Functional integration of quantum dot labeled mesenchymal stem cells in a cardiac microenvironment.

Bone marrow derived multipotent mesenchymal stem cells (MSCs) have the potential to differentiate into bone, cartilage, fat, and muscle cells and are being investigated for their utility in cell-based therapies. Stem cell transplantation therapy represents a novel and innovative approach with the promise to restore function to diseased or damaged heart muscle. Transplanted MSCs are expected to engraft, differentiate, and remodel in response to the surrounding cardiac microenvironment significantly changing the therapeutic approach for heart disease. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. Here, we describe in vitro QD labeling of MSCs, MSC integration in a cardiomyocyte co-culture microenvironment, and a fluorescent recovery after photobleaching (FRAP) technique to assess functional cell-cell communication. FRAP techniques establish an optical record of dynamic cellular interactions with high spatial and temporal resolution and can be used to successfully evaluate dynamic changes in cellular coupling in multicellular preparations.

Labeling and imaging mesenchymal stem cells with quantum dots.

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, -cartilage, adipose, and muscle cells. Adult derived MSCs are being actively investigated because of their potential to be utilized for therapeutic cell-based transplantation. Methods to track MSCs in vivo are -limited, preventing long-term functional studies of transplanted cells. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. Nanoparticles are resistant to chemical and metabolic degradation, demonstrating long-term photostability. Here, we describe the technique to label MSCs with QDs and demonstrate intracellular QD distribution in the labeled MSCs with laser scanning confocal fluorescent microscopy.

Quantum dot labeling of mesenchymal stem cells.

BACKGROUND: Mesenchymal 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. RESULTS: A 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. CONCLUSION: Fluorescent 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.

Beating and arrested intramyocardial injections are associated with significant mechanical loss: implications for cardiac cell transplantation.

BACKGROUND: Cellular cardiomyoplasty is emerging as a potentially novel therapeutic option for heart failure and typically involves direct intramyocardial injection of donor cells into a beating heart. Yet, limited rates of cell engraftment remain an obstacle to be overcome before cell therapy is fully recognized. Mechanical and biological mechanisms may account for observed donor cell loss. This study examines acute mechanical loss during intramyocardial injections in beating and arrested hearts. MATERIALS AND METHODS: A porcine cardiopulmonary bypass model was used. Animals underwent either beating (n = 5) or arrested (n = 5) intramyocardial injections into the left ventricle. Fluorescent microspheres were used in lieu of cells because they are biologically inert. Thirty minutes after delivery, animals were euthanized. Microspheres in cardiac and peripheral tissues were quantified using flow cytometry. RESULTS: Approximately 10% of microspheres were retained within the site of injection in both groups. There was no statistical difference between microsphere retention rates in either the beating or the arrested heart group. Microspheres were found in peripheral organs, pericardial fluid, and the delivery device. CONCLUSIONS: The majority of microspheres injected intramyocardially are lost in both beating and arrested hearts. Cardiac standstill does not enhance microsphere retention. Possible mechanisms include leakage from the injection site and washout via the cardiac venous/lymphatic system. Delivery strategy will need to be modified if more cells are to be retained within the target organ.