ABSTRACT
Stem cell transplantation is a promising therapeutic approach for several neurological disorders. However, it has yet to fulfill its high expectations, partially due to the lack of a reliable noninvasive method for monitoring the biodistribution of the grafted stem cells in vivo. We have used high-resolution magnetic resonance imaging (MRI) at 17.6 T, combined with efficient magnetic labeling of the stem cells with iron oxide nanoparticles, in order to assess the in vivo detection limit in small animal models. Injection of different concentrations of magnetically labeled stem cells in gel phantoms led to significant reductions in image intensity from small cellular clusters of less than 10 cells. To determine the detection limit in vivo, various numbers of both labeled and unlabeled cells were injected stereotactically into the striatum of rats. Significant hypointense signal changes were observed for 100 labeled cells. After injection of approximately 20 labeled cells, signal reduction at the injection site was observed but could not be assigned unambiguously to the cells. Our results show that high-field MRI allows tracking of a minimal number of cells in vivo, well below the number used in previous studies, opening the possibility of gaining new insights into cell migration and differentiation.
Subject(s)
Brain/cytology , Magnetic Resonance Imaging/methods , Stem Cell Transplantation/methods , Stem Cells/physiology , Animals , Cell Count , Cell Differentiation/physiology , Cell Survival/physiology , Cells, Cultured , Ferric Compounds , Magnetics , Mice , Microscopy, Confocal , Microspheres , Models, Anatomic , Neostriatum/cytology , Rats , Spectrophotometry, Atomic , Stereotaxic TechniquesABSTRACT
Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.