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BackgroundMesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, fat and muscle cells and are being investigated for their utility in cell-based transplantation therapy. Yet, adequate methods to track transplanted MSCs in vivo are limited, precluding functional studies. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. These nanoparticles are resistant to chemical and metabolic degradation, demonstrating long term photostability. Here, we investigate the cytotoxic effects of in vitro QD labeling on MSC proliferation and differentiation and use as a cell label in a cardiomyocyte co-culture.ResultsA dose-response to QDs in rat bone marrow MSCs was assessed in Control (no-QDs), Low concentration (LC, 5 nmol/L) and High concentration (HC, 20 nmol/L) groups. QD yield and retention, MSC survival, proinflammatory cytokines, proliferation and DNA damage were evaluated in MSCs, 24 -120 hrs post QD labeling. In addition, functional integration of QD labeled MSCs in an in vitro cardiomyocyte co-culture was assessed. A dose-dependent effect was measured with increased yield in HC vs. LC labeled MSCs (93 ± 3% vs. 50% ± 15%, p < 0.05), with a larger number of QD aggregates per cell in HC vs. LC MSCs at each time point (p < 0.05). At 24 hrs >90% of QD labeled cells were viable in all groups, however, at 120 hrs increased apoptosis was measured in HC vs. Control MSCs (7.2% ± 2.7% vs. 0.5% ± 0.4%, p < 0.05). MCP-1 and IL-6 levels doubled in HC MSCs when measured 24 hrs after QD labeling. No change in MSC proliferation or DNA damage was observed in QD labeled MSCs at 24, 72 and 120 hrs post labeling. Finally, in a cardiomyocyte co-culture QD labeled MSCs were easy to locate and formed functional cell-to-cell couplings, assessed by dye diffusion.ConclusionFluorescent QDs label MSC effectively in an in vitro co-culture model. QDs are easy to use, show a high yield and survival rate with minimal cytotoxic effects. Dose-dependent effects suggest limiting MSC QD exposure.

The gap junction connecting cardiac myocytes is voltage and time dependent. This simulation study investigated the effects of dynamic gap junctions on both the shape and conduction velocity of a propagating action potential. The dynamic gap junction model is based on that described by Vogel and Weingart (J. Physiol. (Lond.). 1998, 510:177-189) for the voltage- and time-dependent conductance changes measured in cell pairs. The model assumes that the conductive gap junction channels have four conformational states. The gap junction model was used to couple 300 cells in a linear strand with membrane dynamics of the cells defined by the Luo-Rudy I model. The results show that, when the cells are tightly coupled (6700 channels), little change occurs in the gap junction resistance during propagation. Thus, for tight coupling, there are negligible differences in the waveshape and propagation velocity when comparing the dynamic and static gap junction representations. For poor coupling (85 channels), the gap junction resistance increases 33 MOmega during propagation. This transient change in resistance resulted in increased transjunctional conduction delays, changes in action potential upstroke, and block of conduction at a lower junction resting resistance relative to a static gap junction model. The results suggest that the dynamics of the gap junction enhance cellular decoupling as a possible protective mechanism of isolating injured cells from their neighbors.

The mechanisms underlying stem cell acquisition of a cardiac phenotype are unresolved. We studied early events during the acquisition of a cardiac phenotype by a cloned adult liver stem cell line (WB F344) in a cardiac microenvironment. WB F344 cells express a priori the transcription factors GATA4 and SRF, connexin 43 in the cell membrane, and myoinositol 1,4,5-triphosphate receptor in the perinuclear region. calcium signal transduction ͉ cardiomyogenesis ͉ gap junctions E xtracardiac stem cell use in early phase clinical trials and animal experiments has raised the possibility that stem cell therapy may be able to repair the damaged heart (for review see refs. 1 and 2). The mechanisms underlying the acquisition of a cardiac phenotype by stem cells have not been elucidated. In some cases, fusion between stem cells and cardiomyocytes has been proposed as the basis for the apparent ''transdifferentiation'' (3). We (4) and others (5, 6) have demonstrated that extracardiac stem cells can acquire a cardiac phenotype when placed in a cardiac microenvironment without the benefit of fusion with surrounding myocytes.We have found that cells from a stem cell line (WB F344) derived from a cloned single nonparenchymal epithelial cell isolated from the liver of a young adult rat (for review see ref.7) respond in vivo (8) and in culture (4) to signals from a cardiac microenvironment and acquire a cardiomyocyte phenotype. Understanding the mechanisms that regulate the acquisition of a cardiac phenotype by a clone-derived stem cell (for example, WB F344 cells) may prove valuable in selecting and manipulating stem cells from other sources, e.g., autologous stem cells, and so enhance their potential for successful use in cell therapy.In the current study we examined the earliest events associated with the acquisition of a cardiomyocyte phenotype by WB F344 cells cocultured with rat neonatal heart cells, a cardiac environment model system (4). We find that cell-cell communication through shared and functional gap junction channels is estab- ] nu oscillations require myoinositol 1,4,5-triphosphate receptor (IP3R) stimulation and are associated with de novo expression of cardiac transcription factors in the WB F344 cells. We hypothesize that collectively these events usher in the acquisition of a cardiac phenotype in the WB F344 stem cell line. Results Early Intercellular Communication Between Neonatal Myocytes andWB F344 Cells. Because WB F344 cells express connexin 43 (Cx43), the predominant isoform expressed in ventricular myocytes, we examined whether myocytes and WB F344 cells develop shared Cx43-derived gap junctions. Twenty-four hours after dsRed fluorescent WB F344 cells were cocultured with heart cells, functional cell-cell communication between their cytoplasm and juxtaposed neonatal cardiomyocytes was demonstrated by fluorescence recovery after photobleaching (Fig. 1). When in older cocultures (4-6 days old) WB F344 cells had acquired a cardiac phenotype, Cx43 was preferentially localized at the interface between neonata...

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