高速分化技術によりヒトiPS細胞から作成したアストロサイトの神経細胞サポート機能の評価

2P-056Functional evaluation of iPSC-derived astrocytes generated by a rapid differentiation method○和田唯奈1*, 矢本梨恵1, 瀬尾学2, 細谷俊彦1 1株式会社リコー, 2Elixirgen Scientific, Inc.

Abstract

1Ricoh Company, Ltd., 2Elixirgen Scientific, Inc.
○Yuina Wada1*, Rie Yamoto1, Manabu Seo2, Toshihiko Hosoya1*e-mail; yuina.wada@jp.ricoh.com

The Quick-Tissue™ technology (Elixirgen Scientific, Inc.) is a transcription factor-based method for rapid differentiation of human induced pluripotent stem cells (iPSCs) into desired cell types.
The method generates a pure population of neurons (Quick-Neuron™ Excitatory) within 10 days and astrocytes (Quick-Glia™ Astrocyte) within 28 days, respectively. Recent studies haverevealed that astrocytes play important roles in the brain function and nervous system diseases. The neuron-astrocyte co-culture system is an in vitro tool to evaluate the supportivefunctions of astrocytes and therefore is an attractive test bed for drug screenings, toxicological assays, and disease research. In the present study, we examined whether the iPSC-derivedastrocytes provide supportive functions. We co-cultured the iPSC-derived neurons with the iPSC-derived astrocytes and characterized their responses to various drugs using calcium imaging.
The co-cultured neurons responded to antagonists and agonists for receptors of various neurotransmitters as expected. Calcium oscillations of the co-cultured neurons exhibited higher
frequency and stronger synchrony compared to that of neuronal cultures without astrocytes, suggesting supportive functions of the astrocytes. These results suggest that the iPSC-derived
astrocytes provide neuron‐supportive functions that are similar to those of in vivo astrocytes and that they are useful for pharmacological assays of glial functions.

Introduction◆ iPSC-derived neural and glial cells generated by the Quick-Tissue™ technology
Quick-Glia™ Astrocyte

Quick-Neuron™ Excitatory• Differentiate within 28 days
• Pure populations
ALDH1L1 /Hoechst• Differentiate within 10 days
• Positive for neuronal markers
TubβIII/vGLUT1/Hoechst

S100β/Hoechst

Source;Company websites

The Quick-TissueTM technology is a transcription factor-based
method for rapid differentiation of stem cells (iPSCs or ESCs) into
desired cell types. With this method, a pure population ofdifferentiated cells can be produced typically within 10 days.

Source:Elixirgen ScientificwebsiteImmunofluorescence of Quick-Glia™ Astrocyte shows the expressionof the astrocytic markers ALDH1L1 and s100β (scale bar = 100 μm).

◆ Aim of this study

Immunofluorescence staining of Quick-Neuron™ Excitatoryshows the expression of the pan-neuronal marker TUBB3 andthe glutamatergic neuron marker vGLUT1 (scale bar = 100 μm).

To assess the neuronal-supportive functions of the iPSC-derived astrocytes (Quick-Glia™ Astrocyte),
we examined the calcium spikes in excitatory neurons cultured with these iPSC-derived astrocytes.

Materials and Methods⚫ Cultured period: 7 weeks⚫ Calcium imaging:⚫ Cells：Human iPSC-derived astrocytes (Quick-Glia™ Astrocyte) and iPSC-derived excitatoryneurons (Quick-Neuron™ Excitatory) from Elixirgen Scientific⚫ Plate: 96-well plate (Corning) or 384-well plate (Thermo Fisher Scientific)⚫ Medium conditions: Neurobasal Plus Medium, B-27™ Plus Supplement, Neuron Culture
Medium (FUJIFILM Wako Pure Chemical Corporation)-Instrument: FDSS/μCell system
Ca2+ indicator: Cal-520AM (f.c. 2 μM)
Measurement time: 20min
Data acquisition interval: 0.1 secResults◆ Evaluation of neuronal-supportive functions in 96-well plates
w/oastrocytes

16:1

neurons : astrocytes
4:1
2:1

8:1

1:1

Calcium spikes of the co-cultured neurons:
・exhibited stronger synchrony and higher frequency
・supported robustly under a wide range of mixing ratios

1:2

Fluorescentintensity

Δ700

iPSC-derived astrocytes provide neuron-supportivefunctions

20 min◆ Evaluation of drug responses in the co-culture in 384-well plates

Neurons : astrocytes = 4:1
× means 0.
*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.

Log (Ratio of baseline amplitude)

Baseline amplitude [10 min after drug administration]
CNQX

D-AP5

AMPA/Kainate
receptor antagonist

Glutamate

NMDA receptor
antagonist

Kainic acid

AMPA/NMDA
receptor agonist

Amoxapine

Chlorpromazine

Kainate receptor
selective agonist

DR, 5HTR
antagonist

PTZGABA antagonist

SSRI

Linopirdine

Carbamazepine

4-AP

Selective KCNQ
channel blocker

Sodium channel
blocker

Potassium channel
blocker****** *****[μM] 3.7[N]6**

***

**

**
*

**
*

*

*

*

*

****

*

*DMSO

*

11.1

33.3

100

3.7

11.1

33.3

100

1.1

3.3

1030

0.4

1.1

3.3

10

3.7

11.1

33.3

100

0.7

2.2

6.7

20

37

98

10

7

10

7

4

10

97

6

7

10

4

7

5

6

1

4

4

5

6

7

4

111.1 333.3
6

6

1000

3.7

11.1

33.3

100

3.7

11.1

33.3

100

1.1

3.310305887957577712111722

Change ratio of spike number

Spike number [10 min after drug administration]
CNQX

100D-AP5Glutamate

Kainic acid

Amoxapine

Chlorpromazine

PTZ

Linopirdine

Carbamazepine

4-AP

***

***

10DMSO

1***

***

*

*** ***

0.1***** ***

0.01[μM] 3.7[N]

611.1×33.3

×100

9810*** *** ***3.7

×11.1×710733.3×***** ****** ***

0.4

1.1

3.3

103.7

710475*** ***××30
61001.1

3.3

1041097××××***

**

11.1

33.3

1000.72.2

6.7203761445674××*********××111.1 333.3
66×1000

3.7

11.1

33.3

100

3.7

11.1

33.3

100

1.1

3.3

1030

5887

95

75

7

7

7

12

111722

CNQX, D-AP5, Glutamate, Chlorpromazine, Amoxapine, Carbamazepine, Linopirdine and 4-AP: Pharmacological effects detected
Amoxapine: Toxicity detected, Pentylenetetrazole (PTZ) : No effects

Summary•••iPSC-derived astrocytes are useful for HTS pharmacological assays

iPSC-derived astrocytes (Quick-Glia™ Astrocyte) provide neuron-supportive functions.
These astrocytes are useful for pharmacological assays.
The system is suited for HTS assays in 384-well plates.
(C)2022 Ricoh Company, Ltd. All rights reserved.

NEURO2022

Disclosure of Conflict of Interest
Name of first author: Yuina Wada

I have no COI
with regard to the presentation.