10 research outputs found
Treatment of 3D neuro-spheroids with BACE1 and γ-secretase inhibitors.
<p>3D neuro-spheroids differentiated from five AD patients’ iPSC lines were treated with either BACE1 (BI, 1 μM; blue) or γ-secretase inhibitor (g-SI, 0.5 or 1 μM; red) for two days. Media were collected for Aß 40 (top) and 42 (middle) ELISA quantification. A. Lower levels of Aß 40 were found in media after treatment with either BACE1 or γ-secretase inhibitors. B. Aß42 levels from AD1 remained unchanged after being exposed to BACE1 inhibitor. Aβ42 levels from other AD subjects were reduced. C. The drug levels of BACE1 inhibitor (blue, dosing at 1 μM) and γ-secretase inhibitor (red, dosing at 1 μM) in 3D neuro-spheroids were quantified by LC-MS/MS. The graph shows Mean±SEM; * represents p< 0.05, comparison of inhibitor vs. DMSO.</p
Relative protein levels in 3D neurons differentiated from five AD subject-derived iPSC lines.
<p>Relative protein levels in 3D neurons differentiated from five AD subject-derived iPSC lines.</p
Treatment of 2D cell culture by a BACE1 inhibitor.
<p>2D neurons differentiated from five AD patients’ iPSC lines were treated with increasing concentrations of BACE1 inhibitor (BI, 0.1, 0.5 and 1 μM; blue) or DMSO as control (grey) for two days. Conditioned media were collected and Aß 40 and 42 were quantified by ELISA. Both Aß 40 (A) and 42 (B) levels were significantly reduced in all lines from 5 AD subjects after the treatment with the BACE inhibitor at all doses tested. Levels of Aβ42 were undetectable in 1 μM BI-treated neurons derived from AD5. The graph shows Mean±SEM; * represents p< 0.05, comparison of inhibitor vs. DMSO.</p
Structures of the BACE1 and γ-secretase inhibitors used to treat 3D neurons.
<p>A. Immunocytochemical staining of 3D neuro-spheroid section for Tau protein using antibody BT-2. B. ICC of neuro-spheroid section for phosphorylated Tau. Antibody AT270 specifically stains phosphor-Tau at residue Thr181. C. LY2886721, a potent and selective BACE1 inhibitor (left), and Compound E, a cell permeable non-competitive inhibitor of γ-secretase (right), are used to treat neurons.</p
Characterization of neural stem cells by protein markers Sox1 and PAX6.
<p>iPSC-derived neural stem cells were identified by different protein markers, Sox1 (green) and PAX6 (red). Sox1 and PAX6 expression was higher in lines N3 and N4 compared to lines N1, N2 and N5. Merged images are illustrated in yellow. Scale bar: 50 μm.</p
Treatment of 2D cell culture by a γ-secretase inhibitor.
<p>2D neurons differentiated from five AD patients’ iPSC lines were treated with increasing concentrations of γ-secretase inhibitor (g-SI, 0.1, 0.5 and 1 μM; red) or DMSO as control (grey). A. Aβ40 levels in conditioned media from cells treated with γ-secretase inhibitor were significantly reduced. B. Aβ42 levels were significantly decreased in all 5 AD subjects after treatment with the γ-secretase inhibitor at all doses tested. The graph shows Mean±standard error of means (SEM); * represents p< 0.05, comparison of inhibitor vs. DMSO.</p
Methods for clinical and/or pathological diagnosis of five AD patients.
<p>Methods for clinical and/or pathological diagnosis of five AD patients.</p
Characterization of 3D neuro-spheroid cultures by NeuN and GFAP.
<p>Neuro-spheroids (3DS1-5) were characterized by immunofluorescence staining of different protein markers, NeuN (green) and GFAP (red). Neuro-spheroids were positive for both NeuN and GFAP in all 5 cell lines. Merged and magnified images illustrate NeuN positive nucleus (green) and GFAP positive cells (red) localized in the filaments of astrocytes. Representative bright field (BF) images are presented on the left column. Scale bar: 50 μm.</p
Characterization of 2D neuronal culture differentiated from neural stem cells.
<p>2D neurons were immunostained with antibodies against the markers NeuN, GFAP, BT3 and MAP2. Differentiated neurons from all five subjects showed NeuN-, BT3- and MAP2-positive cells. Cells positive for the astrocyte marker GFAP were also presented. The left column of images is an illustration of merged NeuN (red) and GFAP (green) staining. NeuN (red) is highly expressed in the nucleus of differentiated neurons whereas GFAP (green) is expressed in the filament of the astrocytes. Nuclei staining was illustrated in blue across all images. Scale bar: 100 μm.</p
Characterization of neural stem cells by protein markers Nestin and Sox2.
<p>Induced pluripotent stem cell-derived neural stem cells were identified by different protein markers, Nestin (green) and Sox2 (red). All 5 AD patients’ iPSC-derived neural stem cells were Nestin- and Sox2-immunoreactive. The expression of both protein markers was higher in N3 and N4 and lower in N1 and N5. Merged images are illustrated in yellow. Scale bar: 100 μm.</p