Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants

Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs) are promising candidates for this type of therapy, because they (1) have migratory properties, enabling migration after transplantation, (2) can differentiate into sensory neurons and glial cells, and (3) can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP). Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 μm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair

Impact of Age on the Cerebrovascular Proteomes of Wild-Type and Tg-SwDI Mice

The structural integrity of cerebral vessels is compromised during ageing. Abnormal amyloid (Aβ) deposition in the vasculature can accelerate age-related pathologies. The cerebrovascular response associated with ageing and microvascular Aβ deposition was defined using quantitative label-free shotgun proteomic analysis. Over 650 proteins were quantified in vessel-enriched fractions from the brains of 3 and 9 month-old wild-type (WT) and Tg-SwDI mice. Sixty-five proteins were significantly increased in older WT animals and included several basement membrane proteins (nidogen-1, basement membrane-specific heparan sulfate proteoglycan core protein, laminin subunit gamma-1 precursor and collagen alpha-2(IV) chain preproprotein). Twenty-four proteins were increased and twenty-one decreased in older Tg-SwDI mice. Of these, increases in Apolipoprotein E (APOE) and high temperature requirement serine protease-1 (HTRA1) and decreases in spliceosome and RNA-binding proteins were the most prominent. Only six shared proteins were altered in both 9-month old WT and Tg-SwDI animals. The age-related proteomic response in the cerebrovasculature was distinctly different in the presence of microvascular Aβ deposition. Proteins found differentially expressed within the WT and Tg-SwDI animals give greater insight to the mechanisms behind age-related cerebrovascular dysfunction and pathologies and may provide novel therapeutic targets

Erfassung subjektiver Theorien von Hauptschullehrern, des Unterrichtsklimas sowie berufsbezogener Einstellungen Dokumentation und Beschreibung der von der Forschungsgruppe 'Sozialpsychologie der Schule' entwickelten Skalen

SIGLEUuStB Koeln(38)-880106436 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

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

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

<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