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

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

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

<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

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 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

<i>In Vivo</i> Non-Invasive Tracking of Macrophage Recruitment to Experimental Stroke

<div><p>Brain-infiltrating monocyte-derived macrophages are one of the key players in the local immune response after stroke. It is now widely accepted that the inflammatory response is not an exclusively destructive process. However, the underlying molecular mechanisms needed for proper regulation still remain to be elucidated. Here, we propose an <i>in vitro</i> labelling strategy for multimodal <i>in vivo</i> observation of macrophage dynamics distinguished from brain-residing microglia response. Prior to intracerebral transplantation into the striatum of recipient mice or systemic administration, monocytes and macrophages, isolated from luciferase-expressing mice, were labelled with superparamagnetic iron oxide particles. Temporo-spatial localization was monitored by magnetic resonance imaging, whereas survival of grafted cells was investigated using bioluminescence imaging. The labelling procedure of the isolated cells did not significantly influence cell characteristics and resulted in detection of as few as 500 labelled cells <i>in vivo</i>. Two weeks after stereotactic transplantation, the luciferase signal was sustained traceable, with approximately 18% of the original luciferase signal detectable for monocytes and about 30% for macrophages. Hypointensity in MRI of the graft appeared unaltered in spatial location. In a therapeutically relevant approach, systemic cell administration after stroke resulted in accumulation mostly in thoracic regions, as could be visualized with BLI. For detection of homing to ischemic brain tissue more cells need to be administered. Nevertheless, during parallel MRI sessions recruitment of i.v. injected cells to the lesion site could be detected by day 2 post stroke as scattered hypointense signal voids. With further increase in sensitivity, our multi-facetted labelling strategy will provide the basis for <i>in vivo</i> tracking and fate specification of tissue-infiltrating macrophages and their distinct role in stroke-related neuro-inflammation.</p></div

<i>In vivo</i> longitudinal detectability of grafted monocytes and MΦ using MRI at 9.4 T.

<p>(A) Qualitative MR images (MGE sequence) of graft titration with different numbers of SPIO labelled monocytes (WT), with the cell numbers indicated below both hemispheres, n = 1. Transplanted cells are visible as small hypointense clusters. (B) Imaging time course of transplanted WT monocytes and MΦ for days 1, 3 and 12 post transplantation. Left hemisphere 1500, right hemisphere 500 cells, n = 1 per cell type. (C) Representative PB staining of brain tissue section indicating grafted monocytes as PB⁺ (blue) cells. Scale = 50 μm.</p

<i>In vitro</i> detectability of SPIO labelled monocytes and MΦ (WT).

<p>T2-weighted MR images at 11.7 T (TE/TR = 11.25/5,000 ms) of control cells (unlabelled), and of 1x10³, 5x10³, 1x10⁴ and 5x10⁴ labelled cells (168 μg Fe/ml; upper row) with corresponding quantitative T2 maps (lower row). Next to the probes in the upper left image corresponding cell numbers are noted. The cell numbers also apply for phantoms loaded with MΦ. Images were acquired with a MSME sequence. T2 values (ms) are displayed in the color scale bar below the images.</p