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2003; 18: 593–595.
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et al. J Am Acad Child Adolesc Psychiatry 2008; 47: 673–681.
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Psychopharmacol 2009; 17: 374–383.
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Hum Genet 2009; 125: 265–279.
pluripotent stem cells
derived from schizophrenia
patients with a DISC1
Molecular Psychiatry (2011) 16, 358–360; doi:10.1038/
mp.2011.13; published online 22 February 2011
Schizophrenia, a severe brain disease with a promi-
nent genetic basis, has been suggested to have a
neurodevelopmental origin.1Although many candi-
date susceptibility genes for schizophrenia have been
identified,2access to human neural progenitors and
neurons to study the biology of disease genes is very
limited. Induced pluripotent stem cells (iPSCs),
reprogrammed from somatic cells of healthy subjects
and patients,3offer an unprecedented opportunity to
recapitulate both normal and pathologic human
development, thereby enabling a new approach to
understanding human disease mechanisms.
Rare, large pedigrees in which a single genetic
locus is likely to confer disease susceptibility have
proven to be invaluable for the study of complex
disorders.4Disrupted-in-Schizophrenia 1 (DISC1) was
initially identified at the breakpoint of a balanced
segregates with schizophrenia, bipolar disorder and
family.5,6Linkage and association studies of addi-
evidence to support the DISC1 locus as a risk factor
for major mental disorders.2How DISC1 contributes
to a spectrum of mental disorders is unknown.
Studies from rodents have implicated DISC1 in
many neurodevelopmental processes.7The splicing
and expression of DISC1 in postmortem brains
from individuals with schizophrenia is abnormal.8
Little is known about the function of human
in alarge Scottish
DISC1, which is significantly different from the
rodent gene.8We have previously identified an
American family with schizophrenia and a 4bp
deletion in DISC1 that appears to result in diminished
DISC1 expression.9Here, we report the derivation of
multiple iPSC lines from two affected siblings of this
family with the DISC1 mutation, and from a healthy
We collected skin biopsies from a male patient
(III:5; named D1) and his sister (III:7, named D2) who
were diagnosed with chronic undifferentiated schizo-
phrenia and chronic paranoid schizophrenia, respec-
tively.9Both patients experienced auditory and visual
hallucinations, multiple delusions and had formal
thought disorder.9Male neonatal foreskin fibroblasts
from American Type Culture Collection were used in
parallel (CRL-2097; named C1). We confirmed normal
karyotypes of all fibroblasts and the presence of the
4bp deletion at the exon-intron 12 region in D1 and
D2, but not C1 fibroblasts (Supplementary Figure 1;
Supplementary Table 1).
To avoid integration of foreign DNA into the host
genome associated with classic virus-based methods
of iPSC derivation,3we explored the episomal vector
approach, which was previously used to generate
integration-free iPSCs from fetal human skin fibro-
blasts.10Because of its low efficacy, no iPSC lines
have yet been reported to be derived from adult
patients using this approach. We transfected a
mixture of reprogramming plasmids into fibroblasts
by electroporation and plated them onto freshly
prepared feeder layers (see Supplementary Informa-
tion). Colonies of iPSCs were manually picked after
3–6 weeks for expansion and subjected to extensive
and a minimum of two lines from each fibroblast that
exhibited characteristic properties of iPSCs were
obtained (Figure 1).
All iPSCs exhibited typical morphology of human
embryonic stem cells (hESCs), normal karyotypes and
expression of a panel of markers for human pluripo-
tent stem cells, including Nanog, Oct4, Sox2, SSEA3,
SSEA4, TRA-1-60, TRA-1-81 and TRA-2-49 (Figures
1a–c; Supplementary Figure 2). Successful repro-
gramming requires demethylation of CpGs in promo-
ter regions of pluripotent genes, which are normally
heavily methylated in fibroblasts.3Bisulfite sequen-
cing analysis of genomic DNA from iPSCs demon-
strated the largely unmethylated status of CpGs in
Nanog and Oct4 promoters (Figure 1d). The teratoma
assay was used to determine the pluripotency of
iPSCs after injection into adult SCID/Beige mice.
Tumors were harvested 1–3 months later and histo-
logical analysis showed the differentiation of iPSCs
into ectodermal, mesodermal and endodermal tissues
(Figure 1e; Supplementary Figure 3). Sequencing
analysis confirmed the 4bp deletion in D1 and D2,
but not C1 iPSCs (Figure 1f). Using an established
PCR-based detection method,10no signal for repro-
gramming plasmids was found in genomic DNA
of iPSCs (Figure 1g). Together, our analyses demon-
lines were examined
Letter to the Editor
strated that these cell lines are bona fide iPSCs
that maintain genetic characteristics of original
To our knowledge, this study presents the first
iPSC lines derived from schizophrenia patients.
In addition, it presents the first cases of generation
(a) Sample phase images of iPSCs derived from two schizophrenia patients with a DISC1 mutation (D1 and D2) and from a
control subject (C1). Scale bar: 100mm. (b) Sample confocal images of immunostaining of pluripotency markers, Nanog and
Oct4, and DAPI. Scale bar: 100mm. (c) Normal karyotypes of derived iPSCs. (d) Methylation status of CpGs in the promoter
regions of Nanog and Oct4. Open and close circles represent unmethylated and methylated CpGs, respectively. (e) Sample
H&E staining images of teratomas formed by iPSCs injected into adult SCID/Beige mice. Scale bar: 100mm. (f) Genomic
sequencing analysis of the 4bp deletion in iPSCs. (g) PCR-based demonstration of the lack of plasmid sequences in the
genomic DNA of iPSCs.
Characterization of iPSC lines from two schizophrenia patients with a DISC1 mutation and a healthy subject.
Letter to the Editor
of integration-free iPSCs from adult patients using the Download full-text
episomal vector approach.10Although significant
effort has been made towards the derivation of iPSCs
free of foreign DNA integration to avoid the disrup-
tion of host genome and potential reactivation of
oncogenes used for reprogramming, these methods
are generally much less efficient.10Our study demon-
strates the feasibility of generating high quality
integration-free iPSCs from adult patients. These
established iPSC lines from schizophrenia patients
with a defined DISC1 mutation will provide a useful
resource for investigating the function of DISC1
in human neurodevelopment. Future studies using
these iPSCs may serve as an entry point to clarify
the molecular and cellular pathogenesis of schizo-
Conflict of interest
The authors declare no conflict of interest.
C-H Chiang1,2, Y Su1,3, Z Wen1,3, N Yoritomo4,
CA Ross2,3,4,5, RL Margolis3,4,5, H Song1,2,3,5
and G-l Ming1,2,3,5
1Institute for Cell Engineering, Johns Hopkins
University School of Medicine, Baltimore, MD, USA;
2The Solomon H. Snyder Department of
Neuroscience, Johns Hopkins University
School of Medicine, Baltimore, MD, USA;
3Department of Neurology, Johns Hopkins University
School of Medicine, Baltimore, MD, USA;
4Department of Psychiatry, Johns Hopkins University
School of Medicine, Baltimore, MD, USA and
5Graduate Program in Cellular and Molecular
Medicine, Johns Hopkins University School of
Medicine, Baltimore, MD, USA
or email@example.com or firstname.lastname@example.org
1 Weinberger DR. Arch Gen Psychiatry 1987; 44: 660–669.
2 Ross CA, Margolis RL, Reading SA, Pletnikov M, Coyle JT. Neuron
2006; 52: 139–153.
3 Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A
et al. Cell 2008; 134: 877–886.
4 McClellan JM, Susser E, King MC. Br J Psychiatry 2007; 190:
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G et al. Lancet 1990; 336: 13–16.
6 Millar JK, Wilson-Annan JC, Anderson S, Christie S, Taylor MS,
Semple CA et al. Hum Mol Genet 2000; 9: 1415–1423.
7 Chubb JE, Bradshaw NJ, Soares DC, Porteous DJ, Millar JK. Mol
Psychiatry 2008; 13: 36–64.
8 Nakata K, Lipska BK, Hyde TM, Ye T, Newburn EN, Morita Yet al.
Proc Natl Acad Sci USA 2009; 106: 15873–15878.
9 Sachs NA, Sawa A, Holmes SE, Ross CA, DeLisi LE, Margolis RL.
Mol Psychiatry 2005; 10: 758–764.
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Science 2009; 324: 797–801.
Supplementary Information accompanies the paper on the Molecular
Psychiatry website (http://www.nature.com/mp)
studies of ZNF804A locus
Molecular Psychiatry (2011) 16, 360–361; doi:10.1038/
mp.2010.55; published online 11 May 2010
Recently, O’Donovan et al.1genome-wide association
study found the single-nucleotide polymorphism
rs1344706, in intron 2 of ZNF804A on chromosome
2q32.1, to be associated (P=1.61?10?7) with schizo-
phrenia (MIM 181500).
Thus, ZNF804A is very likely a true susceptibility
locus for schizophrenia, albeit one that confers a
small increment in risk.1,2We examined rs1344706
within two independent samples of Han Chinese
populations: a population-based sample and a family-
based sample. Our results provide further evidence
for a positive association between the ZNF804A locus
The study was approved by the local psychiatry
research ethics committees and informed consent was
obtained from all subjects. The population-based
sample included 566 schizophrenics (295 males,
mean age=35.2±11.9; 271 females, mean age=
32.6±13.7) and 574 healthy control subjects (329
males, mean age=29.0±14.1; 245 females, mean
age=29.3±13.5). The family-based sample consisted
of 101 schizophrenia probands and their biological
parents. The probands included 39 males (mean age=
25.3±7.5) and 62 females (mean age=22.2±6.8). All
patients were diagnosed by the psychiatrists of the
First Affiliated Hospital of Xi’an Jiaotong University
School of Medicine according to Diagnostic and
Statistical Manual of Mental Disorders (DSM-IV)
criteria for schizophrenia. The diagnosis was checked
and verified by two independent senior psychiatrists
who reviewed the psychiatric case records. The
controls were drawn from local volunteers and blood
transfusion donors, and the subjects with a personal
or family history of mental illness were excluded by
Cases and controls were mixed on the same plates,
and double-blind procedure was performed. Genotyp-
ing was accomplished by allele-specific PCR, in
which methods have been described elsewhere.3To
ensure that the obtained genotypes were valid,
re-genotyping was performed on 80 random DNA
samples for rs1344706. All genotypes were in agree-
ment with the first round of genotyping, and no
genotyping errors were found. Hardy–Weinberg equi-
librium of rs1344706 was assessed using the software
program Finetti (http://ihg2.helmholtz-muenchen.de/
cgi-bin/hw/hwa1.pl). For the case–control analysis,
Letter to the Editor