10 research outputs found
Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen
This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O(2)) level of 3% and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O(2), where they acquire a neuroepithelial identity over two weeks. This timescale is comparable to that at 20% O(2), but survival is enhanced. Sequential application of retinoic acid (RA) and purmorphamine (PM), from day 14 to 28, directs differentiation towards spinal motor neurons. Alternatively, addition of FGF-8 and PM generates midbrain dopaminergic neurons. OLIG2 induction in motor neuron precursors is 2-fold greater than at 20% O(2), whereas EN1 is 5-fold enhanced. 3% NPCs can be differentiated into all three neural lineages, and such cultures can be maintained long-term in the absence of neurotrophins. The ability to generate defined cell types at 3% O(2) should represent a significant advance for in vitro disease modelling and potentially cell-based therapies
Neuregulin and BDNF Induce a Switch to NMDA Receptor-Dependent Myelination by Oligodendrocytes
<div><p>Myelination is essential for rapid impulse conduction in the CNS, but what determines whether an individual axon becomes myelinated remains unknown. Here we show, using a myelinating coculture system, that there are two distinct modes of myelination, one that is independent of neuronal activity and glutamate release and another that depends on neuronal action potentials releasing glutamate to activate NMDA receptors on oligodendrocyte lineage cells. Neuregulin switches oligodendrocytes from the activity-independent to the activity-dependent mode of myelination by increasing NMDA receptor currents in oligodendrocyte lineage cells 6-fold. With neuregulin present myelination is accelerated and increased, and NMDA receptor block reduces myelination to far below its level without neuregulin. Thus, a neuregulin-controlled switch enhances the myelination of active axons. <i>In vivo</i>, we demonstrate that remyelination after white matter damage is NMDA receptor-dependent. These data resolve controversies over the signalling regulating myelination and suggest novel roles for neuregulin in schizophrenia and in remyelination after white matter damage.</p></div
NRG specifically increases NMDA receptor currents in oligodendrocyte lineage cells.
<p>(A–F) Images of Lucifer yellow in patch-clamped cells (left), antibody labelling for identification (middle), and merged images (right). (A) NG2-expressing OPC. (B) CNPase-expressing myelinating oligodendrocyte, with axons labelled for NF 160/200. (C) Myelinating oligodendrocyte expressing MBP. (D) NF 160/200-expressing DRG neuron. (E) GFAP-expressing astrocyte. (F) SCIP-expressing satellite cell. (G–J) Specimen currents and mean currents (number of cells shown on bars) evoked at −64 mV by 60 µM NMDA and 30 µM kainate in the absence or presence of NRG, in (G–H) oligodendrocyte precursor cells and (I–J) differentiated oligodendrocytes in the process of myelinating neurons. (K–N) Mean currents in (K) DRG neurons, (L) interneurons, (M) astrocytes, and (N) satellite cells. Internal solution had E<sub>Cl</sub> = 0 mV. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001743#pbio.1001743.s004" target="_blank">Figure S4</a>.</p
Signalling underlying the effects of NRG.
<p>(A) Effect of blocking integrin function with an antibody to the β<sub>1</sub> subunit (β1) in the absence and presence of NRG (number of experiments shown on bars). ANOVA across all conditions gave <i>p</i><0.0001. The <i>p</i> values above each bar compare with control. (B–C) Western blot of control (Con) and NRG cocultures (numbers of co-cultures shown on bars), in the absence and presence of MK-801, for phosphorylated (pAkt, pERK) and total Akt and ERK, with level of phosphorylated enzyme normalized to total enzyme in bar charts below. (D) Cocultures labelled for nuclei with DAPI, and with antibodies to Nkx2.2 (for late OPCs/immature oligodendrocytes) and pCREB (arrows show cells expressing both). (E) Percentage of cells expressing and not expressing Nkx2.2, which label for pCREB (in 29 control and 29 NRG fields of view, including 26,261 control and 21,518 NRG cells). (F) Change in immediate early gene expression mRNA level (by qPCR) in NRG-treated cocultures, normalized to control levels. (G) Effect of BDNF and MK-801 on myelination. One-way ANOVA gave <i>p</i><0.0001 (the potentiation of myelination by BDNF here cannot be directly compared with that for NRG in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001743#pbio-1001743-g002" target="_blank">Figure 2A</a> because the experiments were not done on the same set of cocultures). (H–I) Western blot of control (Con) and BDNF cocultures, for phosphorylated (pAkt, pERK) and total Akt and ERK, with level of phosphorylated enzyme normalized to total enzyme in bar charts below. (J) Western blots of control (Con) and BDNF-treated cocultures for NR1 and NR3A, with densitometric quantification of subunit protein levels in bar graph below. The <i>p</i> values over single bars compare with control (Con). The <i>p</i> values are from Holm–Bonferroni corrected <i>t</i> tests in (A and G), Dunnett's post hoc tests in (B and C), and Student's <i>t</i> tests in (H–J). Number of cultures are shown on bars.</p
NRG does not affect neuronal activity.
<p>(A) Lucifer yellow filled patch-clamped interneuron, identified after recording by labelling for glutamate decarboxylase (GAD) 65/67, from which action potential evoked synaptic currents were recorded. DAPI labelling shows cell nuclei. (B) Spontaneous synaptic currents at −64 mV recorded in an interneuron with E<sub>Cl</sub> = 0 mV. (C–D) Event frequency (C) and block by 1 µM TTX (D) in control and NRG cultures. (E) Spontaneous synaptic currents at −70 mV recorded with E<sub>Cl</sub> = −88 mV, during application of 25 µM NBQX+50 µM D-AP5, or 10 µM GABAzine, or 1 µM TTX. (F–G) Event frequency (F) and block by NBQX/AP5, GABAzine, and TTX (G) in control and NRG cultures.</p
Effect of NRG and NMDA receptor block on myelination.
<p>(A, B) High-magnification views of a myelinating oligodendrocyte (A) with MBP (green) expressed in processes wrapping around axons expressing NF 160/200 (NF, red), and of a nonmyelinating oligodendrocyte (B) with MBP expressed (in a more patchy and often diffuse manner) in processes that are not aligned with axons. Myelination was quantified as the fraction of all MBP-expressing oligodendrocytes that provided a thick straight myelin sheath to at least one axon. (C–F) Myelinating processes (MBP, green) wrapping DRG axons (NF, red) in control conditions (C), in the presence of MK-801 (D), in the presence of NRG (E), and in the presence of NRG and MK-801 (F). Filled and open arrows show some myelinating and nonmyelinating oligodendrocytes. Graphs show fraction of oligodendrocytes that are myelinating, versus fraction of area occupied by DRG processes, for 30 images of each coverslip from which the specimen images shown were taken, best fit with a linear dependence of myelination on axon density.</p
NRG switches myelination to an activity- and NMDA receptor-dependent programme.
<p>(A) Mean myelination parameter (the value of A from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001743#pbio.1001743.e001" target="_blank">eqn. 1</a> of the Materials and Methods) from experiments as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001743#pbio-1001743-g001" target="_blank">Figure 1</a> for different conditions (number of experiments shown on bars). For <i>No NRG</i>, ANOVA indicated no significant differences across all bars (<i>p</i> = 0.79); <i>p</i> values from <i>t</i> tests are for comparison with control. For <i>With NRG</i>, ANOVA showed significant differences across all bars (<i>p</i><0.0001); <i>p</i> values (from Holm–Bonferroni post hoc test) are for comparison with NRG alone (comparison between conditions with and without NRG: TTX versus NRG+TTX <i>p</i> = 0.29; MK versus NRG+MK <i>p</i> = 6×10<sup>−7</sup>; MK+NBQX versus NRG+MK+NBQX <i>p</i> = 0.14; 7CK versus NRG+7CK <i>p</i> = 0.02; NBQX versus NRG+NBQX <i>p</i> = 0.65; TTX+MK versus NRG+TTX+MK <i>p</i> = 0.72; AP5 versus NRG+AP5 <i>p</i> = 0.85). (B) Axon density (fraction of image pixels labelled for NF 160/200) for the conditions in (A) (ANOVA showed no significant differences, <i>p</i> = 0.19). (C) Myelination in control and NRG at different times after plating OPCs onto DRG cells (5–7 cocultures per point). Plots are myelination (M) as a function of time (t) where M = M<sub>max</sub>.t<sup>n</sup>/(t<sup>n</sup>+T<sup>n</sup>) with n fixed at 4.7 (best fit for control) and best fit values were M<sub>max</sub> = 0.0180 (NRG) or 0.0155 (Con) and T = 1.56 (NRG) or 2.28 (Con) weeks. The <i>p</i> values are shown for the increase of M<sub>max</sub> and decrease of T in NRG compared to control. (D) Potentiation of myelination by NRG as a function of the level of myelination in control conditions (each point is one individual coculture). (E) Fraction of myelination remaining in MK-801 as a function of the level of myelination in control conditions (each point is one individual coculture; variability in the data reflects taking the ratio of two variable levels of myelination), in the absence (Con) and presence of NRG. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001743#pbio.1001743.s002" target="_blank">Figure S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001743#pbio.1001743.s003" target="_blank">S3</a>.</p
Remyelination is dependent on NMDA receptor activation.
<p>(A–B) Semithin sections of lesioned caudal cerebellar peduncle (CCP), 21 d postlesion, infused with saline (A) or MK-801 (50 µM; B) for 18 d. The dotted white lines mark the lesion edge. (C) Ranking of remyelination; each symbol represents one animal. Higher ranks represent more remyelination. The <i>p</i> value from Mann–Whitney U test. (D, E) Higher magnification picture shows that fewer axons are remyelinated in lesions treated with MK-801 (remyelinated axons are coloured green). (F) Mean percentage of axons remyelinated averaged over 20 areas in each of three lesions for each condition. (G) Specimen images of a normal myelinated axon, a demyelinated axon, and remyelinated axons in lesions infused with saline or MK-801. (H–I) Mean g-ratio of all axons, and mean g-ratio at all diameters, are higher with MK-801 present (student <i>t</i> test, <i>n</i> = 3).</p
Expression of NMDA receptors.
<p>(A) Western blots of control and NRG-treated cocultures for NR1, NR2B and its phosphorylated form (pNR2B), NR2C and its phosphorylated form (pNR2C), NR3A and NR3B, as well as for NR2A and NR2D compared to their respective positive controls of rat cortex (Ctx) and thalamus (Th); β-actin acts as a loading control throughout. (B) Densitometric quantification of subunit protein levels in cocultures (normalized to β-actin) in NRG normalized to the levels in control (NR2A and NR2D levels were undetectable). (C) Western blot of control (Con) and NRG-treated pure DRG cultures for NR3A and (below) densitometric quantification of subunit protein levels (normalized to β-actin and then to control). (D) Western blot for NR1 and NR3A of control (Con) and NRG-treated (for 6 d) pure OPC cultures, treated (+) or not treated (−) with 20 min glutamate (Glu, 100 µM) stimulation every day, with densitometric quantification of subunit protein levels (normalized to β-actin and then to control). The <i>p</i> values over the bars, in (B) from Holm–Bonferroni corrected <i>t</i> tests and in (C and D) from one-sample Student <i>t</i> tests, compare with control; numbers of experiments shown on bars.</p
Derivation of neural precursor cells from human ES cells at 3% O2 is efficient, enhances survival and presents no barrier to regional specification and functional differentiation
In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O2, demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O2 can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O2 environment results in significant cell death, while maintenance in 3% O2 is protective. Together these findings support 3% O2 as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury