Newly generated hNSCs.

<p>(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</p

Histological analysis of graft survival and immune response of host tissue.

<p>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.</p

Longitudinal evaluation of cell viability by <i>in vivo</i> BLI.

<p>(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.</p

<i>In vivo</i>¹⁹F MRI.

<p>(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.</p

Immunohistochemistry validation of grafted hNSCS.

<p>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.</p

<i>In vitro</i> detectability of ¹⁹F labeled hNSCs by means of ¹⁹F MRI and ¹⁹F MRS.

<p>(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).</p

Effect of the transduction and ¹⁹F labeling on hNSCs.

<p>(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) <i>In vitro</i> BLI signal from transgenic unlabeled hNSCs compared to ¹⁹F labeled cells. (D) <i>In vitro</i> BLI signal is displayed for a dilution series of cells (labeled and unlabeled) in 6 independent experiments. (+) outliers at least 1.5x interquartile range.</p

List of primary (1°) and secondary (2°) antibodies used for ICC and IHC, with corresponding dilutions and distributers.

<p>List of primary (1°) and secondary (2°) antibodies used for ICC and IHC, with corresponding dilutions and distributers.</p

Effect of iron labelling on cell viability.

<p>(A) Representative BLI images of an <i>in vitro</i> BLI dilution series with unlabelled (control) and SPIO labelled (168 μg Fe/ml) luc⁺ monocytes (upper images) and MΦ (lower images). Values (ph/s/cm²/sr) are indicated in the color scale bars below the images. (B) Quantitative plot of mean total flux (ph/s). Values are normalized to photon flux of either 1x10⁴ control (light grey) or labelled (dark grey) cells (for both cell types n = 2, including 3 samples for each cell number). (C) Viability of luc⁺ monocytes (light grey) and MΦ (dark grey) was estimated via Presto Blue reaction for different SPIO concentrations. Values (relative light units–RLUs) were normalized to unlabelled control cells and are displayed as percentage for both cell types (n = 3, with 3 samples for each concentration).</p

Representative MR images of i.v. injected WT monocytes and luc⁺ MΦ.

<p>Displayed are T2*-weighted images (second echo; TE = 8.5ms), acquired with a MGE sequence at 11.7T directly after MCAO surgery and before cell injection (upper row, d0), on day 2 as well as day 3 post stroke (1 and 2 days post cell injection) with corresponding T2 maps calculated for better stroke visualization. T2 relaxation times are displayed with the color scale bar below the images. Left column: contrast agent labelled WT monocytes (2.64x10⁶); center column: contrast agent labelled luc⁺ MΦ (2.5x10⁶); right column: unlabelled control luc⁺ MΦ (2.5x10⁶). Hypointense signal voids evoked by cells reaching the ischemic brain territory are indicated by red arrows.</p