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.

Cancer detection and biopsy classification using concurrent histopathological and metabolomic analysis of core biopsies.

BACKGROUND: Metabolomics, the non-targeted interrogation of small molecules in a biological sample, is an ideal technology for identifying diagnostic biomarkers. Current tissue extraction protocols involve sample destruction, precluding additional uses of the tissue. This is particularly problematic for high value samples with limited availability, such as clinical tumor biopsies that require structural preservation to histologically diagnose and gauge cancer aggressiveness. To overcome this limitation and increase the amount of information obtained from patient biopsies, we developed and characterized a workflow to perform metabolomic analysis and histological evaluation on the same biopsy sample. METHODS: Biopsies of ten human tissues (muscle, adrenal gland, colon, lung, pancreas, small intestine, spleen, stomach, prostate, kidney) were placed directly in a methanol solution to recover metabolites, precipitate proteins, and fix tissue. Following incubation, biopsies were removed from the solution and processed for histology. Kidney and prostate cancer tumor and benign biopsies were stained with hemotoxylin and eosin and prostate biopsies were subjected to PIN-4 immunohistochemistry. The methanolic extracts were analyzed for metabolites on GC/MS and LC/MS platforms. Raw mass spectrometry data files were automatically extracted using an informatics system that includes peak identification and metabolite identification software. RESULTS: Metabolites across all major biochemical classes (amino acids, peptides, carbohydrates, lipids, nucleotides, cofactors, xenobiotics) were measured. The number (ranging from 260 in prostate to 340 in colon) and identity of metabolites were comparable to results obtained with the current method requiring 30 mg ground tissue. Comparing relative levels of metabolites, cancer tumor from benign kidney and prostate biopsies could be distinguished. Successful histopathological analysis of biopsies by chemical staining (hematoxylin, eosin) and antibody binding (PIN-4, in prostate) showed cellular architecture and immunoreactivity were retained. CONCLUSIONS: Concurrent metabolite extraction and histological analysis of intact biopsies is amenable to the clinical workflow. Methanol fixation effectively preserves a wide range of tissues and is compatible with chemical staining and immunohistochemistry. The method offers an opportunity to augment histopathological diagnosis and tumor classification with quantitative measures of biochemicals in the same tissue sample. Since certain biochemicals have been shown to correlate with disease aggressiveness, this method should prove valuable as an adjunct to differentiate cancer aggressiveness.

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.