Application Note
Human iPSC-derived excitatory neurons and astrocytes differentiated by Quick-Tissue™
technology for functional drug screening on MEAs
Tatsuya Sameshima1, Michaela Kilander2
1. Ricoh Co., Ltd., Kawasaki, Kanagawa, Japan, 2. Elixirgen Scientific, Inc, Baltimore, MD, US

Introduction
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 anddifferentiated into a variety of cell types, thereby providinga valuable and reproducible supply of trillions of cells forextended research. Such hiPSCs can be establishedfrom healthy or diseased individuals to study normal
physiology as well as specific diseases and contributetowards drug discovery and new therapies. We havedeveloped a rapid differentiation method, namely
Quick-Tissue™ technology, resulting in the conversion ofhiPSCs to highly homogenous neurons in 7 to 10 days1
and functional astrocytes in 1 month. Based on theexpression of markers, these neurons and astrocytes
display high levels of purity and low batch-to-batch
variation and have been consistently produced fromdozens of different hiPSC lines, including lines derived
from patients and lines with known mutations. We findthat these hiPSC-derived neurons and astrocytes exhibit
expected responses to known drugs when they aretested using the micro- (or multi-) electrode array (MEA)
system. These hiPSC-derived cells thus have great
potential to revolutionize high-throughput phenotypic andtoxicology screening through establishment of 2D or 3Dmodels (e.g., spheroids, organoids, and organ-on-a-chip)of entirely human origin, thereby accelerating theadvancement of regenerative medicine, tissue
engineering and personalized therapeutic strategies.

Results and Discussion
Excitatory Neurons
Quick-Tissue™
technology,
Elixirgen
Scientific’s
proprietary transcription factor-based technology, allows
rapid and reproducible differentiation of hiPSCs into
neurons without sacrificing the purity of the cells. We
havecharacterized
ourneurons
using
immunofluorescent staining for marker expression andMEA
with
human
primary
astrocytes2.
Our
Quick-Neuron™ Excitatory - Human iPSC-derived
Neurons exhibit typical morphology with neurite
outgrowth and express a variety of neuronal markers,

such as the pan-neuronal marker tubulin beta 3 class III(TUBB3) and the glutamatergic neuron marker vesicularglutamate transporter 1 (vGLUT1/SLC17A7) by day 10 ofdifferentiation (or by day 7 post-thaw: Figure 1A).
Excitatory neurons can be maintained in culture for atleast 7 weeks while exhibiting stable firing activity and aresuitable for a variety of applications such as neurotoxicity
assays.
Astrocytes
Quick-Tissue™ technology also allows rapid andreproducible differentiation of hiPSCs into astrocytes.
We have characterized our astrocytes using
immunofluorescent staining for marker expression,
glutamate uptake assay, and MEA with iPSC-derived
Excitatory Neurons. Our Quick-Glia™ Astrocyte - Human
iPSC-derived Astrocytes exhibit typical morphology
including the characteristic spiky star-shape, and express
a variety of astrocyte markers, such as the Aldehyde
Dehydrogenase 1 Family Member 1L (ALDH1L1), Glial
Fibrillary Acidic Protein (GFAP) and S100 Calcium
Binding Protein B (S100B) by day 49 of differentiation (orby day 7 post-thaw: Figure 1B). At day 56 ofdifferentiation Quick-Glia™ Astrocyte - Human
iPSC-derived Astrocytes show a level of glutamate
uptake comparable to that of human primary astrocytes(Figure 1C). These astrocytes slowly proliferate and canbe maintained in mono-culture in our recommended
medium conditions for several weeks or can bemaintained in co-culture with excitatory neurons in aneuronal culture medium for weeks for the formation of amature neural network.
MEA-drug responses
HavingestablishedthatElixirgen
Scientific’s
Quick-Neuron™ Excitatory - Human iPSC-derived
Neurons and Quick-Glia™ Astrocyte - HumaniPSC-derived Astrocytes display physiologically relevantmarkers and expected functionality, we next exploredtheir utility in MEAs. MEAs offer a powerful, scalable, andhighly sensitive platform to investigate the formation andthe maturation of neural networks by hiPSC-derived
neurons and astrocytes in a high-throughput manner andwere previously demonstrated with hiPSC-derived
neurons and human primary astrocytes2. Unlike

Last revised: March 14, 2023

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1traditional Ca2+ oscillation assays, MEAs offer the uniqueadvantage of allowing the evaluation of dose-dependent
responses of neurons to specific drugs in the same well.
Elixirgen Scientific’s Quick-Neuron™ Excitatory Human iPSC-derived Neurons and Quick-Glia™
Astrocyte - Human iPSC-derived Astrocytes werecocultured on MEA plates for 43 days post-thaw and theirspontaneous firing activity was monitored. Among thewells prepared, these neurons and astrocytes were
uniformly plated and did not show any sign of cell growthor proliferation, or peeling (Figure 2A). We found thatthey showed stable firing and frequent strong network
bursts over the duration of recording (Figure 2B),
indicating that these cells formed mature neuronal
networks and that the network formation is reproducible
across the wells (Table 1). Next, they were exposed toknown compounds at 3 increasing concentrations
sequentially. The compounds used are categorized asreceptor agonists, antagonists, or ion channel blockers.
Analysis of spontaneous firing activity (i.e., spikes)
indicated that these neurons displayed expected
responses to each of the compounds examined (Table2). Acetaminophen served as a negative control andinduced only moderate changes in the spike count atconcentrations of 1-100 μM. Interestingly, with thehistamine receptor agonist histamine, the number ofspikes increased at low dosages of 0.1-1 μM but did notchange beyond the range of the negative control at thehigher dose of 10 μM. When a kainate receptor agonist,kainic acid, was applied at low dosages of 0.1-1 μM, thenumber of spikes showed moderate changes comparable
to those found in the negative controls, but decreased at
10 μM. Glutamate had a similar effect - causing
suppression at a higher dosage. Additionally, when
GABA and dopamine were applied to the cultures at
sufficient concentrations, the number of spikes
decreased, indicating the presence of inhibitory synapses
among the network of these neurons. An AMPA/Kainatereceptor antagonist CNQX and an NMDA receptor
antagonist D-AP5 suppressed neuronal activity at 0.5-50
μM and 1-100 μM, respectively, whereas theadministration of a GABA receptor antagonist picrotoxin
resulted in only small changes in the firing frequency at0.1-10 μM as were found in the negative controls.
Antiepileptic drug Carbamazepine, a sodium channel
blocker, serves as a suppressor of neuronal activity and,as expected, the number of spikes decreased whenadministered at 10 and 100 μM. When a potassiumchannel blocker 4-Aminopyridine (4-AP) was added at 3μM or greater,
the number of spikes increased.
Collectively these data confirm the expression of various
receptors (e.g., NMDA, AMPA, and Kainate) in
Quick-Tissue™ iPSC-derived neurons (co-cultured with
Quick-Tissue™ iPSC-derived astrocytes) that arefunctional and demonstrate their feasibility in MEA-based
drug-screening.
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Figure 1: Characterization of Quick-Tissue™ Excitatory
Neurons and Astrocytes. (A & B) Fluorescence images ofiPSC-derived neurons at Day 10 (A) and iPSC-derived
astrocytes at Day 49 (B). Scale bars = 100 µm. Nuclei werestained with Hoechst 33342. (C) Assessment of glutamate
uptake over 60 minutes in iPSCs, iPSC-derived astrocytes 42
and 56 days after differentiation, and human primary astrocytes.

Conclusion2Animal models have not fully been able to predict drug
responses in humans. Attrition of drugs due toneurotoxicity and problems in the central nervous system
is a serious and expensive issue for pharmaceuticalcompanies. hiPSC-derived neurons and astrocytes
possess great potential to establish human cell-based invitro platforms to screen out toxic small compounds, andmay serve as better alternatives for the study of drug
efficacy and toxicity3-6 without batch-to-batch varianceand ethical issues. 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 andtherapies. The results presented here demonstrate that
Elixirgen Scientific’s hiPSC-derived Quick-Neuron™
Excitatory Neurons are physiologically relevant, havecharacteristic gene expression profiles, and are
amenable to toxicity screening, pharmacogenomics,
drug discovery, disease modeling, and regenerative
medicine. When they are combined with Elixirgen
Scientific’s hiPSC-derived Quick-Glia™ Astrocytes,
assays like MEAs will become more physiologically
relevant to humans without ethical and technicalchallenges in obtaining primary cells and can bepersonalized, particularly when the same hiPSC line isused to differentiate the two cell types

Figure 2: Quick-Neuron™ Excitatory - Human iPSC-derived Neurons cocultured with Quick-Glia™ Astrocyte - Human iPSC-derived
Astrocytes on a CytoView MEA plate. (A) A representative well on the MEA plate shows cells plated homogeneously as seen on days 22(left) and 43 (right). Scale bars = 1 mm. (B) A raster plot of the MEA recording for the representative well shown in A demonstrates thatduring a 10-minute recording, there are three strong synchronized network bursts (pink rectangles) detected without compound
treatment.
Table 1. Well-to-well consistency in parameters during 10-minute recording on day 43
Measured parameters
Coefficient of variance
Number of
Number of
Number of Spikes Number of Active Number of Network Number of
Number ofwells (n)Active(±SD)
Electrodes (±SD)
Bursts (±SD)
SpikesNetwork Bursts
Electrodes
Plate 148
17,899 ± 5,177
14.7 ± 1.06
2.5 ± 0.71
0.29
0.071
0.28
Plate 247
17,570 ± 4,260
14.9 ± 0.94
2.6 ± 0.570.240.062
0.21
Table 2. Summary of drug responses measured by the MEA system.
Mechanism

Receptor/Channel

DrugConc. Range

Relative # of spikes*

Vehicle control
Negative control

DMSO
0.2, 0.3, 0.4%
0.4
0.3
0.4
0.7
0.9
0.7
Acetaminophen 1, 10, 100 μM
0.3
-0.7
-1.7
Glutamate
Glutamate0.3, 3, 30 μM
-0.7
-7.5
-12.1
GABA
GABA
0.1, 1, 10 μM
-2.5
-2.5
-2.1
AgonistDopamine
Dopamine
0.1, 1, 10 μM
1.5
1.7
-0.8
Histamine
Histamine
0.1, 1, 10 μM
0.60.3-3.0
KainateKainic acid0.1, 1, 10 μM-3.0-3.0-3.0
AMPA/Kainate
CNQX
0.5, 5, 50 μM
0.4
0.6
1.0
Antagonist
GABA(A)
Picrotoxin
0.1, 1, 10 μM-2.3-3.0-3.0
NMDA
D-AP5
1, 10, 100 μM
0.0-2.0-3.0
SodiumCarbamazepine 1, 10, 100 μM
Ion Channel Blocker
0.8
2.3
2.6
Potassium
4-AP
0.3, 3, 30 μM* The number of spikes relative to that of a baseline (before DMSO administration) is shown as a value in a log2 scale. The gradient of redindicates increase in the number of spikes, whereas the gradient of blue indicates decrease in the number of spikes.

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3Materials and methods
Cell Culture
Elixirgen Scientific’s Quick-Neuron™ Excitatory - Human
iPSC-derived Neurons (Catalog No. EX-SeV-CW50065)
and Quick-Glia™ Astrocytes - Human iPSC-derived
Astrocytes (Catalog No. AS-SeV-CW50065) were
thawed and maintained following the instructions in theuser guides (https://www.elixirgensci.com/resources/).
Immunofluorescence Staining
Elixirgen Scientific’s Quick-Neuron™ Excitatory - Human
iPSC-derived Neurons and Quick-Glia™ Astrocytes Human iPSC-derived Astrocytes were maintained for 7days post-thaw under medium conditions instructed by
the user guides prior to immunofluorescence staining.
Glutamate uptake assay
Human primary astrocytes (ScienCell ResearchLaboratories, Catalog number: 1800) and Quick-Glia Astrocyte SeV kit cultures, harvested at either 28 days or
42 days post SeV infection, were plated at 7,800 cells/cm2
in a Geltrex-coated 96-well plate (Corning, Catalog
number: 353072) and grown in ScienCell Astrocyte
medium (ScienCell Research Laboratories, without FBS,Catalog number: 1801) for 14 days. As a negative control,
human iPSCs were plated in StemFit Basic04 medium(Ajinomoto, Catalog number: ASB04-C) two days before
assay measurement using the plating density statedabove. 30 minutes prior to assay execution, culture
medium was replaced with Hanks' Balanced Salt Solution(HBSS) without phenol red (Fisher Scientific, Catalog
number: 14025092). Subsequently, cells were exposed
to 100 µM L-glutamate (Tocris, Catalog number: 0218) inHBSS for 60 minutes. Immediately after the assay,
solution was collected and cells were dislodged using a
1:1 mixture of TrypLE Select Enzyme (Fisher Scientific,Catalog number: 12563029) and 0.02% EDTA (Lonza,Catalog number: 17-711E). The numbers of live cells
were determined using a NucleoCounter NC-200(Chemometec, Catalog number: 900-0200) cell counter.
Glutamate concentration was measured by abioluminescence-based Glutamate-Glo assay kit(Promega,Catalog
number:J7021)
andaSPECTRAFluor Plus microplate reader (Tecan). To
determine the amount of glutamate cleared, all values
were first background signal-subtracted using a sample
treated with HBSS only and concentrations werethereafter acquired from the glutamate titration curve
according to manufacturer’s instructions.
MEA
Quick-Neuron™ Excitatory Neurons were plated at 0.8 x
10⁵ cells/well of a CytoView 48-well MEA plate with 16
electrodes per recording area in each well (1.21 mm2;elixirgensci.com

Axion Biosystems), after mixing with Quick-Glia™
Astrocytes at 0.2 x 10⁵ cells/well and 0.2 μg/well laminin
similarly to our Application Protocol of the Excitatory
Neurons and human primary astrocytes except thatastrocytes were thawed using Medium N. Cultures weremaintained at 37°C, 5% CO2 for 49 days with 50% of themedium changed twice a week. Observation of thecultures and recording were done once a week. Cells ineach well were exposed to a diluent of drugs (i.e., 0.1 %DMSO) first and then drugs in 3 increasing doses
sequentially with intervals of 10-min recording followedby 10-min resting under 37°C, 5% CO2. Data werecollected immediately after administration of drugs using
MaestroPro (Axion Biosystems) at a sampling rate of12.5 kHz per electrode. After a series of recordings wascompleted, treated cells were washed with medium andincubated overnight. Finally, spontaneous spike firing
activity was measured again to confirm that cells were notdamaged by the treatment. Drug response was evaluatedby measuring spike counts. Data were extracted by
AxISNavigator 2.0.4 and analysis was performed using
Neural Metric Tool.
References
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neurons by mRNAs encoding transcription
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hiPSC-derived Excitatory Neurons Differentiatedby Quick-Neuron™ Technology(https://www.elixirgensci.com/applications/)
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& Suzuki, I. Toxicological evaluation of
convulsant and anticonvulsant drugs in humaninduced pluripotent stem cell-derived corticalneuronal networks using an MEA system. Sci.
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M. J., de Groot, A. & Westerink, R. H. S. Towards
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seizure liability assessment. ALTEX 37, 121–135(2020).
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neuronal models for in vitro neurotoxicity
assessment. Neurotoxicology 67, 215–225(2018).
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