Figure 1.
Diagram illustrating the steps involved in this study.
Overview of the lentiviral vector constructs used to induce reporter gene expression and the experiments performed with these respective vectors.
Table 1.
Vector constructs used for gene transfer.
Figure 2.
Results of uptake experiments with 99mTcO4− in HEK293T cells after the induction of hNIS expression.
Uptake ratios resulting from the transduction with a unicistronic LV (b) and a bicistronic LV (c). Significantly higher uptakes were observed in cells expressing hNIS. **: p<0.01; ***: p<0.001; ****:p<0.0001.
Figure 3.
Optimization of LV constructs and selection.
Different promoters were tested with FACS (AU: arbitrary units) for eGFP expression in MSCs transduced with different LV driven by different human or viral promoters (a). On day 38, FACS selection of the highest expressors was done (red arrow on panel (a)), and this population was also monitored (b). 99mTcO4− uptake experiment in Fluc-hNIS or Fluc expressing MSCs with or without puromycin selection (c). ****:p<0.0001.
Figure 4.
Validation of the multicistronic LV contruct.
99mTcO4− uptake kinetics in MSCs transduced with the two different multicistronic LV constructs or wild type MSCs (a). Tracer elution from Fluc-hNIS expressing MSCs (b). The effect of different concentrations of NaClO4 on the uptake of 99mTcO4− in Fluc-hNIS expressing MSCs (c). Immunocytochemistry against hNIS on Fluc-hNIS expressing MSCs and DAPI (d), against 3flag in 3flagFluc-hNIS expressing MSCs and DAPI (e) and negative control (f). Immunocytochemistry against 3flag in 3flagFluc expressing MSCs and DAPI (g) and negative control (h).
Figure 5.
In vitro optical validation of the imaging reporter genes.
In vitro BLI (a) and CLI (b) of Fluc-hNIS expressing MSCs and Fluc expressing MSCs. ****: p<0.0001.
Figure 6.
Adipogenic, osteogenic and chondrogenic differentiation capacity of Fluc-hNIS expressing MSCs, Fluc expressing MSCs and wild types.
Extraction of the Oil red O dye following adipogenic differentiation measured at 490(a). Extraction of the Alizarin red S dye from differentiated osteoblast matrix, measured at 560 nm (b). Extraction of Alcian Blue dye from differentiated chondrocyte matrix, measured at 595 nm (c). *: p<0.05, **: p<0.01. Images of the Oil red O stainings (d,g,j), the Alizarin red S stainings (e,h,k) and the Alcian Blue stainings (f,I,l) for all conditions: Fluc-hNIS, Fluc expressing cells and wild types.
Figure 7.
BLI data of intravenously injected Fluc-hNIS expressing MSCs.
An accumulation of cells could be visualized in the lungs. Different cell numbers were injected, and total flux was measured. Both parameters were strongly correlated with an R2 of 0.97.
Figure 8.
Visualization and follow-up of MSC xenografts.
Fluc expressing MSCs were injected on the left flank of the body, and Fluc-hNIS expressing MSCs were engrafted on the right side of the body. Xenografts of 10,000 injected cells were injected near the front, and 1,000,000 cells were injected at the back for both conditions. Time activity curves of the ratios comparing the Fluc-hNIS expressing xenograft to background up tissues, showing higher expressions in the Fluc-hNIS expressing xenograft compared to background signals (a,d). BLI was also performed to follow the xenografts over time, with robust expression within all xenografts (b,e). CLI was performed to follow up the mice after 124I injection. The Fluc-hNIS expressing MSCs could be visualized and monitored over time (c,f).