U/ml. The CMs were washed and resuspended in M199 medium with Earl's salts and Hepes (Gibco) by adding 100 U/ml penicillin, 100 µg/ml streptomycin, 5% fetal bovine serum and 10% horse serum. The cells were plated at a density of 104/cm2 in gelatine-coated cover slips.

Human BMCs were aspirated and afterwards flushed with PBS-EDTA (phosphate buffer salines, pH 7.4, EDTA 2 mM) to obtain a maximum amount of cells. Cells were washed in PBS-EDTA and BMCs were isolated by Ficoll density gradient centrifugation according to Tomita [2]. After two washing steps BMCs were resuspended in 12 ml DMEM (Sigma) by adding 10% fetal bovine serum, 100 U/ml penicillin G, 100 µg/ml streptomycin (Sigma). To induce differentiation, cells were cultured with 10 µM 5-azacytidine for 24 h (Sigma). The cells were seeded on gelantine-coated Petri dishes (Falcon BD Bioscience).

The isolated cell suspension contains a heterogenous population of mononuclear cells, which were characterised by FACS analysis (Becton Dickinson FACS Calibur System) prior to transplantation. More than 96% of the cells were CD45+; overall 1.9% of CD45/CD34-positive cells were identified. The fraction of viable cells was about 97%.

Each cell type was cultured at 37 °C in a 5% CO2-humidified atmosphere and tested to ensure freedom from mycoplasma contamination.

2.2 Cell labeling
For tracking the fate of BMCs in co-culture with CM, the donor and host cells were labeled with two different fluorescent dyes—Vybrant DiO and Vybrant DiI (Molecular Probes). These fluorochromes are lipophilic carbocyanine dyes binding to intracellular phospholipid bilayer membranes. They are non-translocating, low cytotoxic dyes which make them particularly suitable for long-term labeling and tracking of cells. Due to their high molecular weight they are considered to be non-gap junction permeable. Vybrant DiO and Vybrant DiI have different absorptions and fluorescence emissions maxima separated by about 65 nm enabling two-color labeling (absorption/fluorescence emission maximas; DiO: 484/501 nm and DiI: 549/566 nm). Vybrant DiO is a fluorochrome emitting light of green and Vybrant DiI of red wavelength. The fluorochrome solution is a dye delivery solution that can be added directly to normal culture media to uniformly suspended or attached culture cells and allow cell population to be marked in distinctive fluorescent colors for identification after mixing. Cells that have been fused or that formed stable clusters can be identified by double labeling [21,22].

Cells were labeled according to the manufacturers instructions. Briefly, before transplantation BMCs were incubated with 2.5 µL/ml Vybrant DiO cell solution and CMs were stained with 2.5 µL/ml Vybrant DiI cell solution (Molecular Probes) for 30 min protected from light at 37 °C in a 5% CO2-humidified atmosphere followed by three washes with PBS.

Following the cell labeling procedure the DiO-marked BMCs were detached with PBS containing 0.6 mM EDTA and 500 units trypsin/ml over a 15 min period at 37 °C. The detached cells were centrifugated at 170 g for 5 min, washed twice with PBS, resuspended in culture medium M199 and mixed with recipient cardiomyocytes at a ratio of 1:4. After 2 days, the medium was changed to 10% horse serum and renewed every 3 days. Co-cultures were maintained in a humidified incubator with an atmosphere of 5% CO2 and 95% air at 37 °C.

2.3 Laser scanning cytometry
After 1, 7, 14 and 28 days the co-culture coverslips with human BMCs and rat CMs were fixed with 2% paraformaldehyde in PBS for 30 min at room temperature and than embedded using fluorescent mounting medium (DAKO). Thereafter cells were measured using laser-scanning-cytometry (LSC, CompuCyte Corp./Olympus), a subtype of slide-based cytometry. Briefly, LSC is a slide based analytic technique that allows rapid quantitative analysis of cells and other specimens tagged with fluorescent dyes. The LSC set-up is built around a conventional epi-fluorescence microscope. Fluorescence is exited by laser light that is reflected by mirrors onto the specimen. The laser sources used are an argon laser for blue light (488 nm) excitation and red emitting helium–neon laser (630 nm). The object is scanned by the laser light, whereby the emitted fluorescences are collected by up to four photomultiplier tubes set up in order to detect only light of a certain color, i.e. from a defined fluorochrome with optical filters. Additionally, the light scatter signal is measured using a photodiode. The measured signals of a scanned field are reconstructed to images for all measured colors. These images are used to define the objects to be measured (trigger) and to acquire different informations from this and the other light signals for a certain object. After one field has been analyzed by the software the microscope stage moves to the next field. The LSC performs the analysis again until the whole area to be analyzed has been scanned. In comparison with conventional cytometry, the LSC technique allows to re-localize and to visualize each event that has been measured. The major advantage of LSC is the combination of two features: The minimal sample volume needed and the connection of fluorescence and morphological informations [21,22].

2.4 Single-cell PCR
Before single cell PCR was carried out, class coverslips containing co-cultured BMCs and rat CMs were transferred to the 1 ml perfusion chamber of a patch clamp inverted microscope (Axiovert 25, Zeiss, Jena, Germany) and superperfused with modified Tyrode's solution with an extracellular tyrode solution containing (in mM; NaCl 135, KCl 4, CaCl2 2, MgCl2 1, NaH2PO4 0.33, Hepes 10, glucose 10, pH 7.4) at room temperature as described previously [12]. Cell detection was performed by morphology and fluorescent phenotype as well as whole cell patch clamp configuration employing micropipettes of 4–5 M{Omega} resistance made with a vertical puller (PIP5, Heka, Lambrecht, Germany) in a two step procedure from 1.5 mm borosilicate capillary tubes (GB150F-8P, Science Products, Hofheim, Germany) [12]. Morphologically identified isolated fluorescent double positive cells were harvested by using micropipettes followed by incubation with a buffer prior to single-cell PCR. The isolated whole cell was reversely transcribed into cDNA using Sensiscript Kit (Qiagen, Hilden, Germany) according to the instructions of manufacturers. The following realtime-PCR reactions were performed using a LightCycler (Roche Biochemicals) with identical cardiomyocyte specific primers for ß-MHC for both species (human and rat). Because the abundance of ß-MHC in a single cell is low, the RT-PCR was performed as nested-PCR. The quality of the reverse transcription reaction was determined by the PCR for the high abundant transcript GAPDH (glyceralaldehyde-3-phosphate dehydrogenase). Table 1 characterizes the used primers.


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  		Table 1. Characteristics and sequences of the used primers
 
2.5 Statistical analysis
All data are represented as mean ± standard error of mean. Statistical evaluation was performed by using Mann-Whitney test (two groups) or one-way analysis of variance (ANOVA). Values of p<0.05 were considered statistically significant. Cell morphology and quantity performed by LSC technique was analysed using WinCyte Analysis Software (CompuCyte).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A
 References
 
3.1 Tracking of transplanted fluorescent labeled BMCs in co-culture
To determine the fate of transplanted BMCs with spontaneous beating rat cardiomyocytes cultures we used the technique of two-color fluorescence labeling performed by LSC. With LSC we tracked transplanted non-selected BMCs in the simulated natural environment of cardiomyocytes to study the crucial interactions between donor and host tissue types. The co-cultures were monitored on days 1, 4, 7, 14 and 28, respectively, to investigate the influence of time to fluorescence cell phenotype. Therefore, cells were imaged by brightfield laser scatter and two-color fluorescence (virtual CompuColorTMimage) to quantify the relationship between morphometric and fluorescence measurements. Fig. 1 shows the qualitative fate of donor and host cells using brightfield laser scatter (Fig. 1a,d and g) and fluorescence cell phenotyping (Fig. 1b,c,e,f,h and i). Morphologically, we were able to distinguish between two different phenotypes. First, we observed isolated Vybrant DiO labeled BMCs without any contact or with direct cell contact to Vybrant DiI labeled recipient CMs (Fig. 1a–f). Secondly, some BMCs and CMs formed spontaneous two-color positive cell formations—also described as hybrid formations (Fig. 1g–i). The double fluorescence phenotyping performed by virtual CompuColorTMimage allows to visualize the two different fluorochromes and to discriminate between both cell types (Fig. 2 ). Due to registration of the exact position of each event, the data files can be combined. Thus, the obtained merged data files contain the color-coated information of both fluorochromes. Thus, two different morphological structures were observed—a merge of both dyes Vybrant DiO and Vybrant DiI in one well structured cell formation (Fig. 2d–f) and a phenomenon, where donor cells are situated on the top of recipient cells-may be simulating a hybrid formation due to overlapping of fluorescences (Fig. 2a–c). Fig. 3 shows the quantitative distribution of donor cell population according to the single DiO-fluorescence and the double positive cell phenotype of all detected events (100%) including the fraction of DiI positive CMs. Within the first 4 days a significant increase of about 18% (p = 0.023) of double labeled cell structures, followed by a slight decrease (no significance) of this cell population to day 28, could be observed. In contrast, the DiO positive BMC population decreased significantly during the first 4 days in co-culture (15%, p = 0.012). Subsequently, there was a slight detectable decrease of DiO positive events. During the cell tracking no significant alterations of the DiI positive CM cell population could be observed (data not shown).


Figure 1
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  		Fig. 1. Fluorescence phenotyping of BMCs and CMs in co-culture, day 28, the left column shows the scatter equivalent to transmission image (morphology), the middle column displays the DiO phenotype and the right column represents the DiI fluorescence; (a–c): DiO positive BMCs; (d–f): DiI positive staining for recipient rat CMs and (g–i): Hybrid cell formation that is positive for DiO and DiI. Images were acquired using LSC, magnification x400.

 

Figure 2
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  		Fig. 2. Cell-fusion and fluorescence overlapping of BMCs with CMs in co-culture—two different virtual phenomenas performed by LSC: Images were obtained 28 days after transplantation: DiO fluorescence: (a), (d); DiI fluorescence: (b), (e) and virtual two-color visualisation of BMCs and CMs by CompuColor images: (c), (f). The left column illustrates the phenomenon of overlapping of two different fluorochromes because a clear morphological differentiation of cells are possible (a–c)—a false positive event of fusion raised. The right column shows one separated fused cell formation and one single DiI positive CM (d–f). Images were acquired using LSC, magnification x400.

 

Figure 3
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  		Fig. 3. Quantitative distribution of donor cell population and hybrid cells according to the fluorescence phenotype of all detected cell events in co-culture (mean ± SEM). (a) Significant decrease of single DiO positive BMC content within the first four days (15%, p = 0.012). (b) The double fluorescence positive cell formations increased significantly to day four (18%, p = 0.023).

 
3.2 Transplanted BMCs express donor and host specific ß-MHC mRNA
To examine and to characterize the gene expression profile of ß-MHC mRNA, isolated two-fluorescence positive cell formations were sampled by emitting fluorescences and patch clamp technique. To ensure that only host and donor specific markers and not the genomic content were investigated we examined the expression of cardiac tissue specific marker ß-MHC mRNA. We analysed 103 isolated double fluorescence positive cell formations. In six of these cell formations we were able to find donor as well as host specific mRNA of ß-MHC and GAPDH as control. These findings can be explained by true cell fusion. Furthermore, nine cells were positive for human ß-MHC mRNA. In order to exclude the event of contamination with recipient nuclear fragments we could show that no rat mRNA expression was observed in the nine transdifferentiated human donor BMCs. The other 88 analyzed hybrid cells showed only rat specific ß-MHC mRNA expression (Table 2 ).


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  		Table 2. Profile of ß-MHC mRNA gene expression
 
Fig. 4 shows species specific melting curves for the ß-MHC mRNA expression obtained from two isolated BMCs and CMs from co-culture. In six of 103 double-fluorescence positive cell formations two different donor and host specific types of melting curves for ß-MHC mRNA were found (Fig. 5 ). The residual 88 cells were only positive for recipient ß-MHC mRNA.


Figure 4
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  		Fig. 4. Melting curve of ß-MHC cDNA (transcription of mRNA) from isolated human BMCs and rat CMs from co-culture; method validation; first peak represents the rat ß-MHC mRNA from two cells, second peak shows the human variant of ß-MHC mRNA for two BMCs.

 

Figure 5
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  		Fig. 5. Melting curve of ß-MHC cDNA (transcription of mRNA) from double positive (blue curve), DiI positive/CM (green curve) and DiO positive/BMC (red curve) co-cultured cells; blue curve with two different melting point maxima represents donor and host specific co-expression of ß-MHC mRNA—cell fusion occurred; the green line shows only rat ß-MHC mRNA content and the red line displays human ß-MHC mRNA in co-culture—transdifferentiation into cardiac lineage.

 
Corresponding to our findings some BMCs have different electrophysiological properties to recipient cardiomyocytes similar to a trial cardiac action potentials in the setting of co-culture [12]. However, in this study no characteristic action potential of a trial ventricular or sinus-like-node different from the environmental neonatal rat cardiomyocytes was obtained in the transdifferentiated or fused cell formations.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A
 References
 
Current experimental data suggest that bone marrow-derived stem cells are able to lead to tissue repair after transplantation to sites of injury. The donor cells either differentiate into the recipient phenotype of the damaged tissue or enhance repair by performing a micro-environment that promotes the regional regeneration of cells endogenous to the recipient cells [1–3,13,14]. The dominant mechanism responsible for tissue repair is currently controversially discussed because the published results are rather confusing and difficult to compare since different isolation and identification methods have been used to determine the cell populations studied.

Using two-color fluorescence tracking of unpurified bone marrow-derived stem cells in co-culture with spontaneous beating neonatal rat cardiomyocytes we could demonstrate that about 18% of the single labeled BMCs rapidly changed the fluorescence phenotype and converted into two-color positive cell formations, particularly within the first 4 days. This unexpectedly high percentage of fluorescence double positive cell formations with donor cell origin and simultaneous expression of host markers-also called hybrid formations-was absolutely unclear. Some, not more closely characterized fractions of these BMCs acquired the typical morphology of cardiomyocytes with a broad cytoplasm and an elevated perinuclear region. Few of the hybrid cell formations were mobile and others were single large flat cells with two nuclei. This is in accordance to our early findings that after 7 days of co-culture some BMCs display typical phenotypic and functional properties of cardiomyocytes [12]. Furthermore, Rastan et al. could show that BMCs and rat CMs exhibited gap junctional communication through dye coupling experiments. In this study we could confirm the coupling process by positive double colour staining [12].

The reason for the observed unexpectedly high level of double positive cell formations performed by LSC-analysis was still unclear. Therefore, we analysed 103 isolated fluorescence double positive cell formations using single-cell PCR to distinguish between species specific expression of ß-MHC mRNA. Analyzing the chromosomal content of cultured stem cells has also been proven to be an effective method in the detection of cell fusion [7,8]. This approach is unable to distinguish stem cell plasticity from cell fusion due to the fact that donor specific markers will be presented in the new fused cells. To address the event of cell fusion we have to track donor as well as host specific cell markers and to show whether these markers are co-expressed in the same cell [20]. The method of single-cell PCR gives an analytic tool to simultaneously monitor cardiac tissue specific markers for transdifferentiation as well as markers for cell fusion. In six of 103 double color positive formations we observed a co-expression of donor and host specific mRNA of ß-MHC (Fig. 5). These findings can be interpreted that a true cell fusion has occurred. Additionally, only nine cells of the hybrid formations were positive for human ß-MHC mRNA and no rat mRNA expression was detectable. This observation strongly supports their identity as cardiomyocyte-like-cells and transdifferentiation of BMCs have occurred. The other 88 analyzed cell formations showed only rat specific ß-MHC expression. The explanation for the observed high degree of fluorescence double positive cell formations by LSC could be as follows (I–IV): Labeling donor cells with fluorescence cell-tracking dyes prior to transplantation has been used to monitor their survival and fate. In general, the dyes do not transfer from labeled to unlabeled cells, although some transfer may have occurred when the cell membranes were disrupted (I). Usually there is a high degree of donor cell death that possibly accounts for the dye transfer (II). Müller-Ehmsen and colleagues demonstrated that only 5% of transplanted cells survive during the acute phase of grafting into the zone of acute myocardial infarction [17]. Nygreen et al. showed only a transient survival of the transplanted cells outside the infarcted myocardium [6]. Therefore, caution must be applied to ensure that the visualized fluorescence signals arise from the living donor cell and not from the host cell, which have acquired dye fragments from dying donor cells. This phenomenon could be a possible explanation to overestimate the event of cell fusion, or could incorrectly suggest transdifferentiation by dye transfer. Furthermore, multylayering (III) of uncoupled donor and host cells may contribute to the overestimation of cell fusion using fluorescence cell-tracking dyes in vitro. While this paper was under review Dimmler and colleagues [23] demonstrated the evidence for a novel transient nanotubular connection with a diameter of 50–800 nm between human EPC's and rat CM's in co-culture allowing the transport of labeled structures (IV). This form of intercellular communication contributes to the acquisition of cardiomyogenic phenotype independent of permanent cellular or nuclear fusion. However, the generation of two-color fluorescence positive hybrid cells may also be due to of a transient transmembrane exchange of proteins or organelles between cells [23]. The membrane nanotubes could transfer fluorescence tagged cell surface proteins and may be responsible for the high percentage of double positive cells within the first 4 days [23].

Using different approaches to monitor the fate of bone marrow cells co-cultured with beating rat cardiomyocytes, we showed that the change of donor cell phenotype were derived not only from transdifferentiation of bone marrow cells but also from cell fusion. This was suggested in previous studies using ischemic and non-ischemic animal models [6,7,9,10,14]. The findings that transplanted bone marrow-derived cells express cardiospecific markers, observed in a very low frequency, were generated either by transdifferentiation or cell fusion. Recent studies observed that the process of cell fusion between bone marrow-derived cells and cardiomyocytes could generate new myocardial tissue [10]. Additionally, cardiac progenitor cells are isolated from adult myocardium and are able to form new cardiomyocytes through differentiation and cell fusion [10,19]. A low frequency of hybrid formation in vivo has also been reported between bone marrow cells and hepatocytes, suggesting that the event of cell fusion might occur after different types of injuries [15]. Nygreen and colleagues demonstrated that bone marrow-derived cardiomyocytes were observed at a low frequency and were derived exclusively through cell fusion [6]. However, elegant studies have recently demonstrated that in different tissue types fusion of haematopoietic cells and tissue specific cells seems to be the main mechanism of generation of ‘transdifferentiated’ cells from bone marrow [10,15].

The donor cells adopt the functional features of the recipient tissue by means of cell fusion mediated acquisition determinants rather than by signal mediated differentiation [7,8,15].

The reported low frequency of these different events and the theoretical inability to generate new cardiac tissue by cell fusion, compared with our results, leads to a high degree of uncertainty that these mechanisms are the main process for myocardial regeneration.

However, the functional and long-term properties of bone marrow-derived cardiomyocytes generated through cell fusion and transdifferentiation need to be investigated in more detail.

In addition, beyond the aim of our study, adult stem cell transplantation could potentially contribute to endogenous myocardial repair based on other mechanisms. Therefore, activation of resident cardiac precursor cells or cardiospheres and the influence of paracrine effects to modulate apoptosis and extracellular matrix might be a possibly appropriate source for cardiac regeneration [19]. Another point of view is since myocyte proliferation and death are occurring side by side in the human heart [17], the myocardium has to be considered as a tissue with cell turnover properties. So the hypothesis arises whether new myocardium can be generated by dedifferentiation and cell cycle re-entry of pre-existing myocytes. [18].

4.1 Limitations
The used trans-species in vitro model is an accepted tool to mimic the cardiac environment. The co-culture of human embryonic cells [24], circulating progenitor cells [25] or CD34+ cells [23] with beating neonatal rat cardiomyocytes pushed the expression of cardiac genes of donor cells indicating the influence of the natural environment [7,8,10]. The presence of cell fusion and transdifferentiation in this in vitro trans-species model does not imply that these phenomenas in vivo occurred. Nevertheless the evidence of transdifferentiation on single cell level without any recipient genetic information suggests that BMCs transdifferentiate into cardiac lineage in the natural environment. It cannot be ruled out that the observed cell fusion in the trans-species model is the result of immune rejection or phagocytosis of dying cell fragments. However, the in vitro as well as in vivo appearance of allogenic bone marrow-derived tissue types is due to spontaneous fusion of BMC with tissue they reside in [7–10]. Thus, the incidence of this process must be examined under physiological conditions of single cell level. The results of these experiments do not allow direct transfer from bench to bedside and the long-term effect and the functional properties of transdifferentiation and cell fusion are unknown.

On the basis of the data outlined here, we conclude that transdifferentiation and cell fusion of unselected bone marrow-derived cells in a heterologous co-culture with cardiomyocytes are true but rare entities. The capacity to generate sufficient contractile tissue and the functional properties are debatable. Furthermore, our data demonstrate that direct cell-to-cell contact is essential for BMC transdifferentiation. In the absence of direct fluorescence coupling between donor and host cells no expression of ß-MHC mRNA was achievable. Depending on the methods used, fluorescence labeling to track the fate of stem cells to confirm the presence or absence of donor specific origin can be confused and can be associated with misinterpretations. Therefore, we employed mRNA analysis on single cell basis as the first step for changing of the genetic information and as the favourable method to investigate the fate of transplanted cells.

Our findings in association with other publications should enhance the extensive experimental research to investigate the crucial mechanisms responsible for myocardial regeneration for further cell-based clinical trails.


   Appendix A
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
References
 
Conference discussion

Dr A. Wechsler (Philadelphia, Pennsylvania, USA): Would it have been possible to do this study in vivo rather than in vitro? That's the first question.

And the second is, won't you have to do it in vivo in order to be certain that what you observed is not a peculiarity of the in vitro co-culture system?

Dr Garbade : The first question, now we tried to do that study in vivo, but at this time we have no results, sorry.

And I think it's very difficult to realize this project in vivo because it's known that many cells are dying following transplantation. And the number of cells that we have after 4 or 8 weeks in vivo are very rare, I think, that's why it's difficult to transfer this model in an in vivo model.

Dr H. Vanermen (Aalst, Belgium): Do you think your study tells us something about the best way to do the administration of stem cells in the infarcted areas, for example?

Dr Garbade : No. It presents only that both mechanisms, plasticity as well as cellfusion are possible mechanisms, but the dominant mechanism is still unclear.

Dr G. Gerosa (Padova, Italy): I take your point on cell survival after transplantation, in an animal model. Do you think it is applicable to your technology the transfection of these cells so that they can overexpress for example grp 94 in order to increase cell survival after transplantation?

Dr Garbade : May be, I can only speculate. At this time, we are trying to transfer this model to our tissue engineering project, but I don't know if we will have success.


   Acknowledgments
 
This work is supported by the German Heart Foundation ‘Deutsche Stiftung für Herzforschung’ by grant F/08/03.


   Footnotes
 
{star} Presented at the joint 18th Annual meeting of the European Association for Cardio-thoracic Surgery and the 12th Annual meeting of the European Society of Thoracic Surgeons, Leipzig, Germany, September 12–15, 2004. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A
 References
 

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A. Orlandi, F. Pagani, D. Avitabile, G. Bonanno, G. Scambia, E. Vigna, F. Grassi, F. Eusebi, S. Fucile, M. Pesce, et al.
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