EGF signaling deregulates human anterior NPC identity.

<p>(<b>A</b>)<b>:</b> Schematic of the experimental outline. Human PSCs were neuralised at 21% O₂ and dissociated neural rosettes were propagated at 3% or 21% O₂ with mitogens as shown. EGF/FGF propagation at 21% O₂ was described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085932#pone.0085932-Koch1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085932#pone.0085932-Falk1" target="_blank">[17]</a>. (<b>B–D</b>)<b>:</b> Immunofluorescence analysis of neurosphere cryosections before platedown. Radially organised neuroepithelia express FOXG1, OTX2 and NESTIN, and display uniform staining of EGFR1. Scale bars are 20 µm (<b>E</b>)<b>:</b> aNPCs maintained in FGF2 or EGF/FGF2 containing media proliferate at similar rates (n = 3, cumulative cell count from 5 passages shown). (<b>F</b>)<b>:</b> Propagation of aNPCs in EGF/FGF2 results in down regulation of anterior marker <i>OTX2</i> by passage15, while the expression of neural progenitor marker <i>NESTIN</i> remains unchanged as determined by qRT-PCR relative expression analysis, n = 4, <i>P</i><0.05, un-paired <i>t</i>-test. (<b>G</b>)<b>: </b><i>OTX2</i> relative expression analysis by qRT-PCR of aNPC cultures established in FGF2 (F) and propagated in EGF/FGF2 (E/F) or EGF/FGF2/EGFR-blocker (E/F/PD) for five passages. Relative <i>OTX2</i> expression is significantly down-regulated in E/F cultures compared to F and E/F/PD, n = 4, <i>P</i><0.05, ordinary ANOVA with Tukey’s multiple comparison test.</p

Functional characterization of cortical neurons differentiated from 3% O₂ FGF2-propagated aNPCs.

<p>(<b>A</b>)<b>:</b> 3% O₂ aNPC-derived neurons were subjected to Fluo-3 Ca²⁺ imaging before and during treatment with elevated K⁺ (50 mM final) in the presence of FPL 64176 (5 µM). For each cell, the fold-increase in cytoplasmic Ca²⁺ concentration was calculated (n = approximately 350 cells from n = 7 independent differentiations; passages 10–20). (<b>B</b>)<b>: </b><i>FOS</i> mRNA fold induction in 3%-O₂ aNPC-derived neurons in response to membrane depolarisation as determined by qRT-PCR. Expression is normalised to <i>GAPDH</i>, n = 7. (<b>C</b>)<b>:</b> Development of induced AP activity in 3% O₂ aNPC-derived neurons over 5 weeks (n = 61 from 5 <i>de novo</i> derivations). (<b>D</b>)<b>:</b> Developmental increase in current density of voltage-gated ion channels (<i>Na_V</i>, <i>I_K</i>, <i>I_A</i>) in 3% O₂ aNPC-derived neurons. All current density values for each ion channel at week 3 are significantly higher (significance not indicated for clarity) than week 1 (<i>P</i><0.001; Kruskal-Wallis test with <i>post hoc</i> Dunn’s test; n = 19–30 for each week, from 3 <i>de novo</i> derivations). (<b>E</b>)<b>:</b> Current-clamp recording of a 3% O₂-derived neuron that exhibited sustained repetitive firing at a holding potential of –45 mV.</p

Cortical neurons derived at 3% O₂ display uniform and enhanced functional maturation.

<p>(<b>A</b>)<b>:</b> Phase-contrast images of aNPCs derived from the same rosette-isolation, propagated in FGF2 at 21% and 3%. aNPCs proliferated at 21% O₂ with FGF2 show rosette-like morphology at early passages (21% O₂ p5) but display increased differentiation and altered morphology with successive passaging (21% O₂ p10). aNPCs propagated at 3% O2 with FGF2 show stable cellular morphology (3% O₂ p10). Scale bars 200 µm. (<b>B</b>)<b>:</b> Comparison of <i>OTX1</i>, <i>OTX2</i>, <i>LHX2</i> and <i>VEGF</i> relative expression levels between 3%- and 21%-O₂ derived aNPCs at passage 5 as determined by qRT-PCR, n = 4, <i>* P</i><0.05, ns: non-significant, un-paired <i>t</i>-test. (<b>C</b>)<b>:</b> Quantitative immunohistochemical analysis of CTIP2 expression after four weeks of differentiation of aNPCs derived at 3%- and 21%-O₂ at passage 5. (<b>D</b>)<b>:</b> Example current-clamp recordings of activity induced by a depolarising current pulse (+30 pA) from a potential of –74 mV. From <i>left</i> to <i>right</i>, the categorised responses depict; (top panels) <i>no response</i>, <i>failed initialisation</i>; (bottom panels) <i>single AP</i>, <i>train of APs</i>. (<b>E</b>)<b>:</b> Bar graph showing the cumulative distribution of activity response of 3%- and 21%-O₂ aNPC-derived week 5 neurons from three independent <i>de novo</i> aNPC derivations. (<b>F</b>)<b>:</b> Bar graph summarising mean (± s.e.m.) percentage of active 3% and 21% O₂ aNPC-derived week 5 neurons per <i>de novo</i> batch of aNPC paired derivations (n = 3 batches; <i>P</i><0.05; unpaired <i>t</i>-test). Mean input resistance measurements were not different between conditions, but a difference (p<0.05) in whole-cell capacitance was observed (21%: 12.8 pF vs 3%: 16.3 pF). (<b>G</b>)<b>:</b> Comparison of BDNF exon IV transcription induction between 5 week old neurons differentiated at 3%- and 21%-O₂ in response to membrane depolarisation with K⁺ in the presence of FPL 64176 (5 µM) as determined of qRT-PCR. Expression is normalised to β-<i>ACTIN</i> and fold induction normalised to untreated respective control cultures is shown (n = 3, <i>P</i><0.05, unpaired <i>t</i>-test).</p

aNPCs give rise to glutamatergic neurons that can form functional excitatory synapses.

<p>(<b>A</b>)<b>:</b> Examples of whole-cell currents recorded from 3% O₂ aNPC-derived neurons in response to bath application of NMDA (100 µM) in the presence of glycine (100 µM), AMPA (50 µM), or GABA (100 µM). Immunohistochemical staining against glutamate transporter VGLUT1 and β-3 tubulin (<b>B</b>) and post-synaptic density protein (PSD-95), Synaptophysin I (SYN) and β-3 tubulin (<b>C</b>) in aNPC-derived cortical neurons. (<b>D</b>)<b>:</b> The apposition of SYN and PSD-95 on MAP2⁺ processes (<b>D</b>) mark putative synapses. Images B and D are 0.5 µm thick single optical sections acquired by confocal microscopy. Scale bars are 20 µm. (<b>E</b>)<b>:</b> Example of miniature EPSCs recorded from a week 5 neuron held at –84 mV and recorded in the presence of TTX (300 nM), strychnine (20 µM) and picrotoxin (50 µM), plus MgCl₂ (2 mM) to block NMDA receptor-mediated currents. All events were blocked by CNQX(5 µM).</p

Characterisation of aNPCs in long-term culture.

<p>(<b>A</b>)<b>:</b> Expression of pluripotency markers <i>NANOG</i> and <i>OCT4</i> are not detectable by RT-PCR in 3% O₂ aNPCs while anterior neuroectoderm marker <i>OTX2</i> expression is maintained (p:passage). (<b>B</b>)<b>:</b> Proliferating aNPCs display uniform NESTIN expression and mosaic OTX2 expression (scale bar 50 µm). (<b>C</b>)<b>:</b> Immunohistochemical staining against p75 (green), NESTIN (red) and DNA (blue) in proliferating 3% O₂ aNPCs (scale bar 20 µm). (<b>D</b>)<b>:</b> RT-PCR analysis of rostral markers <i>DACH1</i>, <i>LHX2</i>, <i>OTX1</i>, <i>OTX2</i> and caudal markers <i>HOXA2</i>, <i>HOXB4</i> and <i>HOXC4</i> in passage 15 and 30 aNPCs maintained in FGF compared to isolated neural-rosettes (Ros) or RA-patterned aNPCs (cont), respectively. (<b>E</b>)<b>:</b> Representative chromosome analysis of a H9 hESC-derived aNPC line (passage 25) by G-banding showed that long-term propagating NPCs maintained a normal karyotype.</p

aNPCs maintained in FGF give rise to cortical neurons by default and are responsive to patterning cues.

<p>(<b>A</b>)<b>:</b> Upon withdrawal of FGF2, aNPCs display time-dependent upregulation of dorsal telencephalic marker <i>EMX2</i> as determined by qRT-PCR (D = days <i>in vitro</i> differentiation. <i>EMX2</i> expression levels are normalised to levels detected in proliferating aNPCs ( = 1), β-actin is used as housekeeping control. <i>EMX2</i> D10 expression = 130.4±29.2, n = 4. (<b>B</b>)<b>:</b> Gene expression in human fetal brain (FB) and aNPC cultures differentiated for 6 days. All expression levels are normalized to levels detected in human tissue ( = 1). Data are represented as mean ± SEM, n = 4 for differentiated aNPCs, passage >20. For tissue, n = 1. aNPC derived neurons express REELIN by 4 weeks of differentiation as determined by qRT-PCR (<b>C</b>; aNPCs vs neurons: 0.7±0.2 vs 35.3±5.4, n = 4, <i>P</i><0.001, un-paired <i>t</i>-test) and immunofluorescence (<b>D</b>). (<b>I</b>)<b>:</b> Quantitative immunohistochemical analysis of neurons differentiated from aNPCS revealed expression of both deep-layer and upper-layer cortical neuronal markers (CTIP2, BRN2, CUX1, SATB2) (<b>E–H</b>). Data from n = 4–7 differentiation experiments from three H9 hESC- (passages >20) and three hIPSC-derived (passages 10–28) aNPC lines shown. (<b>J</b>)<b>:</b> aNPCs are responsive to patterning cues, sequentially upregulating HOXB4, OLIG2, ISL1 and HB9 expression in response to treatment with RA and SHH agonist purmorphamine during motor neuron differentiation. (<b>K</b>)<b>:</b> Quantitative immunohistochemical analysis of OLIG2 induction efficiency in early and late passage aNPCs upon treatment with RA and purmorphamine (n = 3 differentiation experiments from independent, early (<15) and late (>25) passage aNPC lines). Data are represented as mean ± s.e.m. Scale bars are 20 µm.</p