Content uploaded by Norihiro Suzuki
Author content
All content in this area was uploaded by Norihiro Suzuki on Feb 25, 2016
Content may be subject to copyright.
Modeling familial Alzheimer’s disease with induced
pluripotent stem cells
Takuya Yagi1, Daisuke Ito1,∗, Yohei Okada2,3, Wado Akamatsu2, Yoshihiro Nihei1,
Takahito Yoshizaki1, Shinya Yamanaka4, Hideyuki Okano2and Norihiro Suzuki1
1
Department of Neurology,
2
Departments of Physiology and
3
Kanrinmaru Project, School of Medicine, Keio University,
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan and
4
Center for iPS Cell Research and Application, Kyoto
University, Kyoto 606-8507, Japan
Received June 3, 2011; Revised August 1, 2011; Accepted August 29, 2011
Alzheimer’s disease (AD) is the most common form of age-related dementia, characterized by progressive
memory loss and cognitive disturbance. Mutations of presenilin 1 (PS1) and presenilin 2 (PS2) are causative
factors for autosomal-dominant early-onset familial AD (FAD). Induced pluripotent stem cell (iPSC) technol-
ogy can be used to model human disorders and provide novel opportunities to study cellular mechanisms
and establish therapeutic strategies against various diseases, including neurodegenerative diseases. Here
we generate iPSCs from fibroblasts of FAD patients with mutations in PS1 (A246E) and PS2 (N141I), and char-
acterize the differentiation of these cells into neurons. We find that FAD– iPSC-derived differentiated neurons
have increased amyloid b42 secretion, recapitulating the molecular pathogenesis of mutant presenilins.
Furthermore, secretion of amyloid b42 from these neurons sharply responds to g-secretase inhibitors and
modulators, indicating the potential for identification and validation of candidate drugs. Our findings demon-
strate that the FAD-iPSC-derived neuron is a valid model of AD and provides an innovative strategy for the
study of age-related neurodegenerative diseases.
INTRODUCTION
Alzheimer’s disease (AD) is one of the most common neu-
rodegenerative disorders of the elderly, characterized by pro-
gressive memory disorientation and cognitive disturbance.
The pathological profile of AD is neuronal loss in the cere-
bral cortex accompanied by massive accumulation of two
types of amyloid fibril seeding senile plaques and hyperpho-
sphorylated tau forming paired helical filaments. The
amyloid fibril is mainly composed of b-amyloid (Ab) pep-
tides, the 40 and 42 amino acid forms (Ab40 and Ab42),
that are derived by proteolytic cleavages from the amyloid
precursor protein (APP) by b- and g-secretase activity
(1,2). According to the amyloid cascade hypothesis, a pre-
vailing theory of AD pathology, accumulation of Ab,
mainly Ab42, in the brain is the initiator of AD pathogen-
esis, subsequently leading to the formation of neurofibrillary
tangles containing hyperphosphorylated tau protein, and con-
sequently neuronal loss (3–5).
Presenilin 1 (PS1) and presenilin 2 (PS2) genes encoding
the major component of g-secretase have been identified as
the causative genes for autosomal-dominant familial Alzhei-
mer’s disease (FAD). Mutations in the PS1 gene, located on
chromosome 14, occur most frequently in FAD (6,7).
Ala246Glu (A246E) in PS1 is a well-characterized FAD mu-
tation that shows typical phenotypes of AD with complete
penetrance. Mutations in the PS2 gene on chromosome 1 are
a relatively rare cause of FAD and are variably penetrant.
Asn-141 substitutions by Ile (N141I) in the PS2 gene was
the first identified causative mutation of PS2 in affected
patients from the now famous Volga German families (8,9).
Mutations in the PS1,PS2 and the APP gene account for
most of the familial early onset cases of AD either by enhan-
cing the production of pathological Abor especially Ab42,
which has a greater tendency to form fibrillary amyloid depos-
its. These findings support b-amyloid as the common initiating
factor in AD in the amyloid cascade hypothesis (10,11). Both
A246E in PS1 and N141I in PS2 are reported to induce
∗
To whom correspondence should be addressed at: Department of Neurology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan. Tel: +81 353633788; Fax: +81 333531272; Email: d-ito@jk9.so-net.ne.jp
#The Author 2011. Published by Oxford University Press. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
Human Molecular Genetics, 2011, Vol. 20, No. 23 4530–4539
doi:10.1093/hmg/ddr394
Advance Access published on September 7, 2011
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
elevation of Ab42 levels in human plasma, patient-derived
fibroblasts, forced-expressed cells and, in mice, showing
strong toxicity (10–13).
Generation of human iPSCs provides a new method for elu-
cidating the molecular basis of human disease (14,15). An in-
creasing number of studies have employed disease-specific
human iPSCs in neurological diseases, and a few have demon-
strated disease-specific phenotypes to model the neurological
phenotype (16–24). Here, we report the generation of iPSC
from fibroblasts of FAD with the PS1 mutation A246E and
the PS2 mutation N141I, and differentiation of these cells
into neurons. We demonstrate that patient-derived differen-
tiated neurons increase Ab42 secretion, recapitulating the
pathological mechanism of FAD with PS1 and PS2 mutations.
Our findings demonstrate that the FAD – iPSC-derived neuron
is a valid model for studying AD, and provides important clues
for the identification and validation of candidate drugs.
RESULTS
Generation of iPSC with presenilin mutations
We established two clones of iPSCs with the PS1 mutation,
A246E (PS1-2 iPSC and PS1-4 iPSC) and with the PS2 muta-
tion, N141I (PS2-1 iPSC and PS2-2 iPSC) by retroviral trans-
duction of primary human fibroblasts with the five factors
OCT4, SOX2, KLF4, LIN28 and NANOG. Fibroblasts were
obtained from the Coriell Cell Repository (AG07768 and
AG09908). The 201B7 iPSC line (14) and the sporadic Parkin-
son disease (PD)-derived iPSC lines (PD01-25 and 26) were
reprogrammed by an original method (14) with four transcrip-
tion factors (OCT4, SOX2, KLF4 and cMYC) and were used
as the controls in this study. Genotyping of the established
iPSC lines was confirmed by PCR– RFLP and sequencing
(Fig. 1A and B). All PS1 and PS2 iPSC clones demonstrated
typical characteristics of pluripotent stem cells: similar morph-
ology to ESCs, expression of pluripotent markers including
Tra-1-60, Tra-1-81, SSEA3 and SSEA4 (Fig. 1C), silencing
of retroviral transgenes and reactivation of genes indicative
of pluripotency (Fig. 1D). The differentiation ability of PS1
and PS2 iPSC was also confirmed in vivo by teratoma forma-
tion (Fig. 2), and in vitro by the formation of three germ layers
via embryoid bodies (Supplementary Material, Fig. S1). To
validate our reprogramming technique, we performed compre-
hensive analysis of two PS2 iPSCs. Heat map analysis showed
that global gene expression profiles, including the critical
genes for pluripotency, were similar between the iPSC lines
established with four transcription factors (201B7 and
PD01-25) and the PS2 iPSC clones established with five tran-
scription factors (Supplementary Material, Fig. S2). In add-
ition, there were no significant differences in the expression
of AD-related molecules between PS2 iPSCs and control
iPSCs (Supplementary Material, Fig. S3). Array comparative
genomic hybridization (aCGH) analysis on PS2-1, PS2-2
iPSC and AG09908 fibroblasts showed that the total
number of copy number aberrations were 52, 61 and 102 out
of 17 000 locations, respectively (Supplementary Material,
Table S1), and no aberrations were detected in APP,PS1
and PS2 genes.
Differentiation of PS1 iPSC and PS2 iPSC into neurons
Differentiation of FAD patient-specific iPSCs towards neurons
enables modeling the disease pathogenesis in vitro. To estab-
lish whether the presenilin mutations may affect neuronal dif-
ferentiation, both PS1 and PS2 iPSC lines, as well as control
iPSC lines, were induced to differentiate into neural cells
(25,26), and cultured on Matrigel-coated dishes for 2 weeks
to induce terminal differentiation (Fig. 3). We confirmed neur-
onal differentiation by the expression of neuronal markers,
bIII-tubulin, and MAP-2 (Fig. 3A and B). As shown in
Figure 3C, no obvious differences in the ability to generate
neurons (80% bIII-tubulin-positive cells) were observed
among control, PS1 and PS2 iPSCs. This indicated that PS1
and PS2 iPSCs can generate neurons with almost the same ef-
ficiency as the control iPSCs, suggesting these presenilin
mutations may have no significant effect on neuronal
differentiation.
Production of Absecreted from iPSCs-derived neurons
To analyze the functional aspects of FAD, we investigated Ab
secretion from iPSC or iPSC-derived neurons. The Absecre-
tion in the conditioned medium from control iPSC, PS1 iPSC
and PS2 iPSC was very low; Ab42 secretion especially was
below the detection sensitivity. We therefore could not
compare the ratio of Ab42 to Ab40 among iPSC lines.
However, the Absecretion in the conditioned medium from
the iPSCs-derived neurons was increased and measurable, in-
dicating that Absecretion could undergo significant fluctu-
ation during differentiation. Although the levels of Ab42
and Ab40 in the medium showed some clonal variation
(Fig. 4A), possibly depending on the rate of cell growth and
passage number, the ratio of Ab42 to Ab40 was significantly
elevated in the PS1 and PS2 iPSCs-derived neurons, compared
with the controls (Fig. 4B). Thus, PS1 and PS2 iPSCs show
that living neurons derived from patients with the presenilin
mutations ending at residue 42 that are linked to FAD
secrete more Ab. This result is compatible with previous evi-
dences based on patients’ plasma, fibroblasts and
forced-expressed cells (10–13).
To explore recapitulation of key pathological events in AD,
we investigated whether FAD-iPSC-derived differentiated
neurons exhibit abnormal accumulation of tau and performed
an immunoblot analysis of lysates of FAD-iPSC-derived
neurons with anti-tau antibody. However, as shown in Supple-
mentary Material, Figure S4, no abnormal tau protein accumu-
lation or tangle formation was detected in the FAD-derived
neurons, indicating that recapitulation of tauopathy is difficult
to observe during the short culture period (2 weeks) in the
present protocol.
Pharmacological response to g-secretase inhibitors in PS1
iPSC- and PS2 iPSC-derived neurons
To evaluate the capacity of pharmacological drug screening in
iPSC technology, we assessed whether inhibitors could affect
the secretion of Abin PS1 and PS2 iPSCs-derived neurons.
We first examined the secretion of Abfrom PS1-4 and PS2-2
iPSCs-derived neurons in the presence of Compound E, a
Human Molecular Genetics, 2011, Vol. 20, No. 23 4531
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
potent g-secretase inhibitor (27) (Fig. 5A and B). With the add-
ition of 10 and 100 nMCompound E, the production of both
Ab42 and Ab40 was suppressed in a dose-dependent manner,
when compared with untreated in both of PS1-4 and PS2-2
iPSC-derived neurons. Next, we assessed the ability of Com-
pound W, a selective Ab42-lowering agent, to modulate
g-secretase-mediated APP cleavage (28) (Fig. 5A and B). As
expected, the addition of Compound W caused a drastic de-
crease in the ratio of Ab42 to Ab40 in both neurons.
We also determined the effect of these compounds on the
proteolytic processing that causes a release of an intracellular
domain of Notch, another g-secretase substrate. Western blot-
ting using the anti-S3 cleaved Notch1-specific antibody
demonstrated that productions of Notch intracellular domain
Figure 1. Generation of PS1 and PS2 iPSC from patient fibroblasts. (A) Genotypic analysis of PS1 iPSC by PCR –RFLP and sequencing. A246E genotyping by
PCR– RFLP was performed with restriction enzyme DdeI. The A246E mutation results in fragments of 176 and 58 bp, whereas the control fragment has 234 bp.
(B) Genotypic analysis of PS2 iPSC by PCR– RFLP and sequencing. N141I genotyping by PCR –RFLP was performed with restriction enzyme MboI. The N141I
mutation results in fragments of 102, 58 and 43 bp, whereas the control has fragment lengths of 102 and 58 bp. (C) Both PS1 and PS2 iPSC lines exhibit markers
of pluripotency. All iPSCs express pluripotency markers including Tra-1-60, Tra-1-81, SSEA3 and SSEA4. Nuclei were stained with
4,6-diamidino-2-phenylindole (DAPI). Bar ¼200 mm. (D) RT–PCR analysis of the transgenes OCT3/4, SOX2, KLF4 and the endogenous hESC marker
genes. Patient fibroblasts 6 days after the transduction with the retroviruses are positive for the transgenes.
4532 Human Molecular Genetics, 2011, Vol. 20, No. 23
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
(NICD) from both PS1-4 and PS2-2 iPSCs-derived neurons
exposed to Compound E was inhibited in a dose-dependent
manner. Although high dose (100 mM) of Compound W
seemed to decrease NICD production in PS1-4, both neurons
exposed to Compound W showed that NICD was mostly
maintained (Fig. 5C). Taken together, these data indicate
that both PS1 and PS2 iPSC-derived neurons respond to
drug treatment in an expected manner and might be useful
for drug screening in AD.
DISCUSSION
To the best of our knowledge, this study is the first to demon-
strate a model of FAD using the iPSC technology. Using
human neurons carrying a PS1 mutation and a PS2 mutation,
we observed an elevation of the ratio of Ab42 to Ab40, a hall-
mark feature of FAD with presenilin mutations, in neurons
derived from two clones of PS1 and PS2 iPSCs, when com-
pared with non-AD controls (201B7, PD01-25 and 26)
(Fig. 4). Although an increase in Ab42 levels as a result of
the A246E mutation in PS1 and N141I mutation in PS2 has
been reported in patient-derived fibroblasts (11), the present
study provided the first evidence of increased Ab42 secretion
by living human neurons derived from AD patients, thereby
directly supporting the amyloid cascade hypothesis. To test
the possibility of using the iPSC technology for drug screen-
ing, we checked the pharmacological responses to a known
g-secretase inhibitor and modulator (Fig. 5A and B). Results
showed that Absecretion by adding agents against g-secretase
were inhibited or modulated as expected. Moreover, the Notch
signaling pathway reacted with proteolytic cleavage in the
presence of g-secretase inhibitors (Fig. 5C). Recent studies
have revealed that gsecretase activity is influenced in a
complex manner by several cellular factors, including rafts,
trafficking, expression levels of CD147, numb and gamma-
secretase activating protein (1,2,29 –31). We therefore
propose that living human neurons from patients, i.e.
FAD-iPSC-derived neurons, are very suitable material for
drug development and validation of new drugs.
Previous studies on patient-specific iPSC models have
mostly been limited to genetic congenital disorders
(19,20,22,24,32–35). Congenital disorders may be suitable
for modeling disease-specific phenotypes in the iPSC technol-
ogy, because differentiated cells generated from iPSC could
represent the developmental stages of disease (36). However,
modeling familial PD using iPSC that carry the p.G2019S mu-
tation in the Leucine-Rich Repeat Kinase-2 (LRRK2) gene has
been reported recently (23). DA neurons derived from
G2019S-iPSCs were vulnerable to exposure to stress agents,
such as hydrogen peroxide, MG-132 and 6-hydroxydopamine.
Now we also demonstrate the possibility of modeling the most
common aging-related neurodegenerative disorder, AD, by re-
capitulating the key pathological mechanism (Fig. 4). Many
insights into the molecular pathogenesis in neurodegenerative
diseases have come from investigating post-mortem brain
tissues or transgenic animals, due to the difficulty of invasive
access to the living human central nervous system. With
disease modeling using the iPSC technology, these new
tools will make it possible to analyze living disease-specific
Figure 2. Teratomas derived from SCID mice injected with PS1 and PS2 iPSCs. Gross morphology, hematoxylin and eosin stained representative teratoma
generated from PS 1 (PS1-2 iPSC and PS1-4 iPSC) and PS2-1 iPSC (PS2-1 iPSC and PS2-2 iPSC). Both iPSC shows tissues representing all three embryonic
germ layers, including pigmented epithelium (ectoderm), cartilage (mesoderm) and glandular structure (endoderm). Bar ¼50 mm.
Human Molecular Genetics, 2011, Vol. 20, No. 23 4533
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
neurons in vitro. Moreover, we could graft disease-specific
neurons derived from iPSCs into the brain of immunodeficient
animals and we could investigate the time-dependent patho-
logical changes in vivo in future studies.
FAD iPSCs could be a potential strategy for drug discovery
against AD as described here; however, several limitations
must be addressed in future studies. First, a high-yield of dif-
ferentiated neurons from human iPSCs requires multistep
Figure 3. Differentiation of PS1 and PS2 iPSC into neurons. (Aand B) Neural differentiation of control iPSC (201B7, PD01-25 and PD01-26), PS1 iPSC (PS1-2
iPSC and PS1-4 iPSC) and PS2 iPSC (PS2-1 iPSC and PS2-2 iPSC). Representative pictures of immunocytochemistry for bIII-tubulin (A) and MAP-2 (B) after
neural differentiation. Bar ¼40 mm (A) and 20 mm (B). (C) Graphs indicate the percentage of bIII-tubulin-positive cells relative to cells with DAPI-staining
nuclei. Error bars indicate the SD (n¼3).
4534 Human Molecular Genetics, 2011, Vol. 20, No. 23
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
procedures and prolonged culture. Furthermore, heterogeneity
of differentiated neuronal cell types depending on clonal vari-
ability and culture conditions is inevitable using current differ-
entiation methods. Clonal variation in their characters,
including differentiation efficiency and tumor formation, has
been a problem that needed to be solved thus far (26,37,38).
Development of reliable protocols for more rapid neuronal dif-
ferentiation with minimal clonal variation will be necessary, if
drug discovery using iPSCs is to be fruitful. Secondly, another
defining pathology in AD is an accumulation of hyperpho-
sphorylated tau forming paired helical filaments. Growing evi-
dence reveals that toxic Abdirectly induces tau
hyperphosphorylation and accumulation, leading to neurode-
generation processes in affected neurons in AD (39,40). Patho-
logical observations reveal that tau aggregates, but not
amyloid deposits, actually correlate with dementia severity
and extent of neuronal loss (41,42). Therefore, whether FAD
iPSC-derived neurons exhibit accumulation of phosphorylated
tau during extended culture periods should be addressed, and
future studies must also focus on the biochemical dynamics
of tau protein in iPSC-derived neurons treated with exogenous
Ab. Thirdly, the pathological mechanism of late-onset AD,
sporadic AD and AD harboring the apoE4 allele remains
unclear. Recent studies propose that impaired clearance of
Abmay cause late-onset AD through interactions with
ApoE4, rather than increased Abproduction (43,44).
Late-onset AD is more common and accounts for 90% of
people suffering with Alzheimer’s disease. To establish thera-
peutic strategies targeting the common form of AD, neurons
derived from patient-specific iPSCs should be applied to
investigations into the mechanisms underlying Abclearance.
Recently, a number of clinical trials of drugs targeting the
pathogenesis of AD have reportedly failed in succession. Al-
though future advances in iPSC methods are necessary for
the pharmacological development and clinical application of
iPSCs in neurodegeneration, we hope that our study will con-
tribute significantly towards the identification and validation
of novel candidate drugs against one of the most common
and intractable diseases, AD.
MATERIALS AND METHODS
Cell culture and iPS generation
PS1 A246E fibroblasts (AG07768) and PS2 N141I fibroblasts
(AG09908) were obtained from Coriell Cell Repository.
Human fibroblasts were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM; Gibco) containing 10% fetal
bovine serum, 50 U/ml penicillin, 50 mg/ml streptomycin
and 1 mML-glutamine. PS1 iPSC and PS2 iPSC were gener-
ated using the Human iPS Cell Generation Vector Set
(TAKARA). G3T-hi cells were transfected with the Human
iPS Cell Generation Vector set (pDON-5 OCT3/4-SOX2,
pDON-5 KLF4, pDON-5 LIN28-NANOG) and pGP Vector
and pE-ampho Vector with TransIT-293. Forty-eight hours
after transfection, the medium (virus-containing supernatant)
was collected and filtered through a 0.45 mMpore-size cellu-
lose acetate filter. Next, the retrovirus-containing supernatant
was added to RetroNectin-coated plates for centrifugation at
328C and 2000gfor 2 h to facilitate attachment of the virus
particles onto the RetroNectin. Following this, fibroblasts
were added to the plate and retrovirally transduced. Six days
after transduction, fibroblasts were harvested by trypsinization
and replated at 1 ×10
5
cells per 100 mMdish on mitomycin
C-inactivated SNL cells, and the medium was changed to
hiPSC medium, which consisted of DMEM/F12 medium
(Invitrogen) supplemented with 20% Knock-out Serum Re-
placement (Invitrogen), 1 mML-glutamine, 1 mMnon-essential
amino acids, 0.1 mMb-mercaptoethanol, 50 U penicillin,
50 mg/ml streptomycin (Invitrogen) and 4 ng/ml basic fibro-
blast growth factor (bFGF; WAKO Pure Chemicals). The
hiPSC medium was changed every other day until colonies
were picked. The generated iPSCs were maintained on mito-
mycin C-inactivated SNL cells. The hiPSC-culture medium
was changed every other day, and the cells were passaged
using CTK solution every 6 – 7 days.
Sporadic PD patient fibroblasts were generated from dermal
biopsies following informed consent under protocols approved
by Keio University. Two neurologists diagnosed the patient
with sporadic PD, AD was excluded. Sporadic PD-derived
iPSCs were generated as reported previously (14).
Reverse transcriptase-polymerase chain reaction
Total RNA samples were isolated using RNeasy (Qiagen),
according to the manufacturer’s instructions. The concentration
Figure 4. Characterization of Absecretion in PS1 and PS2 iPSC-derived
neurons. (A) The amount of Ab40 and Ab42 secreted from control iPSC-
derived neurons, PS1 iPSC (PS1-2 iPSC and PS1-4 iPSC) and PS2 iPSC
(PS2-1 iPSC and PS2-2 iPSC)-derived neurons. (B) The ratio of Ab42/
Ab40 from control iPSC-derived neurons, PS1 iPSC-derived neurons and
PS2 iPSC-derived neurons. Note, the ratio of Ab42/Ab40 in both PS1 iPSC-
derived neurons and PS2 iPSC-derived neurons was significantly higher than
that of control iPSC-derived neurons. Significant differences among groups
were examined by Student’s t-test versus the ratio of 201B7 iPSC-derived
neurons (∗P,0.05).
Human Molecular Genetics, 2011, Vol. 20, No. 23 4535
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
and purity of the RNA was determined using the ND-1000 spec-
trophotometer (Nanodrop). The cDNA was synthesized using
the SuperscriptIII First-Strand Synthesis System (Invitrogen).
The transgene primers used in the PCR are listed in Supplemen-
tary Material, Table S2. The endogenous primers have been
described previously (14).
Immunofluorescence staining of iPS and iPSC-derived
differentiated neurons
Immunofluorescence staining was performed using the follow-
ing primary antibodies: anti-SSEA 3 (Abcam), anti-SSEA 4
(Abcam), anti-Tra-1-60 (Millipore), anti-Tra-1-81 (Millipore),
Figure 5. Pharmacological response to g-secretase inhibitors in PS1 and PS2 iPSC-derived neurons. (A) The amount of Ab40 and Ab42 secreted from PS1-4 iPSC-
derived neurons (left graph) and PS2-2 iPSC-derived neurons (right graph) treated with Compound E or W. Significant differences were examined by Student’s t-test
versus Ab40 or Ab42 of untreated, respectively (∗P,0.05). (B) The ratio of Ab42/Ab40 from PS1-4 iPSC-derived neurons (left) and PS2-2 iPSC-derived neurons
(right). Significant differences were examined by Student’s t-test versus the ratio of untreated (∗P,0.05). (C) Western blotting of S3 cleaved NICD ( 110 kDa) and
uncleaved Notch1 transmembrane subunit (120 kDa) in PS1-4 iPSC-derived neurons (left) and PS2-2 iPSC-derived neurons (right) exposed to Compound E or W.
a-Tubulin served as internal loading controls. Error bars in (A–D) indicate SD from three independent experiments.
4536 Human Molecular Genetics, 2011, Vol. 20, No. 23
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
anti-SSEA1 (Abcam), anti- MAP-2 (Chemicon) and anti-tau
(HT7, Thermoscientific). 4,6-Diamidino-2-phenylindole
(DAPI; Molecular Probes) was used for nuclear staining.
The secondary antibodies used were: anti-rat IgG and anti-
mouse IgG, and IgM conjugated with Alexa Fluor 488 or
Alexa Fluor 568 (Molecular Probes).
Microarray analysis
Human genome U133 Plus 2.0 GeneChip arrays carrying 54
690 probe sets (Affymetrix) were used for microarray hybridi-
zations to examine global gene expression. Approximately
150 ng of RNA from each sample was labeled using GeneChip
3′IVT Express (Affymetrix) according to the manufacturer’s
instructions. All arrays were hybridized at 458C for 16 h and
scanned using an AFX GC3000 G7 scanner. The gene expres-
sion raw data were extracted using the AFX Gene Chip Oper-
ation System. Quality control was performed on the basis of
Affymetrix quality control metrics. The data were analyzed
with the Gene Spring GX 11.0 (Agilent). Two normalization
procedures were applied. Initially, the signal intensities with
values ,0.1 were assigned a value of 0.1. Then, each chip
was normalized to the 50th percentile of the measurements
taken from that chip. Each gene was normalized to the
median of that gene in the respective controls, to enable com-
parisons of relative changes in gene expression levels between
different conditions.
Microarray data can be found at the GEO website under ac-
cession number ‘GSE28379’. (The following link has been
created to allow review of record GSE28379: http://www.
ncbi.nlm.nih.gov/geo/query/acc.cgi?token=zlovzka
qqwugkdg&acc=GSE28379.) The gene expression profiles of
BJ fibroblasts (GSM248214) were downloaded from the
NCBI Gene Expression Omnibus (GEO) database.
aCGH analysis
Genomic DNA was isolated using DNeasy (Qiagen), accord-
ing to the manufacturer’s instructions. DNA concentrations
were measured on a Nanodrop ND-1000 spectrophotometer
(Isogen). DNA quality was monitored with the Agilent 2100
Bioanalyzer (Agilent Technologies). DNA (500 ng) was
labeled using the Enzo Genomic DNA Labeling kit. Hybridi-
zations were performed on slides containing four arrays, with
each array containing 622 060 in situ synthesized 60-mer oli-
gonucleotides, representing 170 344 unique chromosomal
locations (Agilent Technologies). Images of the arrays were
acquired using a microarray scanner G2505CA (Agilent tech-
nologies) and image analysis was performed using feature ex-
traction software version 10.7 (Agilent Technologies). The
Agilent CGH-v4_107_Sep09 protocol was applied using
default settings. Oligonucleotides were mapped according to
the human genome build NCBI 36. The obtained data were
imported into Agilent Genomic Workbench using the aberra-
tion detection method 2 (ADM-2) algorithm (10.0 threshold)
for further analysis. The aCGH data have been deposited in
GEO and given the series accession number GSE28450.
(The following link has been created to allow review of
record GSE28450: http://www.ncbi.nlm.nih.gov/geo/query/a
cc.cgi?token=ntsvfkqkucksgrc&acc=GSE28450.)
In vitro differentiation
Cells were harvested using CTK solutions and a cell scraper,
and transferred to a Petri dish in hiPSC medium without
bFGF to form embryoid bodies. After 8 days, embryoid
bodies were plated onto gelatin-coated tissue culture dishes
and incubated for an additional 8 days. The cells were incu-
bated at 378Cin5%CO
2
and the medium was replaced
every other day. The cells were stained with mouse
anti-a-fetoprotein IgG (R&D Systems), anti-smooth muscle
actin (Sigma), anti-bIII-tubulin mouse IgG (Chemicon), to-
gether with DAPI.
Teratoma formation
hiPSCs were injected into the subcutaneous tissue of SCID
mice (CREA). At 8 – 10 weeks post-injection, teratomas
were dissected, fixed in 10% formaldehyde in PBS and embed-
ded in paraffin.
Neural induction
Neural induction of hiPSCs cells was performed as previously
described with slight modifications (Okada et al., in prepar-
ation) (25,26). For terminal differentiation, induced neural
cells were plated onto Matrigel-coated coverslips and cultured
for 2 weeks. This was followed by the addition of
Compound E, 2S-2-{[(3,5-difluorophenyl)acetyl]amino}-N-
[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodia-
zepin-3-yl]propanamide (Calbiochem) or Compound W, 3,5-
Bis(4-nitrophenoxy)benzoic Acid (Tokyo Chemical Industry)
for 48 h.
Quantitation of Abby ELISA
Conditioned media of differentiated neurons were collected
after an incubation period of 48 h and subjected to b
Amyloid ELISA Kits (WAKO), according to the manufac-
turer’s instructions.
Immunoblot analysis
Cells were briefly sonicated in cold lysis buffer (50 mMTris –
HCl, pH 7.4, 150 mMNaCl, 0.5% NP-40, 0.5% sodium
deoxycholate, 0.25% sodium dodecyl sulfate, 5 mMEDTA
and protease inhibitor cocktail from Sigma). Total protein con-
centration in the supernatant was determined using a Bio-Rad
protein assay kit. The proteins were then analyzed by immuno-
blotting as follows: protein samples were separated by redu-
cing SDS– PAGE on a 4 – 20% Tris – glycine gradient gel
(Invitrogen), and then transferred to a polyvinylidene difluor-
ide membrane (Millipore). The membrane was incubated with
primary antibodies and then horseradish peroxidase-
conjugated secondary antibodies. Detection was performed
using enhanced chemiluminescence reagents as described by
the supplier (PerkinElmer Life Sciences). Primary monoclonal
antibodies that were used in this study were: anti-tau (HT7,
Thermoscientific), anti-NICD (Cell Signaling Technology),
anti-Notch1 (D1E11) (Cell Signaling Technology) and alpha
tubulin (Cell Signaling Technology).
Human Molecular Genetics, 2011, Vol. 20, No. 23 4537
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
Statistical analysis
Statistical analysis of the data was performed by Student’s
t-test using JMP 8 (SAS Institute, Inc.).
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG online.
ACKNOWLEDGEMENTS
T.Y. is a research fellow of the Japan Society for the Promo-
tion of Science. This work was supported by grants from Eisai
Co. Ltd (to D.I. and N.S.) and the project for realization of re-
generative medicine from the Ministry of Education, Culture,
Sports, Science and Technology of Japan to H.O. We thank
Mari Fujiwara (Core Instrumentation Facility, Keio University
School of Medicine) for the microarray analysis and Satoko
Iwasawa (Department of Preventive Medicine and Public
Health, School of Medicine, Keio University) for helpful
advice about statistical analysis. We also thank Dr Xu Huaxi
for providing the T44 Tau pSG5 plasmid (Sanford-Burnham
Medical Research Institute).
Conflict of Interest statement. None declared.
FUNDING
This work was supported by grants from Eisai Co. Ltd (to D.I.
and N.S.), the Research Fellowship grant of the Japan Society
for the Promotion of Science (to T.Y.), and the Project for
Realization of Regenerative Medicine, and Support for
Core Institutes for iPS Cell Research from the Ministry of
Education, Culture, Sports, Science and Technology of
Japan (to H.O.).
REFERENCES
1. Vetrivel, K.S. and Thinakaran, G. (2006) Amyloidogenic processing of
beta-amyloid precursor protein in intracellular compartments. Neurology,
66, S69– S73.
2. Thinakaran, G. and Koo, E.H. (2008) Amyloid precursor protein
trafficking, processing, and function. J. Biol. Chem.,283, 29615–29619.
3. Hardy, J. and Selkoe, D.J. (2002) The amyloid hypothesis of Alzheimer’s
disease: progress and problems on the road to therapeutics. Science,297,
353–356.
4. Tanzi, R.E. and Bertram, L. (2005) Twenty years of the Alzheimer’s
disease amyloid hypothesis: a genetic perspective. Cell,120, 545–55.
5. Sisodia, S.S. and St George-Hyslop, P.H. (2002) gamma-Secretase, Notch,
Abeta and Alzheimer’s disease: where do the presenilins fit in? Nat. Rev.
Neurosci.,3, 281– 290.
6. Sherrington, R., Rogaev, E.I., Liang, Y., Rogaeva, E.A., Levesque, G.,
Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K. et al. (1995) Cloning of a
gene bearing missense mutations in early-onset familial Alzheimer’s
disease. Nature,375, 754– 760.
7. Cruts, M., van Duijn, C.M., Backhovens, H., Van den Broeck, M.,
Wehnert, A., Serneels, S., Sherrington, R., Hutton, M., Hardy, J., St
George-Hyslop, P.H. et al. (1998) Estimation of the genetic contribution
of presenilin-1 and -2 mutations in a population-based study of presenile
Alzheimer disease. Hum. Mol. Genet.,7, 43– 51.
8. Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D.M., Oshima, J.,
Pettingell, W.H., Yu, C.E., Jondro, P.D., Schmidt, S.D., Wang, K. et al.
(1995) Candidate gene for the chromosome 1 familial Alzheimer’s disease
locus. Science,269, 973– 977.
9. Jayadev, S., Leverenz, J.B., Steinbart, E., Stahl, J., Klunk, W., Yu, C.E.
and Bird, T.D. (2010) Alzheimer’s disease phenotypes and genotypes
associated with mutations in presenilin 2. Brain,133, 1143–1154.
10. Borchelt, D.R., Thinakaran, G., Eckman, C.B., Lee, M.K., Davenport, F.,
Ratovitsky, T., Prada, C.M., Kim, G., Seekins, S., Yager, D. et al. (1996)
Familial Alzheimer’s disease-linked Presenilin 1 variants elevate Ab1-42/
1-40 ratio in vitro and in vivo. Neuron,17, 1005– 1013.
11. Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N.,
Bird, T.D., Hardy, J., Hutton, M., Kukull, W. et al. (1996) Secreted
amyloid b-protein similar to that in the senile plaques of Alzheimer’s
disease is increased in vivo by the presenilin 1 and 2 and APP mutations
linked to familial Alzheimer’s disease. Nat. Med.,2, 864– 870.
12. Tomita, T., Maruyama, K., Saido, T.C., Kume, H., Shinozaki, K.,
Tokuhiro, S., Capell, A., Walter, J., Gru
¨nberg, J., Haass, C. et al. (1997)
The presenilin 2 mutation (N141I) linked to familial Alzheimer disease
(Volga German families) increases the secretion of amyloid beta protein
ending at the 42nd (or 43rd) residue. Proc. Natl Acad. Sci. USA,94,
2025– 2030.
13. Oyama, F., Sawamura, N., Kobayashi, K., Morishima-Kawashima, M.,
Kuramochi, T., Ito, M., Tomita, T., Maruyama, K., Saido, T.C., Iwatsubo, T.
et al. (1998) Mutant presenilin 2 transgenic mouse: effect on an
age-dependent increase of amyloid beta-protein 42 in the brain.
J. Neurochem.,71, 313– 322.
14. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda,
K. and Yamanaka, S. (2007) Induction of pluripotent stem cells from adult
human fibroblasts by defined factors. Cell,131, 861–872.
15. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane,
J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R. et al. (2007)
Induced pluripotent stem cell lines derived from human somatic cells.
Science,318, 1917– 1920.
16. Dimos, J.T., Rodolfa, K.T., Niakan, K.K., Weisenthal, L.M., Mitsumoto,
H., Chung, W., Croft, G.F., Saphier, G., Leibel, R., Goland, R. et al.
(2008) Induced pluripotent stem cells generated from patients with ALS
can be differentiated into motor neurons. Science,321, 1218–1221.
17. Park, I.H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A.,
Lensch, M.W., Cowan, C., Hochedlinger, K. and Daley, G.Q. (2008)
Disease-specific induced pluripotent stem cells. Cell,134, 877–886.
18. Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G.W., Cook, E.G.,
Hargus, G., Blak, A., Cooper, O., Mitalipova, M. et al. (2009) Parkinson’s
disease patient-derived induced pluripotent stem cells free of viral
reprogramming factors. Cell,136, 964– 977.
19. Ebert, A.D., Yu, J., Rose, F.F. Jr., Mattis, V.B., Lorson, C.L., Thomson,
J.A. and Svendsen, C.N. (2009) Induced pluripotent stem cells from a
spinal muscular atrophy patient. Nature,457, 277–280.
20. Lee, G., Papapetrou, E.P., Kim, H., Chambers, S.M., Tomishima, M.J.,
Fasano, C.A., Ganat, Y.M., Menon, J., Shimizu, F., Viale, A. et al. (2009)
Modelling pathogenesis and treatment of familial dysautonomia using
patient-specific iPSCs. Nature,461, 402– 406.
21. Ku, S., Soragni, E., Campau, E., Thomas, E.A., Altun, G., Laurent, L.C.,
Loring, J.F., Napierala, M. and Gottesfeld, J.M. (2010) Friedreich’s ataxia
induced pluripotent stem cells model intergenerational GAA TTC triplet
repeat instability. Cell Stem Cell,7, 631– 637.
22. Chamberlain, S.J., Chen, P.F., Ng, K.Y., Bourgois-Rocha, F.,
Lemtiri-Chlieh, F., Levine, E.S. and Lalande, M. (2010) Induced
pluripotent stem cell models of the genomic imprinting disorders
Angelman and Prader-Willi syndromes. Proc. Natl Acad. Sci. USA,107,
17668– 17673.
23. Nguyen, H.N., Byers, B., Cord, B., Shcheglovitov, A., Byrne, J., Gujar, P.,
Kee, K., Schu
¨le, B., Dolmetsch, R.E., Langston, W. et al. (2011) LRRK2
mutant iPSC-derived DA neurons demonstrate increased susceptibility to
oxidative stress. Cell Stem Cell,8, 267– 280.
24. Marchetto, M.C., Carromeu, C., Acab, A., Yu, D., Yeo, G.W., Mu, Y.,
Chen, G., Gage, F.H. and Muotri, A.R. (2010) A model for neural
development and treatment of Rett syndrome using human induced
pluripotent stem cells. Cell,143, 527–539.
25. Okada, Y., Matsumoto, A., Shimazaki, T., Enoki, R., Koizumi, A.,
Ishii, S., Itoyama, Y., Sobue, G. and Okano, H. (2008) Spatiotemporal
recapitulation of central nervous system development by murine
embryonic stem cell-derived neural stem/progenitor cells. Stem Cells,26,
3086– 3098.
26. Miura, K., Okada, Y., Aoi, T., Okada, A., Takahashi, K., Okita, K.,
Nakagawa, M., Koyanagi, M., Tanabe, K., Ohnuki, M. et al. (2009)
4538 Human Molecular Genetics, 2011, Vol. 20, No. 23
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
Variation in the safety of induced pluripotent stem cell lines. Nat.
Biotechnol.,27, 743– 745.
27. Beher, D., Wrigley, J.D., Nadin, A., Evin, G., Masters, C.L., Harrison, T.,
Castro, J.L. and Shearman, M.S. (2001) Pharmacological knock-down of
the presenilin 1 heterodimer by a novel gamma-secretase inhibitor:
implications for presenilin biology. J. Biol. Chem.,276, 45394– 45402.
28. Okochi, M., Fukumori, A., Jiang, J., Itoh, N., Kimura, R., Steiner, H.,
Haass, C., Tagami, S. and Takeda, M. (2006) Secretion of the Notch-1
Abeta-like peptide during Notch signaling. J. Biol. Chem.,281, 7890–
7898.
29. Zhou, S., Zhou, H., Walian, P.J. and Jap, B.K. (2005) CD147 is a
regulatory subunit of the gamma-secretase complex in Alzheimer’s
disease amyloid beta-peptide production. Proc. Natl Acad. Sci. USA,102,
7499– 7504.
30. He, G., Luo, W., Li, P., Remmers, C., Netzer, W.J., Hendrick, J.,
Bettayeb, K., Flajolet, M., Gorelick, F., Wennogle, L.P. et al. (2010)
Gamma-secretase activating protein is a therapeutic target for Alzheimer’s
disease. Nature,467, 95– 98.
31. Kyriazis, G.A., Wei, Z., Vandermey, M., Jo, D.G., Xin, O., Mattson, M.P.
and Chan, S.L. (2008) Numb endocytic adapter proteins regulate the
transport and processing of the amyloid precursor protein in an
isoform-dependent manner: implications for Alzheimer disease
pathogenesis. J. Biol. Chem.,283, 25492– 25502.
32. Liu, G.H., Barkho, B.Z., Ruiz, S., Diep, D., Qu, J., Yang, S.L.,
Panopoulos, A.D., Suzuki, K., Kurian, L., Walsh, C. et al. (2011)
Recapitulation of premature ageing with iPSCs from Hutchinson–Gilford
progeria syndrome. Nature,472, 221– 225.
33. Zhang, J., Lian, Q., Zhu, G., Zhou, F., Sui, L., Tan, C., Mutalif, R.A.,
Navasankari, R., Zhang, Y., Tse, H.F. et al. (2011) A human iPSC model
of Hutchinson Gilford progeria reveals vascular smooth muscle and
mesenchymal stem cell defects. Cell Stem Cell,8, 31–45.
34. Yazawa, M., Hsueh, B., Jia, X., Pasca, A.M., Bernstein, J.A., Hallmayer,
J. and Dolmetsch, R.E. (2011) Using induced pluripotent stem cells to
investigate cardiac phenotypes in Timothy syndrome. Nature,471, 230–
234.
35. Carvajal-Vergara, X., Sevilla, A., D’Souza, S.L., Ang, Y.S., Schaniel, C.,
Lee, D.F., Yang, L., Kaplan, A.D., Adler, E.D., Rozov, R. et al. (2010)
Patient-specific induced pluripotent stem-cell-derived models of
LEOPARD syndrome. Nature,465, 808– 812.
36. Mattis, V.B. and Svendsen, C.N. (2011) Induced pluripotent stem cells: a
new revolution for clinical neurology? Lancet Neurol.,10, 383 – 394.
37. Hu, B.Y., Weick, J.P., Yu, J., Ma, L.X., Zhang, X.Q., Thomson, J.A. and
Zhang, S.C. (2010) Neural differentiation of human induced pluripotent
stem cells follows developmental principles but with variable potency.
Proc. Natl Acad. Sci. USA,107, 4335–5340.
38. Boulting, G.L., Kiskinis, E., Croft, G.F., Amoroso, M.W., Oakley, D.H.,
Wainger, B.J., Williams, D.J., Kahler, D.J., Yamaki, M., Davidow, L.
et al. (2011) A functionally characterized test set of human induced
pluripotent stem cells. Nat. Biotechnol.,29, 279–286.
39. Busciglio, J., Lorenzo, A., Yeh, J. and Yankner, B.A. (1995)
Beta-amyloid fibrils induce tau phosphorylation and loss of microtubule
binding. Neuron,14,879– 888.
40. Jin, M., Shepardson, N., Yang, T., Chen, G., Walsh, D. and Selkoe, D.J.
(2011) Soluble amyloid b-protein dimers isolated from Alzheimer cortex
directly induce Tau hyperphosphorylation and neuritic degeneration.
Proc. Natl Acad. Sci. USA,108, 5819–5824.
41. Bierer, L.M., Hof, P.R., Purohit, D.P., Carlin, L., Schmeidler, J., Davis,
K.L. and Perl, D.P. (1995) Neocortical neurofibrillary tangles
correlate with dementia severity in Alzheimer’s disease. Arch. Neurol.,
52, 81–88.
42. Schmitt, O., Eggers, R. and Haug, H. (1995) Quantitative investigations
into the histostructural nature of the human putamen. I. Staining, cell
classification and morphometry. Ann. Anat.,177, 243– 250.
43. Jiang, Q., Lee, C.Y., Mandrekar, S., Wilkinson, B., Cramer, P., Zelcer, N.,
Mann, K., Lamb, B., Willson, T.M., Collins, J.L. et al. (2008) ApoE
promotes the proteolytic degradation of Abeta. Neuron,58, 681–693.
44. Mawuenyega, K.G., Sigurdson, W., Ovod, V., Munsell, L., Kasten, T.,
Morris, J.C., Yarasheski, K.E. and Bateman, R.J. (2010) Decreased
clearance of CNS beta-amyloid in Alzheimer’s disease. Science,330,
1774.
Human Molecular Genetics, 2011, Vol. 20, No. 23 4539
by guest on December 25, 2015http://hmg.oxfordjournals.org/Downloaded from
