Characterization of iPSC-derived
Skeletal Muscle
Generated with Rapid Differentiation Method〇Satoru Nagasawa*1, Tomoaki Nakayama*1 , Motoki Azuma*1
1 Drug Discovery Business Department, Biomedical Business Center, Ricoh Futures BU, RICOH Company Ltd.

Introduction
Recently, iPSC-derived skeletal muscle cells (iSkMC) havebeen used in disease and drug discovery research. However,in order to obtain the desired cells from iPS cells (iPSC), along differentiation process is generally required, which isone of the issues in disease and drug discovery research. In
this study, we characterized iSkMC that were differentiatedusing Quick-TissueTM Technology, which enables celldifferentiation in a short period of time. Specifically, theexpression of representative markers and the function ofacetylcholine receptors were evaluated to verify thepossibility of shortening the time required for disease anddrug discovery research.

Quick-TissueTM technology is atranscription factor-based method for
rapid differentiation of stem cells

Differentiation Protocol

Differentiation
Typically 1-2 weeks

PlatingSeVmRNATransfection

Day0

Day1

Day2

Day3

Day4

AssayDay5~8Medium M

Robustness
Low dependency on
iPSC lines

Day9

Medium M(P)Materials
ü iPSC︓Riken, HPS1005 1383D2
ü Differentiation reagent︓Quick-Muscle™ Skeletal - SeV Kit(Elixirgen Scientific）

Results
1. Characterization of iPSC-derived Skeletal Muscle Cells
Day 1

Replating to96 well plate

Day 4

The iPSC-derived skeletal muscle cells
were characterized for the morphology
and protein expression of skeletal
muscle markers including myosin heavy
chain (MHC) and sarcomeric alphaactinin (α-actinin).

Day 7(a) Spindle-shaped cells were observedon day 4 and myotube-like cells wereobserved on day7 after the start ofdifferentiation induction.
40 μm

40 μm(b) The immunocytochemistry (ICC)
images show the expression of themarker proteins and multinucleation. In
addition, a Z-Disc-like structure wasobserved.

40 μm(a) Morphological changes (Phase contrast images)
MHC/Hoechst

α-actinin/Hoechst

Antibody︓- MHC (MAB4470, R&D Systems)- α-actinin (A7811, Sigma)

Z-Disc like structure
100 μm

100 μm

20 μm(b) Marker expression (Left: MHC, Center and Right: α-actinin)

2. Evaluation of Acetylcholine (ACh) Receptor Functions
Methods:
Equipment: FDSS/μCELL (Hamamatsu Photonics)
Method: ACh was added 1.5 min after the onset ofrecording. Total recording time: 6.5 min.

0.001 μM

0.01 μM

0.03 μM

0.1 μM

0.3 μM

1 μM

3 μM

10 μM

100 μM(a) Ca2+ Intensity after the addition of ACh

Peak fluorescence intensity F/F00 μM

Pretreatment
Ca2+ indicator: EarlyTox Cardiotoxicity Kit (Molecular Device)
Concentration: 50%. Incubation: 2 h at 37°C.

(b) Dependency of the Ca2+ signal intensity on the ACh concentration

Conclusions
In this study, we showed that the iPSC cell-derived skeletal muscle cells (iSkMCs) generated with the QuickTissueTM technology exhibit cellular characteristics expected for human muscle cells including expression ofskeletal muscle markers, the Z-disk like structure, and acetylcholine responses. Therefore, the Quick TissueTM
technology and the human iSkMCs provide simple and quick methods for skeletal muscle research in the fields ofdisease analysis and drug discovery.

(C)2021 Ricoh Company, Ltd. All rights reserved.

Skeletal muscle cells induced by QuickTissueTM technology showed tubularmorphology and multinucleation after
7 days from iPSCs. In addition,
expression of skeletal muscle cellmarkers and formation of the Z-disclike structures were observed. These
results indicate that the cells havecharacteristics of skeletal muscle cells.
The changes of the intracellular Ca2+
density after the addition of ACh weremeasured using Ca2+ indicators.
(a) Ca2+ fluorescence intensity before andafter the addition of ACh. Immediately after
the addition of ACh, the signal intensityincreased and reached its peak in 10
seconds and then slowly returned to thesteady state. The peak intensity depended
on the ACh concentration.
(b) The peak fluorescence intensity
positively correlated with the AChconcentration (blue circle). When the AChreceptor antagonist, Tubocurarine, wasadded in advance, the signal was clearlysuppressed.

Ca2+ signals increased in a concentrationdependent manner and was inhibited by
the antagonist. These results suggest thatthe acetylcholine receptors of iPS cellderived skeletal muscle cells differentiatedby Quick-TissueTM technology function
similarly to the in vivo counterparts.