NotchIC Inhibits Nonneural Differentiation

<div><p>(A–D) R26NotchIC cells (NotchIC) or parental 46C cells (control) cultured under monolayer differentiation conditions and stained for Oct4 (red) to indicate ES cells together with a combination of BLBP and GFP (green) to indicate both types of neural progenitor together.</p> <p>(E) qRT-PCR for various markers in R26NotchIC cells relative to parental 46C cells after 6 d of monolayer differentiation. Data are averaged from at least three independent experiments and normalised to GAPDH. Notch1 primers amplify within the NotchIC and detect both endogenous and exogenous transcripts.</p> <p>(F) R26NotchIC cells (NotchIC) or parental 46C cells (control) cultured under monolayer differentiation conditions and stained for cytokeratin 8, a marker of nonneural differentiation (red) and counterstained with DAPI (blue)</p></div

R26NotchIC Does Not Impair ES Cell Self-Renewal

<div><p>(A) RT-PCR for Notch receptors and ligands in Oct4-GiP ES cells selected in puromycin (“purified ES cells”). E13.5 neural tissue was used as a positive control.</p> <p>(B) FACS analysis of Jagged1 expression in live unpermeabilised 46C ES cells using single-chain antibody to Jagged1. NS cells [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040121#pbio-0040121-b042" target="_blank">42</a>] were used as a positive control and a single-chain antibody to the irrelevant intracellular antigen Desmin was used for the negative control. </p> <p>(C) FACS analysis of CD2 expression in R26NotchIC cells with parental 46C ES cells as controls.</p> <p>(D–F) Analysis of R26NotchIC ES cells or parental control 46C ES cells after 10 to 12 passages in LIF+ serum. (D) R26NotchIC ES cells under phase contrast or stained for ES cell markers as indicated (average of three independent samples). (E) Quantitative RT-PCR analysis of ES cell markers, normalised to GAPDH, and displayed relative to expression in 46C ES cells. (F) FACS analysis of <i>Sox1</i>GFP expression. </p></div

R26NotchIC Cells Lose <i>Sox1</i> and Acquire BLBP Expression

<div><p>R26NotchIC ES cells or parental control 46C cells cultured under monolayer differentiation conditions.</p> <p>(A) Proportion of <i>Sox1</i>GFP+ cells at various time points measured by FACS analysis. </p> <p>(B–G) Cultures at various time points shown in phase contrast or stained for markers as indicated.</p> <p>(H) Quantitative RT-PCR for BLBP normalised to GAPDH and displayed relative to expression in day 7 46C ES cells.</p> <p>(I) R26NotchIC cells at day 7 of monolayer differentiation stained with an antibody to GFP in order to amplify detection of <i>Sox1</i>GFP (green) together with an antibody to BLBP (red). </p> <p>(J) Schematic diagram to illustrate the transition of ES cells into <i>Sox1</i>GFP+ neuroepithelial progenitors and then into BLBP+ bipolar neural progenitors. </p></div

Notch Promotes Neural Differentiation of Human ES Cells

<div><p>(A–I) Human ES cells plated on OP9 feeder cells expressing either GFP only (OP9 EV) or the Notch ligand Delta1 (OP9 Dl1) with γ-secretase inhibitor where indicated (“+inhibitor”) were cultured for 7 d and stained for markers as indicated. (A) Higher magnification picture to indicate the cell morphology: the OP9-Dl1 feeders are green and the dotted line demarcates hES cells adjacent to the feeders that have adopted a neural morphology, while cells more distant from the feeders retain an ES-like morphology.</p> <p>(J and K) Quantification of Pax6 immunostaining (averages and standard deviations shown from four experiments).</p> <p>(L–T) Human ES cells grown under monolayer differentiation conditions in the presence of γ-secretase inhibitor (“inhibitor”) or DMSO vehicle and stained for Pax6, <i>Sox1</i>, or TRA1/81 as indicated. T shows quantification of Pax6 immunostaining (averages and standard deviations from four experiments). </p></div

R26NotchIC Cells Undergo Accelerated Neural Specification

<div><p>R26NotchIC ES cells or parental control 46C cells cultured under monolayer differentiation conditions.</p> <p>(A) Typical FACS profile of <i>Sox1</i>GFP expression after 48 h. <i>Sox1</i>GFP+ cells were scored using gate M1. </p> <p>(B) Proportion of <i>Sox1</i>GFP+ cells at various time points (average of triplicates). </p> <p>(C–H) Intact cultures at 72 h of monolayer differentiation, shown in phase contrast or stained for markers as indicated.</p> <p>(I) Typical FACS profile of <i>Sox1</i>GFP expression after 5 d. M1 is the gate used throughout to quantify the proportion <i>Sox1</i>GFP+ cells: note that this FACS profile indicates a more striking difference between control and NotchIC populations than is reflected by our conservative quantification. </p> <p>(J and K) Quantitative RT-PCR for Oct4 and for FGF5 during monolayer differentiation of R26NotchIC cells (NotchIC) or control parental 46C cells. Levels were normalised to GAPDH and are displayed relative to expression in 46C ES cells.</p> <p>(L and M) FACS analysis of the proportion of <i>Sox1</i>GFP+ cells for R26-NotchIC cells or parental 46C cells at various time points from triplicate cultures at normal density (10⁴ cells/cm²) or higher density (3 × 10⁴ cells/cm²) (average of triplicates). </p></div

Lentiviral transduction and establishment of transgenic hNPCs.

<p>(A) Schematic representation of the experimental procedure. (B) Immunofluorescence labeling of undifferentiated (day 0) and differentiated (day 14) hNPCs, following lentiviral transduction. Antibodies against the viral P (green) or X (green) proteins were used and nuclei were stained with DAPI (blue). Note the localization of the P (nuclear) and the X (nuclear and cytoplasmic) proteins. (C) Evaluation of transduction efficiency based on enumeration of immunostained cells. Results are representative of 3 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. <i>ns</i>, non-significant (p > 0.5). Scale bar, 20 μm.</p

Expression of <i>bdv-p</i> or <i>bdv-x</i> gene does not alter hNPCs at the undifferentiated stage.

<p>RNAs from <i>bdv-p-</i> and <i>bdv-x</i>-expressing hNPCs and their matched NT controls were analyzed by RT-qPCR for expression of (A) Nestin and (B) Sox2. Proliferation of <i>bdv-p</i> and <i>bdv-x</i>-expressing hNPCs was analyzed by BrdU labeling (C) and by a mitochondrial dehydrogenase activity-based assay (D and E) in the presence of growth factors and by enumeration of DAPI-positive cells (F and G) in the absence of growth factors. Results in A and B are representative of two independent experiments performed in triplicate. Results in C represent the mean of two independent experiments performed in quintuplicate. Results in D and E are representative of 2 independent experiments performed in quintuplicate. Results in F and G are from 1 experiment performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. <i>ns</i>, non-significant (p > 0.5).</p

<i>bdv-p</i> alters the expression of <i>ApoE</i> and <i>Noggin</i>.

<p><i>bdv-p</i>- and <i>bdv-x</i>-expressing-hNPCs and their matched NT controls were induced to differentiate for 0, 4 or 14 days before RNA and protein analyses. <i>ApoE</i> expression was measured by RT-qPCR at (A) day 4, (B) day 0 and (C) day 14. (D) Western blot analysis showing ApoE level. It was normalized to actin. <i>Noggin</i> expression was measured by RT-qPCR at (E) day 4, (F) day 0 and (G) day 14. The results are representative of 2 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.001.</p

Expression of the <i>bdv-p</i> but not <i>bdv-x</i> gene in hNPCs impairs neuronal differentiation.

<p>Transduced hNPCs expressing <i>gfp</i>, <i>bdv-p</i> or <i>bdv-x</i> genes and their matched NT controls were induced to differentiate for 14 days. (A) Immunostaining with antibodies directed against βIII-Tubulin, a neuronal marker (red), or GFAP, an astrocytic marker (green). Nuclei were stained with DAPI (blue). For panel homogenization, GFAP immunostaining performed in <i>gfp</i>-expressing hNPCs was re-colored in green. The percentage of neurons and astrocytes was determined based on enumeration of βIII-Tubulin-, GFAP- and DAPI-positive cells in (B) <i>gfp</i>-expressing hNPCs, (C) <i>bdv-p</i>-expressing hNPCs and (D) <i>bdv-x</i>-expressing hNPCs. Results in B, C and D are representative of 2, 5 and 2 independent experiments, respectively. All experiments were performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.001, ns, non-significant (p > 0.5). N, neurons. As, astrocytes. Scale bar, 50 μm.</p

<i>bdv-p</i> alters the expression of <i>TH</i> and <i>Scg10/Stathmin2</i>.

<p><i>bdv-p</i>- and <i>bdv-x</i>-expressing- hNPCs and their matched NT controls were induced to differentiate for 0, 4 or 14 days before RNA and protein analyses. <i>TH</i> expression was measured by RT-qPCR at (A) day 4, (B) day 0 and (C) day 14. (D) Western blot analysis showing TH level. <i>Scg10/Stathmin2</i> expression was measured by RT-qPCR at (E) day 4, (F) day 0 and (G) day 14. (H) Western blot analysis showing SCG10/Stathmin2 level. TH and SCG10/Stathmin2 were normalized to actin. The results are representative of 2 independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.001.</p