Application Note

Genetic and Functional Profiling of hiPSC-derived Excitatory Neurons
Differentiated by Quick-NeuronTM Technology
Human induced pluripotent stem cells (hiPSCs) holdmuch promise in providing unique opportunities toadvance understanding and treatment of human disease.
hiPSCs can be expanded in culture indefinitely and canbe differentiated into cells of all types, thereby providing avaluable source of trillions of reproducible cells forextended research. Such hiPSCs can be established fromhealthy or diseased individuals to study normal physiologyas well as specific diseases and contribute towards drug
discovery and new therapies. We have developed a rapid
differentiation method, namely Quick-NeuronTM technology,resulting in the conversion of hiPSCs to highly homogenous neurons in 7 to 10 days1. These neurons display
high levels of purity and low batch-to-batch variation andhave been consistently produced from dozens of different
hiPSC lines, including patient-specific lines and lines withknown mutations. Our cells function comparably to theirin vivo counterparts and can be used in 2D or 3D (e.g.,spheroids, organoids, and organ-on-a-chip) models for theadvancement of regenerative medicine or for phenotypic,
and high-throughput toxicology screening of drug efficacy
and toxicity.

Merge

TUBB3vGLUT1

HoechstRESULTS

Figure 1. Immunofluorescent staining shows typical neurite
growth and expression of the pan-neuronal marker TUBB3 andthe glutamatergic neuron-specific marker vGLUT1 on day 7 postthaw (scale bar = 100 μm).

Excitatory Neurons

RNA-seq profiles

The Quick-NeuronTM Excitatory Neuron technology,
Elixirgen Scientific’s proprietary transcription factor-based
technology, allows rapid and reproducible differentiationof hiPSCs into neurons without sacrificing the purity of thecells. We have characterized our neurons using immunofluorescent staining for marker expression, RNA sequencing to look at gene expression, as well as through functional assays such as patch clamping and micro- (or multi-)
electrode arrays (MEA). Our Quick-NeuronTM Excitatory
Neurons exhibit typical morphology with neurite outgrowthand express a variety of neuronal markers, such as thepan-neuronal marker tubulin beta 3 class III (TUBB3)and the glutamatergic neuron marker vesicular glutamate
transporter 1 (vGLUT1/SLC17A7) by day 10 of differentiation (day 7 post-thaw: Figure 1). Whole-cell patch-clamp
experiments revealed spontaneous action potentials andspontaneous excitatory postsynaptic current by voltage
clamp measurement at -70 mV, indicating the formation ofmature synapses among neurons (Figure 2A-C). Excitatoryneurons can be maintained in culture for at least 7 weeks
and are suitable for a variety of applications such asneurotoxicity assays.

Human iPSCs were differentiated to excitatory neurons
using our Quick-NeuronTM Excitatory Neuron technology
and maintained in culture for up to 52 days. Gene
expression profiling was performed at several time points
by RNA-seq and similarities of gene expression profiles tohuman brain tissue were assessed by principal component
analysis (PCA). PC1 (the x-axis) clearly separates thegene expression profile of hiPSCs from those of excitatoryneurons and the human brain (Figure 2D). Progression ofneural maturation is observed, such that excitatoryneurons in culture for a shorter time display gene expression profiles similar to that of fetal human brain cells whilecells that remain in culture for a longer period of time moreclosely resemble the gene expression profile of adulthuman brain cells. In particular, the gene expression profile
of our excitatory neurons cocultured with human primary
astrocytes (ThermoFisher) displayed a high similarity tothat of the human brain, with markers of neurons, astrocytes, and microglia as well as markers of inhibitory
neurons including GAD1/GAD67 and GAD2/GAD65
expressed (Figure 2D-E).

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Last revised: Dec 22, 20201AD

BCE

Neurons
Immature

Mature

Gluta

Dopa

Glia
Chol SeroGABA

AstroOligo

MicroFigure 2. Quick-Neuron™ Excitatory Neurons are functional and display gene expression profiles similar to those of human brain.
(A-C) Whole-cell patch clamp of excitatory neurons 5-6 weeks post-thaw. (A) A brightfield image of the neurons measured. (B) Spontaneous action potentials were recorded. (C) Spontaneous excitatory postsynaptic current of neurons was detected by voltage clamp
measurement at -70mV, indicating the formation of mature synapses. (D-E) RNA-seq was performed on undifferentiated iPSCs,excitatory neurons (EX only) cultured for 10 days and 38 days, excitatory neurons cocultured with primary astrocytes (EX+AST)
for 18 and 52 days, and primary fetal and adult brain samples. (D) Principal component analysis indicates that Quick-Neuron™
Excitatory Neurons display gene expression similar to that of human brain, particularly when grown with astrocytes. As cells remain inculture over time they more closely resemble adult human brain cells. (E) A heat map of selected gene expression data from RNAseq. Values represent log10(TPM + 1). Patch clamp and RNA-Seq data courtesy of E-PHY SCIENCE SAS and Ricoh, respectively.

MEA-drug responses
Having established that Elixirgen Scientific’s QuickNeuronTM Excitatory Neurons display physiologically
relevant markers, global gene expression, and electrophysiology, we next explored their utility in MEAs. MEAs
offer a powerful, scalable, and highly sensitive platform toinvestigate functional networks of hiPSC-derived neurons
in a high-throughput manner2. They have been used tostudy neuronal function and brain disorders as well as toevaluate the toxicity of chemical compounds3-4. Elixirgen
Scientific’s Quick-NeuronTM Excitatory Neurons werecocultured with primary human astrocytes on MEA plates
for 51 days post-thaw, and exposed to 3 concentrations
each of compounds categorized as receptor agonists,antagonists, or ion channel blockers. Quick-NeuronTM
Excitatory Neurons displayed expected responses to eachof the compounds (Table 1).
Acetaminophen, at concentrations of 1-100 μM, served asa negative control and resulted in no change in the spikecount upon exposure. On the other hand, administration of
the muscarine receptor agonist pilocarpine hydrochloride
at 0.1-10 μM and the histamine receptor agonist histamine
at 0.3-30 μM to the cultures each resulted in an increasein the number of spikes, indicating that excitation wasinduced in these neurons. In contrast, when the GABA
receptor agonist GABA was applied to the cultures at
0.1-10 μM, it resulted in a decrease in the number ofspikes, indicating the presence of inhibitory synapses
2among the network of these neurons. Interestingly, dopamine decreased the number of spikes when administered
at 0.1 μM but increased it when administered at 10 μM,
while glutamate had the opposite effect - causing
excitement at lower dosages and suppression at a higher
dosage. The AMPA/Kainate receptor antagonist CNQX
suppressed neuronal activity at 0.5-50 μM, whereas GABA
receptor antagonists picrotoxin and gabazine induced
excitement responses in the neurons at 0.1-10 μM and
0.3-30 μM, respectively. After exposure to dopamine D2
receptor antagonist chlorpromazine and histamine receptor antagonist ketotifen, the number of spikes increasedslightly at 0.1 μM but markedly decreased at 10 μM.
Similar to glutamate, the glycine antagonist strychnine
increased activity at lower dosages and suppressed at thehighest dosage. Antiepileptic drug carbamazepine, asodium channel blocker, served as a suppressor ofneuronal activity and, as expected, the number of spikes
decreased at 10 μM. Potassium channel blocker 4-Aminopyridine (4-AP) increased the number of spikes whenadministered at 0.3-30 μM. Importantly, when an MEA
plate seeded with Quick-NeuronTM Excitatory Neurons andprimary human astrocytes and maintained for 51 days wasshipped from Tokyo, Japan to Maryland, USA (a journey of33 hours), the number of active MEA electrodes and thebasal activity remained unchanged and the neurons displayed expected responses to 4-AP (Figure 3), signifyingthat the culture of neurons on MEA plates can be outelixirgensci.com

Table 1. Drug responses of Quick-Neuron™ Excitatory Neurons on MEA. Values are log2(rel # of spikes). Data courtesy of Ricoh.

Mechanism

Receptor/ChannelDrugConc. Range

Negative control-Acetaminophen1, 10, 100 μM

Glutamate

Glutamate

1, 10, 100 μM

GABA

GABA

0.1, 1, 10 μM

Dopamine

Dopamine

0.1, 1, 10 μM

Muscarine

Pilocarpine (HCl)0.1, 1, 10 μM

Histamine

Histamine

0.3, 3, 30 μM

AMPA/Kainate

CNQX

0.5, 5, 50 μM

GABA(A)

Picrotoxin

0.1, 1, 10 μM

GABA(A)

Gabazine

0.3, 3, 30 μM

Dopamine D2

Chlorpromazine

0.1, 1, 10 μM

NMDA

D-AP5

0.5, 5, 50 μM

Histamine H1

Ketotifen

0.1, 1, 10 μM

Glycine, Acetylcholine

Strychnine

0.3, 3, 30 μM

SodiumCarbamazepine

3, 30, 300 μM

Potassium

4-AP

0.3, 3, 30 μM

AgonistAntagonist

Ion Channel Blocker

sourced as a service for researchers and pharmaceuticalcompanies around the globe.

Rel. # of spikes

20-2

Elixirgen Scientific’s Quick-NeuronTM Excitatory Neurons(Catalog No. EX-SeV-CW50065) were thawed and maintained following the instructions laid out in our user guide.

Total RNA was processed using the SMART-seq2,
followed by the library preparation using the Nextera XT
DNA library preparation kit. Paired end sequences of 75
bp were obtained using NextSeq 550 (Illumina) up to 20
million reads. Fastq file was used to map sequences on
the human reference genome assembly 38 after removing low quality reads. The levels of gene expression wereexpressed as the number of transcripts per million transcripts (TPM).

Immunofluorescence Staining

Microelectrode array

Excitatory Neurons were maintained for 7 days post-thaw
under medium conditions instructed by the user guide prior
to immunofluorescence staining.

Quick-NeuronTM Excitatory Neurons were plated at 0.8 x
10⁵ cells/well of a 48-well MEA plate with 16 electrodes
per recording area (1.21 mm2; Axion Biosystems), after
mixing with primary human astrocytes (ThermoFisher,Catalog No. N7805200) at 0.2 x 10⁵ cells/well and 0.2 μg/
well laminin according to our application protocol. Cultures
were maintained for 49 days with medium changes twicea week. Plates were incubated at 37°C, 5% CO2. Cells ineach well were exposed to a diluent of drugs (i.e., 0.1 %DMSO) first and then drugs in 3 increasing doses. Data
were obtained immediately after administration of drugs
using either MaestroPro or Maestro 1 (Axion Biosystems)
and collected with a sampling rate of 12.5 kHz at eachelectrode. After spike firing was recorded, treated cells
were then washed with medium and incubated overnight.
Finally, spontaneous spike firing activity was measured
again to confirm that cells were not damaged by the treatment. Drug response was evaluated by measuring spikecounts. Data were extracted by AxISNavigator 2.0.4 andanalysis was performed using Neural Metric Tool.

METHODS
Cell Culture

Patch clamp
Quick-NeuronTM Excitatory Neurons were thawed andmaintained long-term as instructed by our user guide for
5-6 weeks prior to patch clamp. Spontaneous synaptic
activity and action potentials were measured by E-PHY
SCIENCE SAS.
RNA sequencing
Quick-NeuronTM Excitatory Neurons were lysed for total
RNA extraction on days 7 and 35 after recovery from cryopreservation (i.e., days 10 and 38 after differentiationinduction). Quick-NeuronTM Excitatory Neurons were also
co-cultured with primary human astrocytes (ThermoFisher,
Catalog No. N7805200) on an MEA plate and lysed for
total RNA extraction on days 15 and 49 after plating (i.e.,days 18 and 52 after differentiation induction).
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3A0B

Baseline (before transport)

100200300
Time (sec)

400500600

0Baseline (after transport)

100200300
Time (sec)

400

500

600

CControl (DMSO)

0100

200

300
Time (sec)400

0.3 µM 4-AP

500

600

0100200

300
Time (sec)400

30 µM 4-AP

3 µM 4-AP

500

600

0

100200300
Time (sec)

400

500

600

0100200300
Time (sec)400

500600Figure 3. Quick-Neuron™ Excitatory Neurons seeded onto MEA plates remain functional after transport. Baseline readings of thesame well (A) on day 49 post-thaw before transport and (B) on day 50 after 33 hr transport of an MEA plate seeded with QuickNeuron™ Excitatory Neurons and primary human astrocytes from Tokyo, Japan to Maryland, USA. (C) Response on day 51 to varying concentrations of 4-AP after transport. Data courtesy of Ricoh.

Discussion
Animal models have not fully been able to predict human
drug response. Attrition of drugs due to neurotoxicity andcentral nervous system problems is a serious andexpensive problem for pharmaceutical companies. hiPSCderived neurons possess great potential to establish
human cell-based in vitro platforms to screen out toxic
small compounds, and may serve as better alternatives
for the study of drug efficacy and toxicity4-7. Furthermore,in vitro models developed using patient-derived hiPSCs
with known mutations associated with specific diseases
may allow for prediction of patients’ response to treatments
and therapies. The results presented here demonstratethat Elixirgen Scientific’s hiPSC-derived Quick-NeuronTM
Excitatory Neurons are physiologically relevant, have gene
expression profiles similar to their in vivo counterparts,and may be used for toxicity screening, pharmacogenomics, drug discovery, disease modeling, and regenerative
medicine.

4References
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4.

5.

6.
7.

Goparaju, S. K. et al. Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs
encoding transcription factors. Sci. Rep. 7, 1–12 (2017).
Yuan, X. et al. Versatile live-cell activity analysis platform forcharacterization of neuronal dynamics at single-cell andnetwork level. Nat. Commun. 11, (2020).
Kim, B. W. et al. Human Motor Neurons With SOD1-G93A
Mutation Generated From CRISPR/Cas9 Gene-Edited
iPSCs Develop Pathological Features of Amyotrophic Lateral
Sclerosis. Front. Cell. Neurosci. 14, 1–16 (2020).
Odawara, A., Matsuda, N., Ishibashi, Y., Yokoi, R. & Suzuki,
I. Toxicological evaluation of convulsant and anticonvulsant
drugs in human induced pluripotent stem cell-derived corticalneuronal networks using an MEA system. Sci. Rep. 8, 1–11
(2018).
Tukker, A. M., van Kleef, R. G. D. M., Wijnolts, F. M. J., deGroot, A. & Westerink, R. H. S. Towards animal-free neurotoxicity screening: Applicability of hiPSC-derived neuronalmodels for in vitro seizure liability assessment. ALTEX 37,121–135 (2020).
Tukker, A. M., Wijnolts, F. M. J., de Groot, A. & Westerink, R.
H. S. Human iPSC-derived neuronal models for in vitro neurotoxicity assessment. Neurotoxicology 67, 215–225 (2018).
Grainger, A. I. et al. In vitro models for seizure-liability testing
using induced pluripotent stem cells. Front. Neurosci. 12,
1–14 (2018).

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