Cell-to-Cell Connection of Endothelial Progenitor Cells With Cardiac Myocytes by Nanotubes
A Novel Mechanism for Cell Fate Changes?
The regeneration of new myocardium by stem or progenitor cells is an important therapeutic option. Cellular or nuclear fusion is considered as an alternative to cell reprogramming by transdifferentiation. However, the generation of hybrid cells may also be a consequence of a transient transmembrane exchange of proteins and organelles between cells. Therefore, we investigated the formation of intercellular connections, which may allow the transport of macromolecular structures between labeled adult human endothelial progenitor cells (EPC) and GFP-expressing neonatal rat cardiomyocytes (CM) in a coculture system. FACS analysis revealed that, 6 days after initiation of coculture, 2.1±0.4% of the cells stained positive for GFP and Dil-ac-LDL. 6 hours after initiation of the coculture, ultrafine intercellular structures between Dil-ac-LDL-labeled EPC and GFP-expressing CM were observed. The number of EPC, which established nanotubular connections with CM increased from 0.5±0.2% after 6 hours to 2.6±0.3% after 24 hours of coculture. The intercellular connections had a diameter from 50 to 800 nm, a length of 5 to 120 μm, and were only transiently established. To determine whether the nanotubular structures allowed the transport of organelles, we labeled CM with a mitochondrial live tracker (MitoTracker). Using time-lapse video microscopy, we observed the transport of stained complexes between CM and EPC resulting in up-take of MitoTracker-positive structures in EPC. Thus, the present study shows a novel type of cell-to-cell communication between progenitor cells and CM in vitro, which may contribute to the acquisition of a cardiomyogenic phenotype independent of permanent cellular or nuclear fusion.
The formation of new myocardium by stem or progenitor cells is an important therapeutic option to replace myocardial tissue after myocardial infarction. Different types of progenitor cells including hematopoietic progenitor cells, mesenchymal stem cells, SP cells, or cardiac resident progenitor cells were shown to acquire a cardiomyogenic phenotype after infusion or injection in mice models after myocardial infarction. Despite these findings, recent studies questioned the plasticity of adult hematopoetic progenitor cells (for review, see Anversa et al1).
To determine the molecular mechanism(s) underlying cardiomyogenic differentiation of adult stem or progenitor cells, we used a coculture system of neonatal rat cardiomyocytes to mimic the cardiac environment.2 Coculture of embryonic endothelial cells,2 circulating progenitor cells isolated from human blood,3 or CD34+ cells4 with neonatal cardiomyocytes triggered the expression of cardiac genes within the progenitor cells. Interestingly, the expression of cardiac marker proteins was also detected when fixed, dead cardiomyocytes instead of living cardiomyocytes were used indicating that the presence of myocyte membrane proteins is sufficient for the acquirement of a cardiomyogenic phenotype of human EPC. Although these data support the hypothesis that differentiation of EPC can principally occur in the absence of living cardiomyocytes, the incidence of α-sarcomeric actinin expressing human EPC was significantly lower, when fixed cardiomyocytes were used for the coculture assay.3
Cell fusion is considered an alternative to cell reprogramming by transdifferentiation leading to the generation of hybrid cells with progenitor cell origin and simultaneous expression of myocyte markers.5,6 The coexpression of donor and recipient markers may be explained by cellular or nuclear fusion. Alternatively, a cell expressing hybrid markers may also result from an exchange of marker proteins or signaling molecules between the recipient and donor cells. Recently, Rustom et al described the existence of nanotubular structures transiently connecting neighboring mammalian cells.7 These nanotube-like structures had a diameter of 50 to 200 nm, can stretch more than several cell diameters in length, contain F-actin, and allow the transfer of multiprotein complexes and organelles between cells.7 Similarly, nanotube-like networks between a variety of immune competent cells such as NK-cells, macrophages, and B-cells were described.8 These membrane nanotubes could transfer GFP-tagged cell surface proteins, class I MHC, and GPI-anchored proteins.8 Consequently, nanotubes resemble a type of transient cell fusion, which would allow the intercellular exchange of intracellular protein content between cells and the generation of hybrid cells, without the classical cellular or nuclear fusion events. Therefore, we hypothesized that nanotubular communication is involved in the acquisition of a myocyte-like phenotype in EPC.
Materials and Methods
Neonatal cardiomyocytes were isolated and infected with GFP-encoding adenoviruses (MOI 20). Alternatively, nontransduced cardiomyocytes were stained with MitoTracker. Cardiomyocytes were washed 3 times and used for coculture experiments with isolated Dil-ac-LDL-labeled EPC3 a ratio of 1:3. Cocultured cells were used for confocal microscopy, FACS analysis or time lapse video imaging (for details see online data supplement available at http://circres.ahajournals.org).
To study the intercellular communication of EPC with cardiomyocytes, we used a previously described coculture system2,3 Neonatal cardiomyocytes were transduced with an adenovirus encoding GFP, resulting in 98.8±1.2% positive cardiac myocytes after 6 days of infection. EPCs were labeled with Dil-ac-LDL 30 minutes before coculture with GFP-transduced cardiomyocytes. The coculture was then studied by confocal microscopy.
Approximately 6 hours after initiation of the coculture, ultrafine intercellular structures between Dil-ac-LDL-labeled EPC and GFP-expressing cardiac myocytes were observed (Figure 1A). These structures stained positive for wheat germ agglutinin and were also detectable between EPC and nontransduced cardiac myocytes (online Figure I). The number of EPC, which established nanotubular connections with cardiomyocytes increased from 0.5±0.2% after 6 hours to a maximum of 2.6±0.3% after 24 hours (Figure 1B). The nanotubular structures were only transiently established and their number already declined after 48 hours (Figure 1B). The intercellular connections had a diameter ranging from 50 to 800 nm and a length from 5 to 120 μm (Figure 1C). The nanotubular structures only rarely displayed a branched appearance. The nanotubular intercellular connections were highly sensitive to stress. Particularly the ultrafine nanotubular structures exhibited a sensitivity to prolonged light excitation specifically during the phase of establishment of the connection (Figure 2A). Moreover, “shaking” of the culture dishes disrupted the intercellular connections (Figure 2B).
Six days after initiation of coculture, 2.1±0.4% of the cells stained positive for GFP and Dil-ac-LDL as assessed by FACS. Gentle shaking of the coculture significantly reduced the number of Dil-ac-LDL+/GFP+ cells during the coculture (data not shown). To quantify the number of nuclei within the GFP+/Dil-ac-LDL+ cells, cells were isolated by trypsinization and replated on low density to detect the cells at a single cell level. Immunostaining revealed that the majority (92.9±7.1%) of the GFP+/Dil-ac-LDL+ cells were mononucleated (Figure 2C).
To determine whether the nanotubular structures allowed for the transport of organelles, we labeled cardiomyocytes with the cell-permanent MitoTracker green probe. Thirty minutes after labeling with the MitoTracker, cardiomyocytes were washed and were cocultured with Dil-ac-LDL-labeled EPC. After 6 to 24 hours, nanotubular structures were established between MitoTracker-positive cardiomyocytes and Dil-ac-LDL-labeled EPC (Figure 3). Using time-lapse video microscopy, we observed the movement of MitoTracker-green positive complexes between the cardiomyocyte and the Dil-ac-LDL-labeled EPC (Figure 3) resulting occasionally in the up-take of cardiomyocyte-derived MitoTracker green-stained complexes in the human EPC (online Figure II). However, transport of EPC-derived mitochondria was not detected from the EPC to the cardiac myocytes, when mitochondria of EPC were stained (data not shown).
The data of the present study demonstrate that, on coculture, adult circulating blood-derived progenitor cells can transiently form ultrafine intercellular connections with neonatal rat cardiac myoctes. These nanotubular structures allow for the transport of GFP and organelles from the cardiomyocyte to the human EPC in vitro. This novel type of transient cell fusion resulting in the exchange of macromolecular complexes may contribute to the cell fate change of EPC during coculture.
The mechanisms, by which adult progenitor cells acquire a different cell fate, are discussed controversially. Differentiation, transdifferentiation, and fusion have been proposed as possible modes of cell fate changes. The unambiguous distinction between these possibilities is demanding. Fusion usually is defined as cellular or nuclear fusion leading to a binucleated hybrid cell or a cell with a tetraploid progeny, respectively. The result of the present study now demonstrates an additional option: Progenitor cells can transiently “fuse” to cardiomyocytes allowing the exchange of macromolecules via nanotubular structures. However, as shown by time-lapse video microscopy, no hybrid nuclei progeny is formed. The present study does not elucidate the existence of nanotube-like structures within intact tissue. Therefore, future studies have to evaluate whether transient cell communication by nanotubes is involved in cell reprogramming in vivo.
Long distance cell communication was thought to be predominantly accomplished by secreted substances. However, several recent findings suggest that cells have the capacity to explore the extracellular environment and establish direct contact over long distances. Examples are long actin-based extensions (“cytonemes”) during Drosophila development, actin-based filopodia and myopodia in insect neuronal and muscle cells, respectively,9 or noncell-autonomous long-distance RNA transport systems in plants (“plasmodesmata”).10 Recently, mammalian cells also have been shown to be capable of building nanotubular “highways” between cells.7,8 The macromolecular structure such as the diameter and the length of the nanotubes described in these studies is similar to the ones detected between EPC and cardiomyocytes. However, the structural and functional relationships between these mammalian nanotubes and other intercellular communication structures in primitive organisms are unclear. Moreover, future work needs to identify how these extensions develop and define the mechanisms, by which specific cells allow the establishment of a tunnel for protein/organelle exchange. Of note, although most of the cardiomyocytes and EPC develop filopodia-like extensions in the present study, final communication is only established in 2.6% of the progenitor cells. Visual counting of the nanotubes may slightly underestimate the real existing intercellular communication, because nanotubes between neighboring cells in close proximity may not be detectable. However, the small percentage of human cells, which are positive for GFP overexpressed in cardiomyocytes, suggests that only a small percentage of the progenitor cells are capable to establish functional intercellular connections.
This study was supported by the DFG (Di 600/6-1), the Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad to M.K. We thank Christine Goy for expert technical assistance.
Original received November 16, 2004; resubmission received March 24, 2005; accepted April 25, 2005.
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