Journal of Pharmacological Sciences xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of Pharmacological Sciences

journal homepage: www.elsevier.com/locate/jphs

Full Paper

Evaluation of the susceptibility of neurons and neural stem/progenitor

cells derived from human induced pluripotent stem cells to anticancer
drugs
Hayato Fukusumi a, Yukako Handa b, Tomoko Shofuda a, Yonehiro Kanemura b, c, d, *
a
Division of Stem Cell Research, Department of Biomedical Research and Innovation, Institute for Clinical Research, National Hospital Organization Osaka
National Hospital, Osaka 540-0006, Japan
b
Division of Regenerative Medicine, Department of Biomedical Research and Innovation, Institute for Clinical Research, National Hospital Organization
Osaka National Hospital, Osaka 540-0006, Japan
c
Department of Neurosurgery, National Hospital Organization Osaka National Hospital, Osaka 540-0006, Japan
d
Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Various chemicals, including pharmaceuticals, can induce acute or delayed neurotoxicity in humans.
Received 10 May 2019 Because isolation of human primary neurons is extremely difficult, toxicity tests for these agents have
Received in revised form been performed using in vivo or in vitro models. Human induced pluripotent stem cells (hiPSCs) can be
2 August 2019
used to establish hiPSC-derived neural stem/progenitor cells (hiPSC-NSPCs), which can then be used to
Accepted 9 August 2019
obtain hiPSC-neurons. In this study, we differentiated hiPSC-NSPCs into neurons and evaluated the
Available online xxx
susceptibility of hiPSC-neurons and parental hiPSC-NSPCs to anticancer drugs in vitro by ATP assay and
immunocytostaining. The hiPSC-neurons were more resistant to anticancer drugs than the parental
Keywords:
Human induced pluripotent stem cell
hiPSC-NSPCs. In the toxicity tests, high-dose cisplatin reduced the levels of ELAVL3/4, a neuronal marker,
Neural stem/progenitor cell in the hiPSC-neurons. These results suggest that our methodology is potentially applicable for efficient
Neuron determination of the toxicity of any drug to hiPSC-neurons.
Anticancer drug © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological
Toxicology Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

1. Introduction insufficient sensitivity and efficiency, and animal welfare issues.4,5

Various chemical substances, including pharmaceuticals, pose
potential risks of inducing acute or delayed neurotoxicity in adults
and causing developmental neurotoxicity in fetuses or children.1 To
ensure the safety of chemical substances and drugs, neurotoxicity
risk assessment is critical, and an appropriate evaluation platform
for neurotoxicity is desired.2 At present, most neurotoxicity and
preclinical studies on drugs affecting the central nervous system
(CNS) are typically performed following treatment of experimental
animals, mainly rodents, in vivo.2,3 However, these in vivo assays
have various issues, including species differences between experi-
mental animals and humans, high cost, low throughput,
* Corresponding author. Department of Biomedical Research and Innovation,
Institute for Clinical Research, National Hospital Organization Osaka National
Hospital, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan. Fax: þ81 6 6946 3530.
E-mail address: yonehirok@gmail.com (Y. Kanemura).
Peer review under responsibility of Japanese Pharmacological Society.
Therefore, more effective and reasonable alternatives to in vivo
animal testing are desired.
One promising alternative assay is an in vitro test using primary
neuronal cells. The usefulness of in vitro neurotoxicity tests using
primary neurons has been reported.6,7 In addition, in vitro assays
using mouse embryonic stem cells (ESCs) have also been suggested.
However, the use of human primary neurons for neurotoxicity
studies has been greatly limited because of the low accessibility of
human CNS tissues, the technical difficulties related to neuron
isolation from adult human CNS tissues, and the higher ethical
controversy surrounding the use of human CNS tissues and/or fetal
cells compared to animal tissues or cells. Another option to over-
come these issues involves the use of human pluripotent stem cells,
such as human ESCs and human induced pluripotent stem cells
(hiPSCs). In particular, hiPSCs are promising and ethically less
controversial cell sources for neurotoxicity studies. Several pro-
tocols have already been reported for the neural induction of
hESCs/hiPSCs, and the contributions of these hiPSC-derived neural
cells to neurotoxicity assays have been well discussed.8e10

https://doi.org/10.1016/j.jphs.2019.08.002
1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Fukusumi H et al., Evaluation of the susceptibility of neurons and neural stem/progenitor cells derived from human
induced pluripotent stem cells to anticancer drugs, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.08.002
2 H. Fukusumi et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
Anticancer drugs are broadly used as adjuvant therapies against
various cancers in combination with surgical resection of tumors
and radiation therapy. However, it is recognized that some
chemotherapeutic agents cause neurotoxic damage in the central
and peripheral nervous systems in human patients,11,12 resulting in
discontinuation of treatment. Although these adverse effects are a
result of complex mechanisms, neurons derived from hiPSCs might
serve as efficient in vitro models to elucidate such mechanisms,
thus aiding in the development of less neurotoxic therapies.
In our previous study, we investigated the neurotoxicity of
anticancer drugs in hiPSC-derived neural stem/progenitor cells
(hiPSC-NSPCs), which can proliferate well in vitro and differentiate
into neurons and glial cells under appropriate culture conditions.
hiPSC-NSPCs are sensitive to the cytotoxic drugs cisplatin and
etoposide for typically 48 h post treatment and show delayed
sensitivity to the cytostatic drugs mercaptopurine and metho-
trexate.10 However, the cytotoxic effects of these drugs on mature
neurons differentiated from hiPSC-NSPCs have been poorly
investigated.
In this study, we focused on neurons differentiated from hiPSC-
NSPCs and evaluated their susceptibility to six commonly used
anticancer drugs (temozolomide, nimustine, cisplatin, etoposide,
mercaptopurine, and methotrexate). We evaluated the cytotoxic
effects of these drugs on neurons and their parental hiPSC-NSPCs
in vitro with an ATP assay for live cell estimation and by immuno-
cytostaining of marker proteins for each cell characteristic.
2. Materials and methods
2.1. Ethics statement
This study was conducted in accordance with the principles of
the Declaration of Helsinki. The use of hiPSCs was approved by the
ethics committee of Osaka National Hospital (Nos. 110 and 120).
2.2. Cell culture
We used two hiPSC-NSPC lines, 1210B2 and 1201C1, which were
derived from the same healthy donor. Although the parental hiPSC
clones were established using different methods,13e15 both lines
were induced by the dual SMAD inhibition method.15,16 These
hiPSC-NSPCs were propagated as neurospheres in Dulbecco's
modified Eagle's medium (DMEM)/F12 (D8062; SigmaeAldrich, St.
Louis, MO, USA) with 15 mM HEPES (SigmaeAldrich), epidermal
growth factor (EGF, 20 ng/mL; PeproTech, Rocky Hill, NJ, USA),
fibroblast growth factor 2 (FGF2, 20 ng/mL; PeproTech), leukemia
inhibitory factor (LIF, 10 ng/mL; Millipore, Billerica, MA, USA), B27
supplement (B27, 2%; Thermo Fisher Scientific, Waltham, MA, USA),
and heparin (5 mg/mL; Sigma-Aldrich).17 The medium was changed
every 3e5 days. The cells were passaged every 10e12 days using
TrypLE Select CTS (Thermo Fisher Scientific) at 37  C for 10 min for
single-cell dissociation, after which the cells were resuspended in
100% fresh medium at a density of 1  105 cells/mL.16,18
2.3. Differentiation of hiPSC-NSPCs into hiPSC-neurons
Single-cell dissociated hiPSC-NSPCs were seeded onto
polyornithine-coated 96-well plates (161093; Thermo Fisher Sci-
entific) at a density of 7.5  104 cells/cm2 in Neurobasal Plus me-
dium (Thermo Fisher Scientific) with B27 Plus supplement (B27
Plus, 2%; Thermo Fisher Scientific), GlutaMAX (0.5 mM; Thermo
Fisher Scientific), DAPT (10 mM; Abcam, Cambridge, UK), and BDNF
(20 ng/mL; PeproTech). Half of the medium was changed every 2
days during the 5-week culture period.
Please cite this article as: Fukusumi H et al., Evaluation of the susceptibility of neurons and neural stem/progenitor cells derived from human
induced pluripotent stem cells to anticancer drugs, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.08.002
2.4. Treatment with anticancer drugs
The anticancer drugs were prepared as follows: temozolomide
(SigmaeAldrich) and nimustine (SigmaeAldrich) were dissolved in
distilled water to generate 20 mM stock solutions, while cisplatin
(SigmaeAldrich), etoposide (SigmaeAldrich), mercaptopurine
(SigmaeAldrich), and methotrexate (LKT Laboratories, St. Paul, MN,
USA) were dissolved in dimethyl sulfoxide to generate 100 mM
stock solutions. For hiPSC-NSPCs, the neurospheres were dissoci-
ated into single cells and seeded into 96-well plates at a density of
3  104 cells/well. Beginning the next day, the cells were treated
with the anticancer drugs for 48 h. The hiPSC-neurons derived from
hiPSC-NSPCs were treated with the anticancer drugs for 48 h after 5
weeks of differentiation. The dose of each anticancer drug is listed
in Table S1.
2.5. ATP assay
The total ATP content in viable cells was evaluated using
CellTiter-Glo reagent (Promega, Madison, WI, USA) according to the
manufacturer's instructions. Briefly, 50 mL of CellTiter-Glo was
added to wells containing 50 mL of medium. The plates were shaken
for 2 min and incubated for 20 min at 25  C, and luminescence was
determined on a Wallac 1420 ARVOsx (PerkinElmer, Norwalk,
CT, USA).10,17
2.6. Immunocytostaining
After 48 h of treatment with anticancer drugs, cells were fixed in
4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan) at 25  C, washed with
PBS, and then permeabilized and blocked with blocking buffer
containing PBS, 0.1% Triton X-100, and 10% normal goat serum for
1 h at 25  C. Following incubation, the cells were probed with an-
tibodies (Table S2) in blocking buffer overnight at 4  C. Next, the
cells were washed with PBS and then incubated with AlexaFluor
488econjugated goat anti-mouse IgG antibody (Thermo Fisher
Scientific), AlexaFluor 568econjugated goat anti-rabbit IgG anti-
body (Thermo Fisher Scientific) and DAPI (Dojindo, Kumamoto,
Japan) for 1 h at 25  C. Whole-well images were acquired using a
high-content screening system (ArrayScan XTI HCA Reader, Thermo
Fisher Scientific). Semiautomatic cell counting and fluorescence
intensity determination were performed with ilastik19 and ImageJ
(Fiji package),20 respectively.
2.7. Statistical analysis
The predicted doseeresponse curves for the ATP assay and the
50% inhibitory concentration (IC50) values were obtained using the
four-parameter log-logistic (LL2.4) and ED functions, respectively,
of the drc package21 in R software.22 The data are plotted with 95%
confidence intervals (95% CIs).
3. Results
3.1. Terminally differentiated human neurons from hiPSCs
To evaluate the effects of the anticancer drugs on human neu-
rons, we first differentiated two clones of hiPSC-NSPCs to obtain a
neuron-rich differentiated cell population for 5 weeks (Fig. 1A).
Several bIII-tubulinepositive neurites were bundled together, and
both clones developed long processes in our differentiation system
(Fig. 1B, Fig. S1, S2). The two clones of differentiated cells primarily
included neurons positive for the neuronal markers NeuN (1210B2:
81.0 ± 4.61%, 1201C1: 79.0 ± 3.51%; n ¼ 6), MAP2, and ELAVL3/4
 H. Fukusumi et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx 3

A
hiPSC-NSPC hiPSC-Neuron
5 weeks

Drug treatment (48 h) Drug treatment (48 h)

- ATP assay - ATP assay

- Immunocytostaining

B DAPI/βIII-tubulin/GFAP NeuN/MAP2 ELAVL3/4 C

NeuN ELAVL3/4 GFAP

Proportion of marker protein-positive cells (%)

100
1210B2

80

60

40
1201C1

20

1210B2 1201C1 1210B2 1201C1 1210B2 1201C1

100 μm

Fig. 1. Drug-screening strategies. (A) Schematic of the evaluation of the effects of the anticancer drugs on hiPSC-NSPCs and hiPSC-neurons by ATP assay and the immunostaining
method. (B) Representative fluorescent images of hiPSC-neurons after 5 weeks of differentiation. Scale bar, 100 mm. (C) Dot plots and box plots for the proportions of cells positive
for the marker proteins NeuN, ELAVL3/4, and GFAP (n ¼ 6).

(1210B2: 88.7 ± 7.18%, 1201C1: 88.2 ± 2.98%; n ¼ 6) (Fig. 1B, C). A
few cells were GFAP-positive glial cells in both clones (1210B2:
0.01 ± 0.02%, 1201C1: 0.18 ± 0.13%; n ¼ 6) (Fig. 1C, Fig. S1, S2). These
findings indicated that terminally differentiated human neurons
were sufficiently enriched in our 5-week culture system.
3.2. Cytotoxicity of the anticancer drugs to human neurons and
their parental hiPSC-NSPCs
The IC50 values of temozolomide, nimustine, cisplatin, etopo-
side, mercaptopurine, and methotrexate in hiPSC-neurons and
parental hiPSC-NSPCs were determined via an ATP assay (Fig. 2 and
Table 1). Temozolomide, nimustine, cisplatin, and etoposide
showed marked cytotoxicity toward hiPSC-NSPCs rather than
hiPSC-neurons. Although methotrexate did not display cytotoxic
effects toward either hiPSC-NSPCs or hiPSC-neurons, mercaptopu-
rine decreased the ATP content in both hiPSC-NSPCs and hiPSC-
neurons (Fig. 2).
3.3. Cisplatin, but not mercaptopurine, induced neurotoxicity in
human neurons
The proportion of cells positive for the neuronal marker ELAVL3/
4 was largely maintained at a high level after treatment with the
anticancer drugs (Fig. 3A), consistent with the results of the ATP
assay, except in the case of cisplatin. Treatment with a high dose of
cisplatin decreased the fluorescence intensity of ELAVL3/4 in live
cells (Fig. 3B). Moreover, although mercaptopurine decreased the
ATP content, it did not decrease the proportion of ELAVL3/4-
positive cells (Fig. 3A). These findings suggested that while high-
dose cisplatin induced neurotoxicity in hiPSC-neurons, high-dose
mercaptopurine did not induce neurotoxicity.
4. Discussion
The majority of drugs cannot penetrate the bloodebrain barrier
(BBB) into the CNS, and thus, the CNS has been considered to be less

Please cite this article as: Fukusumi H et al., Evaluation of the susceptibility of neurons and neural stem/progenitor cells derived from human
induced pluripotent stem cells to anticancer drugs, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.08.002
4 H. Fukusumi et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx

Temozolomide Cisplatin Mercaptopurine

100 100 100

ATP levels relative to control (%)

50 50 50

0 0 0

Cont 1 10 100 1000 Cont 0.1 1 10 100 Cont 1 10 100 1000

Nimustine Etoposide Methotrexate

100 100 100

50 50 50

NSPC / Neuron
1210B2 /
0 0 0 1201C1 /

Cont 1 10 100 1000 Cont 0.1 1 10 100 Cont 1 10 100 1000

Concentration (μM)
Fig. 2. Doseeresponse curves of the effects of 6 anticancer drugs on human neurons and their parental hiPSC-NSPCs. The x-axis indicates the drug concentration (mM) on a log scale,
and the y-axis indicates the ATP levels in the drug-treated cells relative to the solvent-treated control cells (%). The colors indicate the cell types. A log-logistic model (LL2.4) was
used. The error bars represent the 95% CI.

Table 1 hiPSC-NSPCs usually differentiate into glial cells in addition to

IC50 values (mM) of the anticancer drugs against hiPSC-NSPCs and hiPSC-neurons.
Drug hiPSC-NSPCs
1210B2
Temozolomide 184.5
Nimustine 10.1
Cisplatin 2.17
Etoposide 0.05
Mercaptopurine 59.4
Methotrexate e e
vulnerable to the neurotoxic effects of chemotherapy.23 However,
neurocognitive dysfunction, which is often referred to as “chemo-
brain”, is commonly observed in cancer patients, impairing the
quality of life of cancer survivors.24 Moreover, various new tech-
niques have been developed to bypass the BBB through convection-
enhanced delivery25 and intrathecal infusion of drugs.26 Therefore,
it is important to evaluate the neurotoxic effects of drugs on healthy
human-derived neurons, which can now be realized using hiPSCs.
In this study, we investigated the susceptibility of hiPSC-
neurons and their parental hiPSC-NSPCs to six anticancer drugs,
most of which are commonly used for the treatment of brain tu-
mors, except for mercaptopurine, which is generally used for
treating leukemia but has also been tested for the treatment of
brain stroke in rat models.27 However, our neurotoxic test is not
limited to these anticancer drugs. The doses of anticancer drugs
were chosen in reference to previously reported in vitro toxicity
tests in cell lines, which also included the drug concentrations in
the blood plasma of patients (Table S1).
neurons in response to components of culture media such as fetal
hiPSC-neurons bovine serum (FBS).28 However, we could generate a highly
1201C1 1210B2 1201C1
enriched population of primarily neurons with an FBS-free culture
system combined with Notch signal inhibition.29 Because the
131.0 e e
population size of GFAP-positive cells was extremely small after 5
18.3 e e
0.82 100< 100< weeks of differentiation, it is suspected that the influence of GFAP-
0.04 e 5< positive cell contamination will be very minor. As a result, we were
16.6 77.6 400< able to evaluate the cytotoxic effects of the drugs primarily against
e e
hiPSC-neurons.
The results of the ATP assay indicated that hiPSC-neurons
were clearly more resistant than hiPSC-NSPCs to temozolo-
mide, nimustine, cisplatin, and etoposide, which are intended to
be cytotoxic drugs. However, a high dose of cisplatin decreased
the ATP content to some extent. This is reasonable because
cisplatin is recognized to induce neurotoxicity in patients.30
Interestingly, the proportion of cells positive for the neuronal
marker ELAVL3/4 was low after a high dose of cisplatin was
administered but not after any doses of the other anticancer
drugs were administered. The fluorescence intensity of ELAVL3/4
decreased in live hiPSC-neurons after high-dose cisplatin treat-
ment. Consistent with this finding, thin bIII-tubulinepositive
neurites seemed to be damaged after high-dose cisplatin treat-
ment compared to the other high-dose drug treatments (Fig. S3).
These phenomena might be very early signs of neurotoxicity
induced by cisplatin. Because the effect of cisplatin continues
after its administration to patients is stopped,30 a time course
neurotoxicity test longer than the typical 48 h might be needed
to analyze the effects of this type of anticancer drug using hiPSC-
neurons.

Please cite this article as: Fukusumi H et al., Evaluation of the susceptibility of neurons and neural stem/progenitor cells derived from human
induced pluripotent stem cells to anticancer drugs, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.08.002
 H. Fukusumi et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx 5

A B

Proportion of ELAVL3/4-positive cells (%)

1210B2 1201C1 Cisplatin
1.00
100 100
Dose
0.75

1210B2
Cont
Drug
80 80 0.50 Low
● Temozolomide Middle

Scaled density
● Nimustine 0.25 High
60 60
● Cisplatin 0.00

● Etoposide 1.00
40 40
● Mercaptopurine 0.75

1201C1
● Methotrexate
20 20 0.50

0.25
0 0
0.00
Cont Low Middle High Cont Low Middle High
500 1000 2000 4000 8000
Dose ELAVL3/4 fluorescent intensity

Fig. 3. Proportion of cells positive for the neuronal marker ELAVL3/4. (A) The x-axis indicates the drug dose, and the y-axis indicates the proportion of cells positive for the neuronal
marker ELAVL3/4. The colors indicate the drugs. (B) Histogram of the fluorescence intensity of ELAVL3/4 after cisplatin treatment. The x-axis indicates the fluorescence intensity, and
the y-axis indicates the scaled density. The colors indicate the drug doses.

Mercaptopurine and methotrexate, which are intended to be National Hospital Organization Osaka National Hospital. This
cytostatic drugs, exert similar effects on both hiPSC-neurons and research was supported by AMED under grant numbers
parental hiPSC-NSPCs. Here, mercaptopurine decreased the ATP JP18bm0204001, JP18bk0104077, and JP17mk0104027 and was also
content, which may be considered an indication of supported by JSPS KAKENHI under grant number JP18K08958.
mercaptopurine-mediated inhibition of de novo purine synthesis in
the absence of cell death,31 in contrast to the other cytotoxic drugs.
In fact, the levels of the neuronal marker ELAVL3/4 remained high Appendix A. Supplementary data
in cells after treatment with mercaptopurine and methotrexate.
However, because mercaptopurine and methotrexate are slow- Supplementary data to this article can be found online at
acting and cause cell death after 48 h of treatment in hiPSC- https://doi.org/10.1016/j.jphs.2019.08.002.
NSPCs, as per our previous report, a more prolonged observation
period might be needed for further evaluation.10
Although cell viability (ATP content) and neuronal status References
(ELAVL3/4 expression) were used as the endpoints of the toxicity
assay, cellular toxicity and neurotoxicity appeared at different time 1. Luz AL, Tokar EJ. Pluripotent stem cells in developmental toxicity testing: a
review of methodological advances. Toxicol Sci. 2018;165(1):31e39.
points based on the results of high-dose cisplatin treatment. There-
2. Masjosthusmann S, Barenys M, El-Gamal M, et al. Literature review and
fore, further investigation using recently developed live-cell imaging appraisal on alternative neurotoxicity testing methods. EFSA Supporting Publ.
techniques is needed to study the toxicity of drugs over time. 2018;15(4):1410E.
Moreover, although we selected the typical neuronal marker ELAVL3/ 3. Westerink RH. Do we really want to REACH out to in vitro? Neurotoxicology.
2013;39:169e172.
4 as an indicator of neuronal status, markers for aberrant neuronal 4. Pound P, Ritskes-Hoitinga M. Is it possible to overcome issues of external
function would be more appropriate for studying the phenomenon validity in preclinical animal research? Why most animal models are bound to
of neurotoxicity. Therefore, future studies should analyze markers of fail. J Transl Med. 2018;16(1):304.
5. Hartung T. Thoughts on limitations of animal models. Park Relat Disord.
aberrant neuronal functions, such as synaptic abnormalities, in 2008;14(Suppl 2):S81eS83.
greater detail using the hiPSC-neuron in vitro model. 6. Dingemans MM, Schutte MG, Wiersma DM, et al. Chronic 14-day exposure to
insecticides or methylmercury modulates neuronal activity in primary rat
cortical cultures. Neurotoxicology. 2016;57:194e202.
Conclusion 7. Rogers A, Schmuck G, Scholz G, Williams DC. c-fos mRNA expression in rat
cortical neurons during glutamate-mediated excitotoxicity. Toxicol Sci.
hiPSC-neurons were more resistant to the anticancer drugs than 2004;82(2):562e569.
8. Brennand KJ. Personalized medicine in a dish: the growing possibility of
hiPSC-NSPCs, although a high dose of cisplatin decreased the levels neuropsychiatric disease drug discovery tailored to patient genetic variants
of the neuronal marker protein ELAVL3/4 after a 48-h drug treat- using stem cells. Stem Cell Investig. 2017;4:91.
ment. We believe that the in vitro neurotoxicity evaluation system 9. Tukker AM, Wijnolts FMJ, de Groot A, Westerink RHS. Human iPSC-derived
neuronal models for in vitro neurotoxicity assessment. Neurotoxicology.
employed in this study can be easily applied for evaluation of the 2018;67:215e225.
toxic potential of any drug in hiPSC-neurons. 10. Fukusumi H, Handa Y, Shofuda T, Kanemura Y. Small-scale screening of anti-
cancer drugs acting specifically on neural stem/progenitor cells derived from
human-induced pluripotent stem cells using a time-course cytotoxicity test.
Conflict of interest PeerJ. 2018;6:e4187.
11. Kerckhove N, Collin A, Conde S, Chaleteix C, Pezet D, Balayssac D. Long-term
The authors declare that they have no conflict of interest. effects, pathophysiological mechanisms, and risk factors of chemotherapy-
induced peripheral neuropathies: a comprehensive literature review. Front
Pharmacol. 2017;8:86.
Acknowledgments 12. Meyers CA. How chemotherapy damages the central nervous system. J Biol.
2008;7(4):11.
13. Okita K, Yamakawa T, Matsumura Y, et al. An efficient nonviral method to
The authors thank all other members of the Department of generate integration-free human-induced pluripotent stem cells from cord
Biomedical Research and Innovation, Institute for Clinical Research, blood and peripheral blood cells. Stem Cells. 2013;31(3):458e466.

Please cite this article as: Fukusumi H et al., Evaluation of the susceptibility of neurons and neural stem/progenitor cells derived from human
induced pluripotent stem cells to anticancer drugs, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.08.002
6 H. Fukusumi et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx

14. Nakagawa M, Taniguchi Y, Senda S, et al. A novel efficient feeder-free culture
system for the derivation of human induced pluripotent stem cells. Sci Rep.
2014;4:3594.
15. Sugai K, Fukuzawa R, Shofuda T, et al. Pathological classification of human iPSC-
derived neural stem/progenitor cells towards safety assessment of trans-
plantation therapy for CNS diseases. Mol Brain. 2016;9(1):85.
16. Fukusumi H, Shofuda T, Bamba Y, et al. Establishment of human neural pro-
genitor cells from human induced pluripotent stem cells with diverse tissue
origins. Stem Cell Int. 2016;2016:7235757.
17. Kanemura Y, Mori H, Kobayashi S, et al. Evaluation of in vitro proliferative
activity of human fetal neural stem/progenitor cells using indirect measure-
ments of viable cells based on cellular metabolic activity. J Neurosci Res.
2002;69(6):869e879.
18. Shofuda T, Fukusumi H, Kanematsu D, et al. A method for efficiently generating
neurospheres from human-induced pluripotent stem cells using microsphere
arrays. Neuroreport. 2013;24(2):84e90.
19. Sommer C, Straehle C, Ko €the U, Hamprecht FA. Ilastik: Interactive learning and
segmentation toolkit. In: Paper presented at: 2011 IEEE International Symposium
on Biomedical Imaging: From Nano to Macro; 30 March-2 April 2011. 2011.
20. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for
biological-image analysis. Nat Methods. 2012;9(7):676e682.
21. Ritz C, Baty F, Streibig JC, Gerhard D. Dose-response analysis using R. PLoS One.
2015;10(12):e0146021.
22. Team RC. R: A language and environment for statistical computing. R Foundation
for Statistical Computing; 2017.
23. Seigers R, Schagen SB, Van Tellingen O, Dietrich J. Chemotherapy-related
cognitive dysfunction: current animal studies and future directions. Brain Im-
aging Behav. 2013;7(4):453e459.
24. Moore HC. An overview of chemotherapy-related cognitive dysfunction, or
'chemobrain. Oncology (Williston Park). 2014;28(9):797e804.
25. Mehta AM, Sonabend AM, Bruce JN. Convection-enhanced delivery. Neuro-
therapeutics. 2017;14(2):358e371.
26. Blakeley J. Drug delivery to brain tumors. Curr Neurol Neurosci Rep. 2008;8(3):
235e241.
27. Chang CZ, Kwan AL, Howng SL. 6-Mercaptopurine exerts an immunomodula-
tory and neuroprotective effect on permanent focal cerebral occlusion in rats.
Acta Neurochir (Wien). 2010;152(8):1383e1390. discussion 1390.
28. Tcw J, Wang M, Pimenova AA, et al. An efficient platform for astrocyte differ-
entiation from human induced pluripotent stem cells. Stem Cell Rep. 2017;9(2):
600e614.
29. Borghese L, Dolezalova D, Opitz T, et al. Inhibition of notch signaling in human
embryonic stem cell-derived neural stem cells delays G1/S phase transition
and accelerates neuronal differentiation in vitro and in vivo. Stem Cells.
2010;28(5):955e964.
30. Kanat O, Ertas H, Caner B. Platinum-induced neurotoxicity: a review of possible
mechanisms. World J Clin Oncol. 2017;8(4):329e335.
31. Dervieux T, Brenner TL, Hon YY, et al. De novo purine synthesis inhibition and
antileukemic effects of mercaptopurine alone or in combination with metho-
trexate in vivo. Blood. 2002;100(4):1240e1247.

Please cite this article as: Fukusumi H et al., Evaluation of the susceptibility of neurons and neural stem/progenitor cells derived from human
induced pluripotent stem cells to anticancer drugs, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.08.002