Application of Rapid iPSC Differentiation
to Neurotoxicity Assays

0015

Rie Yamoto1*, Hikaru Watanabe2, Manabu Seo3, Toshihiko Hosoya1

1Drug Discovery Business Department, Healthcare Business Group, RICOH COMPANY, Ltd. 2Biomedical Research Department,

Healthcare Business Group, RICOH COMPANY, Ltd. 3Elixirgen Scientific, Inc.

Introduction*e-mail; rie.yamoto@jp.ricoh.com

Materials and Methods

The Quick-TissueTM technology is atranscription factor-based method forrapid differentiation of stem cells(iPSCs or ESCs) into desired cell types.
The method generates purepopulations of differentiated cells
within 1-2 weeks.
To investigate the suitability of QuickTissue™ neurons to toxicologicalassays, we analyzed theirpharmacological characteristics using
Ca2+ imaging.
1-2 weeks

Cells: Excitatory neurons were generated using the Quick-TissueTM
technology from iPSCs originating from a healthy donor (Elixirgen Scientific,
Inc. #EX-SeV-CW50065). Primary human astrocytes were obtained fromThermo Fisher Scientific Inc.
Spheroid culture: 96-well plates were seeded with the neurons and precultured astrocytes at the density of 8,000 cells/well. Plates were cultured
in the BrainPhys medium (STEMCELL technologies Inc.) for 6 weeks.
Ca2+ imaging: Spheroids were loaded with a fluorescent Ca2+ reporter dye(MOLECULAR DEVICES, LLC.). Fluorescence intensity (FI) was measured
with the FDSS6000 system (Hamamatsu Photonics K.K.). Drug responses
were analyzed by determining the change in the number of peaks of Ca2+
oscillation and the ratio of FI before and after the drug application.
Statistical significance of the comparison to 0.1 % DMSO was determinedby the Wilcoxon rank sum test.
Drug application

Drug application

Summary of compounds
CompoundGABA
Bicuculline

Function
GABA agonist

Gabazine1
Pentylentetrazole2
Picrotoxin1
Glutamate
Kainic Acid
NMDA
CNQX
D-AP5
Dopamine
Chlorpromazine
Haloperidol
5-HT
Amoxapine
Buspirone
Bethanechol

Terminallydifferentiatedneuron turnaround(weeks)Serotonin agonist
Serotonin H1/2 agonist
Mono amine reuptake blocker
Muscarine agonist
Muscarine antagonist
Nicotine/Gly antagonist
Histamine agonist

1

FI averaged
for the last 1min

Diphenhydramine1
4-Aminopiridine
Linopirdine

FI averaged
for the first 1min

Histamine H1 antagonistSodium channel blocker

10, 100

Valproic acid3

change in the number of peaks
FI Ratio (after/before)(after-before)
Analysis method

1, 10
10, 100
1, 10
10, 100
10, 100
3, 30
10, 100
10, 100
1, 10
1, 10
3, 30
3, 30
1, 10
1, 10
1, 10
3, 30
3, 30
1, 10
1, 10
3, 30
10, 100
10, 100

Potassium channel blocker

Carbamazepine3
Phenytoin3

Source;Company websites

Results

the number of peaks
for 10 min

100, 1000

Dopamine D2 antagonist

Ketotifen1

the number of peaks
for 10 min

10, 100

GABA(A) antagonist

AMPA/NMDA agonist
Kainate agonist
NMDA agonist
AMPA/Kainate antagonist
NMDA antagonist
Dopamine agonist

Pilocarpine2
Atropine
Strychnine
Histamine

Conc. (μM)
0.1, 13, 30

1100, 1000

2

Antibacterial agent

Enoxacin

1, 101; Convulsant, 2; Epileptogenic, 3; Antiepileptic

Ca2+ response to major pharmacological compounds

(a)

GABA

Kainic acid

1μM

10μM

30μM

0.1μM

1μM

3μM

5-HT

Dopamine***: p<0.05

DMSO
Histamine

100μM

10μM

30μM

10μM

1μM

3μM

Diphenhydramine(b)

D-AP5Linpirdine*****Carbamazepine

10μM

100μM

100μM

1μM

10μM

10μM(c)*****: p<0.05
*DMSO

*DMSO

0.1%

*5 min(a) Waveforms of Ca2+ oscillation before and after theapplication of drugs that affect the 6 major types of receptors.
(b) Change in the number Ca2+ oscillation peaks. The peaks
were counted during 10 min before and after the drug
application (n=6). (c) Ratio of the fluorescence intensity (FI)measured during 1 min before and after the drug application(n=6).

Summary of drug response
ReceptorsGABA
Glutamate

Drugs
AgonistAntagonist
AgonistAntagonist

Dopamine
Serotonin
CholinHistamine
Ion channel

AgonistAntagonist
AgonistReuptake blocker
AgonistAntagonist
AgonistAntagonist
blockerall
GABAA
AMPA/NMDA
KainateNMDA
AMPA/Kainate
NMDA
all
D2
all
Muscarine
Muscarine
Nicotine/Glyall
H1
Potassium
SodiumResponse
not determined×
Excitatory〇
Excitatory〇*
Excitatory〇〇*〇
Inhibitory〇*×
Inhibitory〇*
Excitatory〇
Inhibitory〇〇〇
Excitatory〇*〇*〇*〇*The concentration of GABA in this test was lower than IC50
value (2.8 μM) and the data was removed from the analysis.

Functional receptors
Confirmed by the responses to theagonists; Glutamate, Dopamine,Serotonin, Cholin, and Histamine
Functional ion channels
Confirmed by the responses to theion channel blockers; K+ and Na+
Functional Synapses
Confirmed by the responses to theantagonists; Glutamate, Serotonin,Cholin, and Histamine*: p<0.05

*

*

*

*

Summary and Conclusions
 Pharmacological analyses of Quick-Tissue™ neurons
suggest the presence of functional receptors for all the 6major neurotransmitters and functional channels for twocations. The results also suggest synaptic connections forGlutamate, Serotonin, Cholin, and Histamine. Theneurons also responded to convulsant, epileptogenic, andantiepileptic drugs.
 The fluorescent intensity (FI) and the number for Ca2+
oscillation peaks responded to drugs and thus likely
constitute reliable drug response markers.
 These results suggest Quick-TissueTM neurons are suited
to neurotoxicity assays and drug screenings.
COI Disclosure Information
We have the following financial relationships to disclose.
Research funds from: Elixirgen Scientific, Inc.

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