244.02

Functional and pharmacological evaluation of iPSC-derived astrocytes
generated by a rapid differentiation methodYuina Wada1*, Yuki Kaga1, Tatsuya Sameshima1, Michaela Kilander2, Tomoyuki Aratani1, Rie Yamoto1, Toshihiko Hosoya1
1Ricoh Company, Ltd., 2Elixirgen Scientific, Inc.
*e-mail: yuina.wada@jp.ricoh.com

Introduction

The Quick-TissueTM technology isa transcription factor-based
method for rapid differentiation ofstem cells (iPSCs or ESCs) into
desired cell types. With thismethod, a pure population ofdifferentiated cells can beproduced typically within 10 days.

Average 3.8
CV 0.25

Average 6.9
CV 0.39

Average 18.0
CV 0.24•***

Calcium spikes of the co-cultured neurons
exhibited stronger synchrony and higher
frequency.
Co-culture with iPSC-derived astrocytes shows
the same increase in spike number as when cocultured with human astrocytes.

***•

Mono-cultured
neurons

Co-cultured
neurons withhuman primary
astrocytesn ≧60, ***p<0.001. The significant difference for mono-cultured neurons wasanalyzed by unpaired t-test.

Co-cultured
neurons withiPSC-derivedastrocytes

S100β/Hoechst

ALDH1L1/Hoechst

Average 20.5
CV 0.23

Average 19.1
CV 0.17②①
Astrocyte lot

Date④

•

Lot A

Neuron lot
Plate③Lot a
Plate 1

Lot b•

Plate 3

Plate 2

* ***

***

DMSO

****
***

*** ********** ****** ***

1.1 3.3 10 30 0.4 1.1 3.3 10 3.7 11.1 33.3 100 3.7 11.1 33.3 100 0.7 2.2 6.7 20 0.4 1.1 3.3 10 37 111 333 1000 0.4 1.1 3.3 10 1.1 3.3 10 30 3.7 11.1 33.3 100 3.7 11.1 33.3 100Pentylenetetr Picrotoxin
Carbamazepine Linopirdine
Glutamate Kainic acid
CNQX
D-AP5
Amoxapine
GABA4-APazole (PTZ)***Mono-cultured neurons
Co-cultured neurons with human astrocytes
Co-cultured neurons with iPSC-derived astrocytes******DMSO*** *** ***CNQX,
100 μMPTZ,
100 μM↓

Excitotoxicity

CNQX↓Inhibitory

↓〇

↓

〇

D-AP5↓

Inhibitory

↓〇

↓

〇

Amoxapine↑

Excitatory

↑

〇

↑

〇

GABA

↓

Inhibitory↓〇↓〇antagonist

PTZ↑

Excitatory→×*→×*

GABA receptor
PotassiumchannelSodium channelantagonist

Picrotoxin↑

Excitatory→×*→×*

blocker

4-AP↑

Excitatory

↑

〇

↑

〇

blocker

CBZ↓

Inhibitory

↓〇

↓

〇

KCNQ channel

blocker

Linopirdine↑

Excitatory

↑〇

↑

〇

Co-cultured neurons
with human primary astrocytes

4-AP,
30 μM

Co-cultured neurons
with iPSC-derived astrocytes

0.1% DMSO

10 min after drug administration. ×means 0. n = 6.
***p<0.001. Statistical significance of the comparison
to 0.1% DMSO was determined by the Wilcoxon rank
sum test.

(C)2022 Ricoh Company, Ltd. All rights reserved.

human primary

iPSC-derived

0-2.5-5.0

iPSC-derived

5432100(CBZ)

Co-culture with iPSC-derived
astrocytes shows the similar drug
responses in spike number as
when mono-cultured neurons andco-cultured with humanastrocytes.

Co-cultured neurons with astrocytes

[μM]

0.5

5500CNQX

10 min after drug administration. ×means 0. n = 6. *p<0.05, **p<0.01, ***p<0.001. Statistical significance of the comparison to 0.1% DMSO
was determined by the Wilcoxon rank sum test.

Kainic acid,
10 μMExcitotoxicity

Co-cultured neurons with astrocytes******** *** ***↓Log2(Reactive ratio)[MFR]**Log (Change ratio of spike number)

iPSC-derived astrocyte is a pure cell population of astrocytes
expressing astrocytic markers.

Excitatory

Evaluation of drug responses

Comparison of drug responses

More than 80% of cells with high expression of astrocytic markers GFAP, S100β andALDH1L1.

↑iPSC-derived astrocytes provide neuron-supportive functions.

CNQX, D-AP5, GABA, 4-AP, CBZ and Linopirdine: Pharmacological effects detected dose-dependently
Amoxapine >6.7 μM: Pharmacological effects detected, <20 μM: Excitotoxicity detected
Glutamate and Kainic acid: Excitotoxicity detected
PTZ and Picrotoxin: No effects

Immunofluorescence of Quick-Glia™ Astrocyte shows the expression of the astrocytic markers GFAP, S100β and ALDH1L1 on days 7 post-differentiation(scale bar = 100 μm).

Kainic acid

Firing of the co-cultures with astrocytes exhibited higher frequency than its of mono-culture ofexcitatory neurons.
Synchronized network bursts were observed in co-cultures with astrocytes.

YX

***

*** *** ***Excitotoxicity

Mono-cultured neurons******↓GABA receptor

human primary***

Excitotoxicity

Average 18.4
CV 0.28

Drug responses of co-cultured neurons with iPSC-derived astrocytes***↓

Electrical activity

Number of spikes /20min

Average 18.0
CV 0.24

Evaluation of drug responses*Excitatoryn ≧60• Cells in all well exhibited spontaneous Ca2+ spikes.
• No difference in number of Ca2+ spikes between
wells in all conditions (CV < 0.3), and median value
is in the range 18-21.

Log (Change ratio of spike number)

Result 1: The expression of astrocytic markers

Ca2+ spikes in the co-culture of ① detected by using FDSS. iPSC-derived neurons
were cultured with iPSC-derived astrocytes in 384-well plates. 7 weeks after
differentiation. Single squares represent single wells in the 384-well plate.

**↑Result 3: Multielectrode Array (MEA) assay
Evaluation of neuronal-supportive functions

Variation of number of Ca2+ spikes

Difference between wells on one plate

Materials and Methods⚫ Cells：Human iPSC-derived astrocytes (Quick-Glia™ Astrocyte) and iPSC-derived excitatoryneurons (Quick-Neuron™ Excitatory) from Elixirgen Scientific, Primary Human astrocytes fromScienCell Research Laboratories (in 384-well plate assay) and Thermo Fisher Scientific (inMEA assay)⚫ Plate: 384-well plate (Thermo Fisher Scientific) or CytoView MEA 48 (Axion)⚫ Medium conditions: Neurobasal Plus Medium, B-27™ Plus Supplement, Neuron Culture
Medium (FUJIFILM Wako Pure Chemical Corporation)⚫ Culture period: 6 or 7 weeks (post-differentiation)⚫ Calcium imaging:- Coating methods: Ricoh’s method- Instrument: FDSS/μCell system- Neurons (10,000 cells /well)- Ca2+ indicator: Cal-520, AM (f.c. 2 μM)- Astrocytes (2,500 cells /well)- Measurement time: 20 min- Data acquisition interval: 0.1 sec⚫ Monitoring electrical activity (MEA):- Coating materials: PEI / Matrigel- Instrument: Maestro Pro (Axion)- Neurons (80,000 cells /well)- Measurement time: 10min- Astrocytes (20,000 cells /well)

Glutamate

iPSC-derived astrocytes are useful for HTS pharmacological assays.

Evaluation of variability in the co-culture

We evaluate whether the Quick-Glia™ Astrocytes (iPSC-derived astrocytes)
provide supportive functions that are similar to that of human primary
astrocytes and whether their co-culture system is suitable for HTS assays.

Mode*The excitatory neurons contains little GABAergic neurons.

iPSC-derived astrocytes provide neuron-supportive functions.

◆ Aim of this study

Spike number changes in cocultured
neurons with astrocytes
Human primary
iPSC-derived

Expected
Compound changes in spike
numberAMPA/NMDAagonist
receptors
Kainate receptors agonist
AMPA/Kainateantagonist
receptors
NMDA receptors antagonist
Serotonergic
Inhibitor
receptors
GABA receptor
agonistLog2(Reactive ratio)[MFR]

Source;Company websites

Receptor/
Ion channel

Evaluation of neuronal-supportive functions
Number of spikes /20min◆ iPSC-derived neural and glial cells generated by
the Quick-Tissue™ technology

GFAP /Hoechst

Summary of drug responses in 384-well plates

Result 2: Ca2+ transient assay in 384-well plates

0.5

550 [μM]

00.3

330

00.3

330 [μM]4-AP

n = 5, *p<0.01, Statistical significance of the comparison to 0 μM (0.1 % DMSO) was determined by the Wilcoxon rank sum test.

Pharmacological effect was detected dose-dependently when co-cultures with astrocytes wereexposed to drugs.

The cells co-cultured on MEA plates show similar drug responses in384-well plate.

Conclusions••••

iPSC-derived astrocytes (Quick-Glia™ Astrocyte) provide neuronsupportive functions.
These astrocytes are useful for pharmacological assays.
The system is suited for HTS assays in 384-well plates.
The cells co-cultured on MEA plates showed similar results in 384-wellplate.