Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

Abstract

Therapeutic implantation of mesenchymal stem cells (MSCs) is entering the realm of clinical trials for several human diseases, and yet much remains uncertain regarding their dynamic distribution and cell fate after in vivo application. Discrepancies in the literature can be attributed in part to the use of different cell labeling/tracking methods and cell administration protocols. To identify a stem cell detection method suitable for myocardial implantation in a large animal model, we experimented on three different MSC labeling methods: adenovirus-mediated expression of enhanced green fluorescence protein (EGFP) and β-galactosidase (LacZ), and nuclear staining with DAPI. Intramuscular and intracoronary administrations of labeled porcine MSCs identified the nuclear affinity dye to be a reliable stem cell tracking marker. Stem cell identification is facilitated by an optimized live cell labeling condition generating bright blue fluorescence sharply confined to the nucleus. DAPI-labeled MSCs retained full viability, ceased proliferation, and exhibited an increased differentiation potential. The labeled MSCs remained fully active in expressing key growth factor and cytokine genes, and notably exhibited enhanced expression of the chemokine receptor CXCR4 and its ligand SDF1, indicating their competency in response to tissue injury. Histological analysis revealed that approximately half a million MSCs or ϳ2% of the administered MSCs remained localized in the normal pig heart 2 weeks after coronary infusion. That the vast majority of these identified MSCs were interstitial indicated the ability of MSCs to migrate across the coronary endothelium. No evidence was obtained indicating MSC differentiation to cardiomyocyte.Key words: Mesenchymal stem cells; Heart; Tracking; Implantation INTRODUCTIONfusion, and intercellular communication through GAP junctions or nanotubes have been proposed to account for the seeming pluripotency of adult stem cells in variStem cell therapy has in recent years received much attention in the field of regenerative medicine. Theraous implantation studies (26,32,50,53,62). Technically, these discrepancies may be caused in part by the use of peutic implantation of mesenchymal stem cells (MSCs), in particular, has been attempted in several clinical trials different cell labeling/tracking methods, cell administration protocols, timing of cell transplantation after tissue for hematological disorders, cardiovascular diseases, osteogenesis imperfecta, neurological pathologies, and even injury, and possibly misinterpretation of microscopy results (4). cancer therapy (9,14,23). The ease of isolation, capacity for large-scale expansion, and alleged immunoprivileged Many cell labeling techniques have been used for tracking implanted stem cells in vivo. Fluorescent quanstatus of MSCs (3,21,29) are likely to generate more clinical applications in the future. Central to these efforts tum dots and iridium nanoparticles are randomly internalized by cells during culturing (13,41). Membrane is a thorough unders...

Potential of Skin Fibroblasts for Application to Anterior Cruciate Ligament Tissue Engineering

“…The first choice for torn ACL replacement is the bone-ligadiacetate (CFDA) or other fluorescent biomolecules, could be achieved before grafting, to evaluate the fate of ment-bone implantation, using the central portion of the patellar tendon (5,8,19). Thus, the combination of the the labeled cells populating the grafts after implantation (8,17). However, such cell biomarker cannot be traced biodegradable scaffold with two bone anchors seems to be an important feature of any potential ACL substitute after 1-2 months in the grafts postimplantation, because the marker is lost by labeled cells that undergo proliferafor torn ACL replacement.…”

Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

“…However, so far there was no data relating the relationship between GFP and the paracrine actions of MSCs in vivo. This question is particularly relevant because some labels may affect host gene expression in vivo, as observed in a previous report (Leiker et al 2008).…”

Enhancing the efficacy of mesenchymal stem cell therapy

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