Maturation of AMPAR Composition and the GABAAR Reversal Potential in hPSC-Derived Cortical Neurons

Rodent-based studies have shown that neurons undergo major developmental changes to ion channel expression and ionic gradients that determine their excitation-inhibition balance. Neurons derived from human pluripotent stem cells theoretically offer the potential to study classical developmental processes in a human-relevant system, although this is currently not well explored. Here, we show that excitatory cortical-patterned neurons derived from multiple human pluripotent stem cell lines exhibit native-like maturation changes in AMPAR composition such that there is an increase in the expression of GluA2(R) subunits. Moreover, we observe a dynamic shift in intracellular Cl(−) levels, which determines the reversal potential of GABA(A)R-mediated currents and is influenced by neurotrophic factors. The shift is concomitant with changes in KCC2 and NKCC1 expression. Because some human diseases are thought to involve perturbations to AMPAR GluA2 content and others in the chloride reversal potential, human stem-cell-derived neurons represent a valuable tool for studying these fundamental properties

Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy

Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies

Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABA(A) receptors (GABA(A)Rs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC(50) values of 278 μm and 182 μm, respectively, and the presence of a GABA(A)R population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABA(A)R-mediated currents, evoked by EC(50) concentrations of GABA, were blocked by bicuculline and picrotoxin with IC(50) values of 2.7 and 5.1 μm, respectively. hECN GABA(A)Rs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABA(A)Rs. Taken together our data support the notion that hECN GABA(A)Rs have an α2/3β3γ2 subunit composition – a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC(50) of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC(50) = 630 nm) and picrotoxin (IC(50) = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits

Physiological normoxia and absence of EGF is required for the long-term propagation of anterior neural precursors from human pluripotent cells

Widespread use of human pluripotent stem cells (hPSCs) to study neuronal physiology and function is hindered by the ongoing need for specialist expertise in converting hPSCs to neural precursor cells (NPCs). Here, we describe a new methodology to generate cryo-preservable hPSC-derived NPCs that retain an anterior identity and are propagatable long-term prior to terminal differentiation, thus abrogating regular de novo neuralization. Key to achieving passagable NPCs without loss of identity is the combination of both absence of EGF and propagation in physiological levels (3%) of O2. NPCs generated in this way display a stable long-term anterior forebrain identity and importantly retain developmental competence to patterning signals. Moreover, compared to NPCs maintained at ambient O2 (21%), they exhibit enhanced uniformity and speed of functional maturation, yielding both deep and upper layer cortical excitatory neurons. These neurons display multiple attributes including the capability to form functional synapses and undergo activity-dependent gene regulation. The platform described achieves long-term maintenance of anterior neural precursors that can give rise to forebrain neurones in abundance, enabling standardised functional studies of neural stem cell maintenance, lineage choice and neuronal functional maturation for neurodevelopmental research and disease-modelling

Rapid Generation of Ventral Spinal Cord-like Astrocytes from Human iPSCs for Modeling Non-Cell Autonomous Mechanisms of Lower Motor Neuron Disease

Astrocytes play important roles in the function and survival of neuronal cells. Dysfunctions of astrocytes are associated with numerous disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Human-induced pluripotent stem cell (iPSC)-based approaches are becoming increasingly important for the study of the mechanisms underlying the involvement of astrocytes in non-cell autonomous processes of motor neuron degeneration in ALS. These studies must account for the molecular and functional diversity among astrocytes in different regions of the brain and spinal cord. It is essential that the most pathologically relevant astrocyte preparations are used when investigating non-cell autonomous mechanisms of either upper or lower motor neuron degeneration in ALS. Here, we describe the efficient and streamlined generation of human iPSC-derived astrocytes with molecular and biological properties similar to physiological astrocytes in the ventral spinal cord. These induced astrocytes exhibit spontaneous and ATP-induced calcium transients, and lack signs of overt activation. Human iPSC-derived astrocytes with ventral spinal cord features offer advantages over more generic astrocyte preparations for the study of both ventral spinal cord astrocyte biology and the involvement of astrocytes in mechanisms of lower motor neuron degeneration in ALS

An Optimized Workflow to Generate and Characterize iPSC-Derived Motor Neuron (MN) Spheroids

A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. To start, the morphological profiling of the spheroids was achieved by developing a pipeline to obtain measurements of their size and shape. Next, we confirmed the expression of different MN markers at the transcript and protein levels by qPCR and immunocytochemistry of tissue-cleared samples, respectively. Finally, we assessed the capacity of the MN spheroids to display functional activity in the form of action potentials and bursts using a microelectrode array approach. Although most of the cells displayed an MN identity, we also characterized the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed an MN 3D model, and we optimized a workflow that can be applied to perform its morphological, gene expression, protein, and functional profiling over time

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

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