Chemical decontamination of iPS cell-derived neural cell mixtures

Article abstract of DOI:10.1039/c7cc08686e

This report describes the design and evaluation of phosphorylated 7-ethyl-10-hydroxycamptothecin (SN38-P), which selectively eliminates tumor-forming proliferative stem cells, including human induced pluripotent stem cells (hiPSCs) and neural stem cells, from iPSC-derived neural cell mixtures. Results of the present study demonstrate that simple phosphorylation of an anticancer drug can provide a safe, cost-effective, and chemically-defined tool for decontaminating hiPSC-derived neuron.

Full text of DOI:10.1039/c7cc08686e

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Chemical Decontamination of iPS Cell‐Derived Neural Cell
Mixtures
Di Mao,a Watson Chung Xie Khim,a Tomoko Andoh‐Noda,b Ying Qin,a Shin‐ichi Sato,a Yasushi
Received 00th January 20xx,
Accepted 00th January 20xx
Takemoto,a Wado Akamatsu,b Hideyuki Okanob and Motonari Uesugia*
DOI: 10.1039/x0xx00000x
www.rsc.org/
This report describes the design and evaluation of phosphorylated
7‐Ethyl‐10‐hydroxycamptothecin (SN38‐P), which selectively
eliminates tumor‐forming proliferative stem cells, including human
induced pluripotent stem cells (hiPSCs) and neural stem cells, from
iPSC‐derived neural cell mixtures. Results of the present study
demonstrate that simple phosphorylation of an anticancer drug can
provide a safe, cost‐effective, and chemically‐defined tool for
decontaminating hiPSC‐derived neuron.
Human induced pluripotent stem cells (hiPSCs) serve as a highly
valuable resource for both basic research and regeneration therapy.
However, there are a number of limitations for their clinical
application, including the tumorigenic risk of undifferentiated cells
2
during transplantation.1,
Complete differentiation or selective
elimination of the undifferentiated cells is required to ensure the safety
of stem cell therapy. A number of biological strategies for selective
elimination have been reported: antibody-mediated cell sorting and
precipitation,3, 4 specific cytotoxic antibodies,5 cell culture conditions
that limit the growth of iPSCs,6, 7 a lectin-toxin fusion protein,8 TRPV-
1 activation,9 and a microRNA switch,10 and a synthetic peptide that
selectively assembles on the surface of hiPSCs through alkaline
phosphatase-mediated activation.11
Fig. 1 Design of SN38‐P. ALP: alkaline phosphatase; Topo I: topoisomerase I.
hiPSC: Human induced pluripotent stem cell.
Small molecule-based approaches are attractive alternatives to
those strategies, due to easy handling and relatively low cost.
Chemical elimination of hiPSCs has been achieved using a stearoyl-
coA desaturase (SCD1) inhibitor,12 chemical inhibitors of survivin,13
DNA topoisomerase II inhibitors,14, 15 a cytotoxic natural product that
is a substrate for selective ABC transporters,16 and an hiPSC-selective
fluorescent probe that generates reactive oxygen species upon light
irradiation.17 Unfortunately, none of these approaches have reached
the level of clinical application. We previously reported selective
elimination of hiPSCs by a hybrid molecule of an iPSC-selective
fluorescent probe and 7-Ethyl-10-hydroxycamptothecin (SN38), a
clinically used topoisomerase I inhibitor. The mechanistic study
indicated that the high selectivity stemmed from the synergistic
effects of ABC-transporter-mediated efflux and the mode of action of
SN38.18 Although the SN38 hybrid molecule exhibited excellent
selectivity for hiPSCs, it cannot be used for selective removal of
iPSCs from neural cell mixtures, due to the lack of expression of
efflux ABC transporters in neural cells.19
An increasing number of studies indicate that hiPSC-derived
neurons or neural precursors have immediate promise for treatment of
neural diseases and for modeling neurological diseases. 20-23 However,
current differentiation protocols suffer from the residue of a few
differentiation-resistant neural stem cells or hiPSCs. Their rapid
proliferation poses a problem of tumor formation following in vivo
engraftment, or of subsequently dominating the in vitro cell
population.24 Trials for addressing the problem have been proposed:
cell sorting using antibodies for specific markers,25-27 modified cell
a. Institute for Integrated Cell‐Material Sciences (WPI‐iCeMS) and Institute for
Chemical Research, Kyoto University, Uji, Kyoto 611‐0011, Japan. Email:
uesugi@scl.kyoto‐u.ac.jp
b. Department of Physiology, School of Medicine, Keio University, Shinjuku‐ku, Tokyo
160‐8582, Japan.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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differentiation protocol,28, 29 and a cell cycle promoter-driven suicide
gene approach.30 However, no small molecule-based approach has
been developed. There is a pressing demand for a reliable, cost-
effective, and safe chemical tool that eliminates hiPSCs or any highly
proliferative stem cells from neural cell mixtures. Here, we report on
the design and effectiveness of phosphorylated SN38 (SN38-P),
which selectively eliminates proliferative stem cells, including human
iPS and neural stem cells, from neural cell mixtures. The selectivity
takes advantage of the high expression level of alkaline phosphatase
on the membrane of human iPSCs and neural stem cells.31 We
hypothesized that ALP converts non-cell-permeable SN38-P to cell-
permeable SN38 exclusively on the cell membrane of the stem cells,
leading to selective cell death (Fig. 1).
To examine the expression levels of ALP on the membrane of
different cell types, we used colormetric alkaline phosphatase
staining with hiPSCs (201B7), six types of commercially
Fig. 3 Conversion and membrane permeability of SN38‐P in living cells. The
amounts of SN38 and SN38‐P in cell lysates were analyzed with an HPLC at
254 nm and the molecular weights were confirmed with mass spectrometry.
Retention times of SN38‐P and SN38 in PBS are shown in (A) and (B),
respectively. hiPSCs converted SN38‐P to SN38, which was detected in the
cell lysates (C), whereas human primary astrocytes failed to do so (D).
Table 1. IC50 values of SN38 and SN38‐P in human stem cells and primary
somatic cells.
Cell type
iPSC‐201B7
SN38 (µM)
0.005±0.001
0.007±0.001
0.003±0.001
0.106±0.033
0.387±0.090
0.003±0.001
0.134±0.037
0.077±0.045
SN38‐P (µM)
0.007±0.001
0.005±0.001
0.048±0.015
1.794±0.166
5.092±0.946
0.067±0.019
0.940±0.154
0.234±0.069
Fold
1.4
Fig. 2 Colorimetric alkaline phosphatase staining (blue) of living human iPSCs,
human primary somatic cells, and cells derived from human pluripotent stem
cells. hiPSCs and hESC‐derived neural stem cells exhibited highest expression
levels of ALP. Prostate epithelial cells had a lower expression level of ALP.
Other types of cells had no significant expression level of ALP. Alkaline
Phosphatase: ALP; ESC: embryonic stem cell; NSC: neural stem cell. Scale bar:
100 µm.
iPSC‐253G1
0.7
Astrocyte
16
Adrenal microvascular
Brain microvascular
Bronchial epithelial
Hepatocyte
16.9
13.2
22.3
7.0
Prostate epithelial
hESC‐derived neural
stem cell
3.0
0.007±0.001
0.365±0.318
0.015±0.002
0.008±0.003
>10
1.1
>27.4
12.1
available human primary somatic cells (astrocytes, adrenal
microvascular cells, brain microvascular cells, bronchial
epithelial cells, hepatocytes, and prostate epithelial cells), and
three types of human pluripotent stem cell-derived cells (hESC-
derived neural stem cells, hiPSC- derived neurons, and hiPSC-
derived cardiomyocytes). hiPSCs and hESC-derived neural stem
cells had the highest expression levels of ALP. The other cell
types had no significant expression level of ALP, with the
exception of prostate epithelial cells which had a lower, but
detectable, expression level (Fig. 2, Fig. S1).
The synthetic route of SN38-P is shown in scheme S1. We
initially examined whether SN38-P is, in fact, a substrate for
ALP and is significantly less cell-permeable than SN38. When
SN38-P was incubated with purified human placental alkaline
phosphatase, it was almost completely converted to SN38 within
70 min. In contrast, incubation with bovine serum albumin
(BSA) failed to convert SN38-P (Fig. S2). The enzymatic
hiPSC‐derived neuron
hiPSC‐derived
0.181±0.004
cardiomyocyte
Cells were treated with SN38 or SN38‐P for 72 h. Data are mean ± SD, n=3.
Fold increase was calculated as IC50 value of SN38‐P / IC50 value of SN38.
Significantly increased resistances are shown in red.
conversion was inhibited by EDTA, a well-known alkaline
phosphatase inhibitor (Fig. S3). These results collectively
demonstrate that SN38-P is a substrate for the alkaline
phosphatase.
We next compared the cell membrane permeability of SN38
and SN38-P, using a parallel artificial membrane permeability
(PAMPA) assay (Table S1). SN38 showed weak cell
permeability, but SN38-P was 16.9 times less permeable than
2 | J. Name., 2012, 00, 1‐3
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Fig. 4 Selective elimination of hiPSCs and hiPSC‐derived Ki‐67‐positive cells by SN38‐P. (A) Effects of SN38‐P (0.01 μM or 0.1 μM), or a DMSO control (0.1%), on
hiPS colonies and partially differentiated hiPSCs. The cells were treated with SN38‐P for 72 h. Colonies of hiPSCs were visualized by colorimetric alkaline
phosphatase staining (blue). Scale bar: 100 µm. (B, C) Selective elimination of Ki‐67‐positive cells from hiPSC‐derived neural cell mixtures. The hiPSC‐derived
neural cell mixtures were incubated with DMSO (0.1%) or SN38‐P (0.01 µM) for 72 h. The residual cells were fixed and co‐immunostained with an anti‐βIII‐
tubulin antibody (green) for neurons and an anti‐Ki‐67 antibody (red) for proliferating cells, and then analyzed by fluorescent microscopy (B) or flow cytometry
(C). Hoechst 33258 (blue, 10 µg/mL) was used as a nuclear indicator. The proliferating cells (Pink) were indicated with arrowheads. Scale bar: 200 µm.
positive cells tends to lower the selectivity, possibly due to bystander
effects on ALP-negative cells, posing a precaution for future
application.
SN38, indicating that the phosphate group of SN38-P reduced its
membrane permeability.
The poor membrane permeability of SN38-P was further confirmed
by cell-based experiments. When hiPSCs were incubated with SN38-
P for 1 h, SN38, but not SN38-P, was detected in the cell lysate (Fig.
3C). In contrast, when SN38-P was incubated with astrocytes in which
ALP was not detectable (Fig. 3D), a high level of SN38-P was
observed in the extracellular buffer, but not in the cell lysate (Fig. 3A).
SN38 is a topoisomerase inhibitor that induces DNA damage. DNA
damage induced by SN38 and SN38-P was subsequently compared in
ALP-negative astrocytes by measuring γH2AX, a marker for DNA
damage.32 After a 3 h incubation with SN38 or SN38-P, the cells were
fixed and immunostained with an anti-γH2AX antibody. As expected,
SN38 induced DNA damage at lower concentrations than SN38-P
(Fig. S4). These results indicate that the negatively charged phosphate
group reduces the penetration of SN38-P through the cell membrane
of ALP-negative cells.
To evaluate the effect of phosphorylation on selectivity, we
measured IC50 values of SN38 and SN38-P with two human iPSC
lines, six types of primary human somatic cells, and three cell types
derived from human pluripotent stem cells. Fetal bovine serum (FBS)
is known to contain alkaline phosphatase (Fig. S5). Thus, serum-free
primate ES cell medium was selected for the cell-based assays. SN38-
P exhibited highly potent cytotoxcity for ALP-positive cells,
including hiPSCs (201B7 and 253G1) and hESC-derived neural stem
cells (Table 1, Fig. S6). For ALP-negative cells, SN38-P was 3.0 to
>27.4-fold less cytotoxic than SN38. The fold differences and ALP
expression levels are well correlated (Fig. 2). The ALP-dependent
selectivity was further confirmed using HEK293 cells that lack ALP
expression. HEK293 cells were transfected with an ALP-EGFP gene
to yield a mixture of ALP-positive and ALP-negative cells (Fig. S7).
The cell mixture was then treated with SN38 or SN38-P, followed by
FACS analysis at the FITC channel. As expected, SN38-P selectively
reduced the number of ALP-EGFP-positive cells (Fig. S8). These
results support the hypothesis that the selectivity of SN38-P depends
on the ALP expression. However, increasing number of ALP-EGFP
To investigate the ability of SN38-P to eliminate hiPSCs from cell
mixtures, we first incubated SN38-P (0.01 and 0.1 µM) with hiPSCs
or partially differentiated hiPSCs for 3 d. Treatment with SN38-P
removed hiPSCs (stained blue in Fig. 4A) in a dose-dependent manner,
but had little effect on ALP-negative SNL feeder cells (Fig. 4A, top)
or differentiated cells (Fig. 4A, bottom). Selective elimination was
further confirmed by flow cytometric analysis with an antibody
against SSEA4, a cell-surface marker for human pulripotent stem cells.
The partially differentiated hiPSC mixtures treated with DMSO alone
had 40.7% SSEA4-positive cells. Treatment with SN38-P decreased
the number of SSEA4-positive cells to 8.1% (Fig. S9). When the
residual SSEA4-positive cells were incubated in growth medium for
another 5 d, no detectable ALP-positive cell colonies (blue in Fig.
S10) were formed, suggesting that delayed cytotoxicity of SN38
completely eliminated ALP-positive undifferentiated cells in the cell
mixtures (Fig. S11).33, 34
SN38-P showed potent cytotoxicity for the hiPSCs and hESC-
derived neural stem cells, but >1000-times weaker cytotoxicity for
hiPSC-derived neurons (Table 1). This encouraged us to test SN38-P
for elimination of proliferating hiPSCs and neural stem cells in hiPSC-
derived neural mixtures. A mixture of hiPSC-derived neural cells was
treated with SN38-P (0.01 µM) for 72 h. The cells were then fixed and
immunostained with an antibody against βIII-tubulin, a marker for
neurons, and with an antibody against Ki-67, a marker for
proliferative cells. As expected, treatment with SN38-P significantly
reduced the number of Ki-67-positive proliferative cells, but had little
effect on the βIII-tubulin-positive hiPSC-derived neurons (Fig. 4B).
Flow cytometric analysis was carried out for further quantitative
evaluation. Following treatment with DMSO alone, more than 80% of
the hESC-derived neural stem cells and 44.8% of the hiPSC-derived
neural cells were Ki-67-positive. Treatment of the hiPSC-derived
neural cell mixtures with SN38-P reduced the Ki-67-positive cells to
9.4% (Fig. 4C). These results collectively demonstrate that SN38-P
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decontaminates hiPSC-derived neurons by selectively eliminating
proliferating stem cells. However, the potential risk of the leftover Ki-
67-positive cells can not be ignored although those cells express
relatively low levels of Ki-67.
For clinical application, it is also important to ensure that the
decontaminated neurons are alive and functional after SN38-P
treatment. The hiPSC-derived neurons displayed healthy morphology
after 3 d treatment with SN38-P (Fig. 4B). Their functional activity
was assessed by measuring glutamate-induced calcium influx.35
Comparable levels of Ca2+ influx were observed after addition of
glutamate (100 µM) in DMSO-treated and SN38-P-treated hiPSC-
derived neurons (Fig. S12). Thus, 3 d treatment with 0.01 µM of
SN38-P is within an acceptable range for maintaining the function of
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In conclusion, simple phosphorylation of the clinically-used
anticancer drug, SN38, provides a cost-effective, chemically-defined
tool for decontaminating hiPSC-derived neurons. Neural precursor
cells have been reported to express higher levels of ALP than
terminally differentiated neural cells. SN38-P may also be useful for
purification of other types of the terminally differentiated neural and
glail cells such as mature oligodendrocytes.36 Clinical application of
SN38-P requires further safety evaluations due to potential
mutagenesis risks of topoisomerase inhibitors. Nevertheless, similar
phosphorylation strategies might be applied to other chemical iPSC
eliminators to improve their selectivity and safety profiles, with the
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Production and Processing Systems for Commercialization of
Z. Zhou, S. Sato, T. Kobayashi, M. Ohtaka, K. Nishimura, H. Kurosawa,
Regenerative Medicine," from the Japan Agency for Medical
T. Yoshikawa, T. Takahashi, M. Nakanishi, M. Ohyama, N. Hattori, W.
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ChemComm
Neural Cell Mixture
Neuron
SN38-P
P
P
Stem Cell
Neuron
ALP
Survival
Death
simple phosphorylation of an anticancer drug provides a safe, cost-
effective, and chemically-defined tool for decontaminating hiPSC-derived
neurons.