Ex Vivo Labeling of bmdc

Besides the use of bone marrow transplantation to study the role of BMDC in renal disease models, local or systemic infusion of BMDC, or a sub-set of BMDC, has been used. This approach does not require total body irradiation and is more relevant in pre-clinical, rather than experimental, studies. However, since the native bone marrow is not replaced by bone marrow transplantation, the infused BMDC will compete with physiologically recruited BMDC, which might hamper the interpretation of BMDC function.

Infusion of BMDC can be performed using the same genetically labeled reporter cells as used in bone marrow transplantation models, i.e. Y-chromosome, P-galactosidase, EGFP and hPAP. To allow detection of the infused cells in the kidney, non-genetic labeling methods have been applied, e.g. infusion of CFDA fluorescence labeled [64] or iron-dextran labeled cells [65].

Long-Lasting Dyes - CFDA

In a study by Togel [64], carboxy-fluorescein diacetate (CFDA; Vybrant cell tracer kit, Molecular Probes) pre-labeled mesenchymal stem cells were infused in the left carotid artery to study their contribution to post-ischemic renal recovery. CFDA enters the cell by passive diffusion and starts to fluoresce after conversion by intracellular esterases, and is therefore suitable for marking living cells. The label is retained during development, meiosis and in vivo tracing, and is inherited by daughter cells after division or fusion, but is not transferred to adjacent cells in a population. However, after each cell division the fluorescence level is twofold reduced. The major drawback of this labeling method and other comparable fluorescent labels is the limited stability of the fluorescence. In vivo the label could be traced up to 20 days after labeling [66] and is, therefore, not suitable for use in longer-term experiments. The emitted fluorescence of CFDA does not penetrate deep enough through tissue to allow for in vivo detection of the label. However, when the kidney was exposed and renal vasculature was visualized using rhodamine-dextran or FITC-albumin, BMDC within the renal vasculature could be observed in vivo using two-photon confocal microscopy [64].

Particles - Iron-Dextran Labeled Particles

Lange et al. [65] infused mesenchymal stem cells, pre-labeled with carboxy-dextran-coated iron oxide nanoparticles ("Resovist", Schering), into the thoracic aorta via a carotid artery after ischemia. These non-toxic iron-dextran particles are smaller than erythrocytes and are readily taken up by rat mesenchymal stem cells [65]. An advantage of this method is the non-invasive detection of the labeled cells by Magnetic Resonance Imaging (MRI), which allows for real-time imaging in the living animal. However, MRI is a laborious technique which requires equipment and trained personnel. Besides in vivo detection with MRI, ex vivo histological identification of the iron-labeled cells by Prussian blue staining can be used to determine the exact location of the cells. It is unknown how these iron-dextran particles, likely to be cleared by phagocytosis, influence the inflammatory response. In the study by Lange [65], the particles did not elicit an immune response, were stable and detectable in a non-invasive way, therefore, providing an elegant method for ex vivo labeling of cells.

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