Fig 1.
(A) Schematic representation of the designed vector system. The two imaging reporters Luciferase 2 (Luc2) and green fluorescence protein (GFP) are kept under the control of the constitutive active promoter EF1α and are linked via the T2A peptide sequence to ensure equal expression level of the two proteins. (B) Representative microscopic image of transduced and FACS sorted hNSCs. The overlay of the bright-field and fluorescence image is shown right. Scale bar: 50 μm
Fig 2.
Effect of the transduction and 19F labeling on hNSCs.
(A) Cell viability is shown for WT hNSCs and transgenic EF1-Luc2-GFP hNSCs with and without 19F labeling (n = 6–8). (B) Cell proliferation was compared among different cell lines. The values were normalized to the WT hNSCs and expressed in percentage (n = 5). (C) In vitro BLI signal from transgenic unlabeled hNSCs compared to 19F labeled cells. (D) In vitro BLI signal is displayed for a dilution series of cells (labeled and unlabeled) in 6 independent experiments. (+) outliers at least 1.5x interquartile range.
Fig 3.
In vitro detectability of 19F labeled hNSCs by means of 19F MRI and 19F MRS.
(A) 19F MRS of labeled hNSCs and a KF solution as internal standard to quantify the amount of 19F atoms per cell (B) high resolution 1H MR image (left), acquired during the same session of the labeled cells 19F MR image (center). 1H and 19F images are then merged to obtain a correct spatial localization (right).
Fig 4.
Longitudinal evaluation of cell viability by in vivo BLI.
(A) BL images of unlabeled (left) and labeled (right) hNSCs, implanted in the right striatum and longitudinally evaluated from day 0 to day 9 post implantation [19F labeled cells day 0 (n = 9), day 1 (n = 9), day 2 (n = 8), day 5 (n = 8), day 7 (n = 7), day 9 (n = 5) / unlabeled cells day 0–9 (n = 4)]. (B) SBR (signal to background ratio) normalized to the first time point shows a decrease of cell viability within one week. (+) outliers at least 3x interquartile range.
Fig 5.
(A) High resolution 1H MR image (left) was acquired as anatomical reference and 19F MR image (center) was then acquired to localize the implanted cell graft. 1H and 19F images were superimposed to combine anatomical information and spatial graft localization (right). (B) Two animals with quantitative depiction of 19F-labelled detectable cells at both two and eight days post implantation. C) Quantification of hNSCs labelled with PFPE at both, day 2 and day 8.
Fig 6.
Immunohistochemistry validation of grafted hNSCS.
Histology of transplanted H9-EF1-Luc2-GFP cells either labeled with 19F (n = 4) (A) or unlabeled (n = 4) (B) 9 days after transplantation. An overview of the mouse brain slice (scale bar: 400 μm) and higher magnification of the grafted cells verified the localization of the transplanted cells (4x magnification, scale bar: 200 μm / 10x magnification, scale bar: 50 μm / 60x magnification, scale bar: 10 μm). GFP-transgene expression (green) and immunostainings with antibodies against: DCX, neuronal marker; HuNu, human nuclei marker; Mito, human mitochondria; GFAP, astrocyte marker; Luc, luciferase marker.
Fig 7.
Histological analysis of graft survival and immune response of host tissue.
An overview of the mouse brain slice (scale bar: 400 μm) and higher magnification confirm that Iba1 positive cells surround the cell graft (4x magnification, scale bar: 200 μm / 10x magnification, scale bar: 50 μm / 60x magnification, scale bar: 10 μm). GFP-transgene expression (green) and immunostainings with antibodies against: Iba1 (IBA), immunoreaction and HuNu, human nuclei marker. In the lower row 3D images of the IBA staining illustrate the surrounding of the cell graft by the immune cells.