IN VIVO IMAGING OF STEM CELL MEDIATED TREATMENT IN A MOUSE MODEL OF SPINAL CORD INJURY
Creators: name: LUI, RAMONA
Publisher: name: Universit� degli Studi di Milano
Year: 2010

description: Introduction: The use of adult stem cells in cell-mediated therapies is an area of considerable interest within tissue regeneration research. However, important variables such as the distribution of the injected cells, cell survival, target organ localisation cell proliferation and differentiation cannot be evaluated in vivo by using classical imaging approaches. This study propose multiple labelling protocols for in vivo visualisation by MRI, nuclear imaging and BLI of adult murine neural stem cell-mediated therapy, in spinal cord injury animal models. Methods: Murine neural stem cells (mNSCs) were directly labelled with different amounts of SPIOs (0 - 100 - 200 - 400 ?g Fe/ml) in the culture medium and incubated with iron labelled medium for 24, 48 or 72 h in presence of carriers such as poly-L-Lysine (PLL), polybrene (PB) and protamine sulphate (PS). PLL and PS were tested at different ratio (Fe/PLL 1:0,03, 1:0,06 and 1:0,09 and Fe/PS 1:0,025 and 1:0,05). Labelled cells were analysed for viability, iron content (Perl?s Staining and spectrophotometer analysis), morphology, staminality and differentiation capability. After the labelling protocol set up, the loaded cells were injected into the tail vein of a spinal cord injury murine model and their distribution was followed by MRI for two months. Initial cell distribution was also followed by nuclear imaging after cell labelling with 111In-oxine (60 ?Ci/106 cells). Cells localization, distribution e viability, over time, were analysed in vivo by BLI after injection of mNCS infected with a viral vector expressing Luciferase under a PGK constitutive promoter (PLW vector). Results: the iron content/cell increased in proportion to the incubation time and to the iron concentration in the medium and in relation to different carriers (PLL, PB and PS) in labelled mNSCs. Longer incubation time (48 and 72h) and higher iron concentration (400 ?g Fe/ml) resulted in marked toxicity and lower cell viability. The use of PB and PLL, as carriers, didn?t produce any increase of the labelling efficiency. The incubation for 24h with 200 ?g Fe/ml in presence of different amount of PS didn?t influence significantly the cell viability and the proliferation rate. Furthermore, the percentage of iron-positive cells and the iron content/cell increased in proportion to the PS content in the medium even if higher amount of PS (Fe/PS 1:0.05 ratio) resulted in an aberrant morphology. For this reason, 200 ?g Fe/ml incubated with Fe/PS 1:0.025 ratio for 24h, has been chosen as the best labelling condition. Labelled cells were able to form new neurospheres and maintained the nestin expression demonstrating the maintenance of self-renewal capability and stem cell features and were also able to differentiate, as confirmed by ?-tubulin III and GFAP expression analysis. Nuclear imaging confirmed initial distribution to filter organs while MRI allowed to detect the presence of an iron signal due to stem cell localization into the lesion site since 7 days after injection. BLI permitted to demonstrate the viability of PLW infected mNSCs migrated at the lesion site and supported the MRI data. Conclusions: These results permitted to conclude that NSCs can be efficiently labelled with different molecules without significantly perturbing physiological stem cell features and self-renewal capability. These labelling protocols can be applied for the in vivo visualisation by MRI, nuclear imaging, and BLI of the distribution of stem cells after their transplantation into murine model of disease.