HMG Advance Access published June 27, 2013
Human Molecular Genetics, 2013
doi:10.1093/hmg/ddt275
1–12
Modeling Dravet syndrome using induced
pluripotent stem cells (iPSCs) and directly converted
neurons
Jiao Jiao2,3,{, Yuanyuan Yang1,2,{, Yiwu Shi4, Jiayu Chen2, Rui Gao2, Yong Fan1, Hui Yao2,5,
Weiping Liao4, Xiao-Fang Sun1,∗ and Shaorong Gao2,∗
1
Received May 1, 2013; Revised May 29, 2013; Accepted June 11, 2013
Severe myoclonic epilepsy of infancy (SMEI, also known as Dravet syndrome) and genetic epilepsy with febrile
seizures plus (mild febrile seizures) can both arise due to mutations of SCN1A, the gene encoding alpha 1 poreforming subunit of the Nav1.1 voltage-gated sodium channel. Owing to the inaccessibility of patient brain
neurons, the precise mechanism of mild febrile seizures and SMEI remains elusive, and there is no effective
pharmacotherapy. Induced pluripotent stem cells (iPSCs) and induced neurons (iNs) have been successfully
generated from patients and applied for modeling various neuronal diseases. In this study, we established
iPSC lines from one SMEI patient and one mild febrile seizures patient, respectively. Functional glutamatergic
neurons were subsequently differentiated from these iPSCs. Electrophysiological analysis of patient iPSCderived glutamatergic neurons revealed a hyperexcitable state of enlarged and persistent sodium channel
activation, more intensive evoked action potentials and typical epileptic spontaneous action potentials. In consistent with the severity of the symptoms, the hyperexcitability of the neurons derived from SMEI patient was
more serious than that of mild febrile seizures patient. Furthermore, the hyperexcitability of the neurons can
be alleviated by treatment with phenytoin, a conventional antiepileptic drug. In parallel, iNs were directly converted from patient fibroblasts which also showed a delayed inactivation of sodium channels. Our results
demonstrate that both iPSC-derived neurons and iNs from mild febrile seizures and SMEI patients exhibited a
hyperexcitable state. More importantly, patient iPSC-derived neurons can recapitulate the neuronal pathophysiology and respond to drug treatment, indicating that these neurons can be potentially used for screening
appropriate drugs for personalized therapies.
INTRODUCTION
More than 70% occurrence of genetic epilepsy with febrile seizures plus (mild febrile seizures) and severe myoclonic epilepsy
of infancy (SMEI) (also known as Dravet syndrome) arise due to
de novo mutations of SCN1A, the gene encoding alpha 1 pore-
forming subunit of the Nav1.1 voltage-gated sodium channel.
The mild febrile seizures spectrum comprises a range of mild
to severe phenotypes varying from classical febrile seizures to
SMEI (1,2). For the mild classical febrile seizures, the vast majority of patients do not require treatment, whereas the most
severe phenotype of mild febrile seizures, SMEI, is a rare
∗
To whom correspondence should be addressed at: 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, P.R. China.
Tel: +86 1080728967; Fax: +86 1080727535; Email: [email protected] (S.G.); Key Laboratory for Major Obstetric Diseases of Guangdong
Province, The Third Affiliated Hospital, Guangzhou Medical College, Guangdong 510150, P.R. China. Tel: +86 2081292202; Fax: +86 2081292013;
Email: [email protected] (X.-F.S.)
†
These two authors contributed equally to this work.
# The Author 2013. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
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Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of
Guangdong Higher Education Institutes, The Third Affiliated Hospital, Guangzhou Medical College, Guangdong 510150,
P.R. China, 2National Institute of Biological Sciences (NIBS), Beijing 102206, P.R. China, 3College of Life Science, Beijing
Normal University, Beijing 100875, P.R. China, 4Institute of Neuroscience and the Second Affiliated Hospital of
Guangzhou Medical College, Guangdong 510150, P.R. China and 5The Affiliated Bayi Brain Hospital, The Military
General Hospital of Beijing PLA, Beijing 100700, P.R. China
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RESULTS
Derivation of iPSC lines from SMEI and mild febrile seizures
patient fibroblasts
Skin fibroblasts were cultured from one patient who was diagnosed with SMEI, one patient with the mild classical febrile
epilepsy and an unaffected control. The SCN1A gene was
cloned from the genomic DNA of these two patients and the unaffected control. Subsequent sequencing of SCN1A revealed that
SMEI patient has a missense mutation F1415I, which located in
the linker domain of S3 and S4 domain, and the Q1923R mutation was detected in the mild febrile seizures patient, which
located in the C-terminal (Fig. 1A; Supplementary Material,
Fig. S1).
To generate iPSC lines, fibroblasts were infected with retroviruses carrying four factors: OCT4, SOX2, KLF4 and
C-MYC. As expected, round, flattened colonies appeared after
2 weeks and became compact after 1 month. Human embryonic
stem cell (hESC)-like colonies were then picked and passaged
manually. The iPSC lines showed typical morphology of
hESCs (Fig. 1B). Most iPSC lines maintained normal karyotype
(Fig. 1C; Supplementary Material, Fig. S2 and Table S1). They
expressed pluripotency markers including OCT4, NANOG,
SOX2 and surface markers, such as TRA-1-60 and TRA-1-81
(Fig. 1D; Supplementary Material, Fig. S3). Microarray analysis
showed that the global gene expression profile of iPSC lines is
similar to that of hESCs, but distinct from that of human fibroblast cells (Fig. 1E). In vivo differentiation ability of SMEI and
mild febrile seizures patient-specific iPSC lines was assessed
using teratoma formation assay. Teratomas-containing cells
derived from the three embryonic germ layers were observed
in NOD/SCID mice (Fig. 1F; Supplementary Material, Fig. S4
and Table S1). With the criteria of right karyotype, pluripotent
genes expression, capability of generating three germ layers in
teratoma, two iPSC lines from the Q1923R patient, two iPSC
lines from the F1415I patient and two iPSC lines generated
from unaffected human fibroblasts were selected for further
experiments of neuron differentiation and electrophysiological
examinations (Supplementary Material, Table S1). Collectively,
these results showed that pluripotent iPSC lines could be generated from mild febrile seizures and SMEI patient fibroblasts.
SMEI and mild febrile seizures iPSCs can be differentiated
into mature functional glutamatergic neurons in vitro
We next intended to differentiate the selected iPSCs into neurons
as previously described (29). The neurons differentiated from
iPSCs (diN for short) through embryoid body (EB) exhibited
typical neuronal morphology and expressed pan-neuronal
markers TUJ-1 and MAP2 (Fig. 2A; Supplementary Material,
Fig. S5 and S6A). The differentiation efficiency of patient
iPSCs and control iPSCs was comparable (Supplementary Material, Fig. S6B). Electrophysiological test showed that the cells
gained resting membrane potentials between 245 and 260 mV
and were indistinguishable among cell lines (Supplementary
Material, Fig. S7A). Furthermore, the neurons showed sodium
channel activities and the action potentials can be eliminated
with tetrodotoxin (TTX) treatment (Fig. 2B and C).
When co-cultured with human astrocytes, the neurons formed
functional synapses reflected by the expression of Synapsin and
miniature excitatory post-synaptic currents recorded in patch
clamp (Fig. 2D and E). This indicated that the differentiated
neurons could integrate into neural circuit in vitro. Further characterization of the neurons showed that 90% of the neurons
were glutamatergic neurons and the rest were GABAergic
neurons (Fig. 3A). These results indicate that successful
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convulsive disorder that is characterized by febrile seizures with
onset during the first year of life followed by intractable epilepsy,
impaired psychomotor development and ataxia (3,4), and SMEI is
typically resistant to standard anticonvulsant pharmacotherapy.
Exploration of the pathological mechanism of mild febrile seizures and SMEI was difficult due to the inaccessibility of patient
brain neurons. Previous studies using SCN1A knockout mouse
models suggested that reduced sodium current in GABAergic
interneurons might be the cause of SMEI (5). However,
another report detected persistent, non-inactivating sodium
channel behavior in immortalized cell lines expressing a
mutated SCN1A gene (6). Thus, the exact pathological mechanism of mild febrile seizures and SMEI remains elusive.
Induced pluripotent stem cells (iPSCs) and directly induced
neurons (iNs) provide us a direct tool to explore the mechanism
of mild febrile seizures and SMEI, which can circumvent the problems raised by divergence between cell types and species.
iPSCs can be generated from differentiated somatic cells by
ectopic expression of the transcription factors Oct4, Sox2,
Klf4 and c-Myc (OSKM) (7 – 12). The generation of patientspecific iPSC lines has attracted great attention because iPSCs
can potentially be used for cell-based therapies (13– 15).
iPSCs have already provided a tool for modeling diseases in
vitro, exploring their pathology and screening for appropriate
drugs (16– 20). Inspired by iPSC reprogramming technology,
direct conversion of one differentiated cell type to another has
been achieved through ectopic expression of cell-specific transcription factors. To date, direct conversion from fibroblasts
has been achieved for neurons, hepatocytes and cadiomyocytes
(21– 23). Moreover, the functionality of the converted cells has
been verified by in vivo transplantation and in situ transdifferentiation (24). By taking advantage of iPSCs and iNs, many neuronal diseases such as Alzheimer’s disease, Parkinson’s disease,
Rett syndrome and schizophrenia have been successfully recapitulated in vitro (25– 28).
In this study, we reported Q1923R and F1415I missense mutations in mild febrile seizures patient and SMEI patient, respectively. We produced SMEI and mild febrile seizures patient-specific
iPSCs and then differentiated the iPSCs into glutamatergic
neurons, which generated action potentials and formed functional
excitatory synapses. Biophysical characterization of the patientderived neurons demonstrated that patient iPSC-derived
neurons showed enlarged amplitude and delayed inactivation of
voltage-gated sodium channels. Furthermore, SMEI and mild
febrile seizures patient iPSC-derived neurons showed spontaneous action potentials with epileptic characteristics, representing
a hyperexcitable state. Importantly, treatment of SMEI neurons
with antiepileptic drug phenytoin alleviated the enlargement
and persistence of sodium channels and eliminated the epilepticform action potentials. In parallel, we generated iNs directly from
patient fibroblasts. Patient-specific iNs also showed delayed
inactivation of sodium channels.
Human Molecular Genetics, 2013
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differentiation of SMEI and mild febrile seizure patient-specific
iPSCs into functional glutamatergic neurons can be achieved.
Glutamatergic neurons derived from SMEI iPSCs displayed
enlarged sodium currents and delayed inactivation of
sodium channel
In order to decipher the pathological mechanism of SMEI and
mild febrile seizures, we next examined the electrophysiological
properties of voltage-gated sodium channels in neurons differentiated from the SMEI and mild febrile seizures patient iPSCs.
In the voltage clamp mode, current – voltage relationships of
the wild-type and patient-specific iPSC-derived neurons were
examined. We marked the cells recorded in patch clamp with
biocytin and further characterized them by immunohistochemistry identification after patch-clamp experiment. Since 90% of
the iPSC-derived neurons were glutamatergic neurons (Fig. 3A),
all neurons recorded expressed VGLUT1 indicating their identities (Fig. 3B). The I – V curve reflected apparently enlarged
magnitude of sodium currents in the neurons from SMEI and
mild febrile seizures patients with different extent: sodium currents in SMEI showed more severe enlarged sodium currents and
mild febrile seizures showed relatively less severe enlargement
(Fig. 3C).
Besides the magnitude of the sodium currents, sodium
channel inactivation of the iPSC-derived neurons of the
F1415I and Q1923R were apparently delayed in the voltage
clamp (Fig. 3C). Normalized current –voltage relationship
and inactivation of the differentiated neurons were further
examined. First, sodium currents were normalized to the
maximum sodium current, respectively, to show the inactivation of sodium channel. Normalized I– V curves from sodium
channels showed that the reverse potential of F1415I neurons
significantly shifted to depolarization and reverse potential of
Q1923R neurons slightly shifted to depolarization. Therefore,
current– voltage relationships for sodium channels showed
incomplete inactivation in the F1415I iPSC-derived neurons,
and relatively minor incomplete inactivation of sodium
channels in Q1923R iPSC-derived neurons was observed
(Fig. 3D).
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Figure 1. Generation of iPSC lines from SMEI patient fibroblasts. (A) Sequencing of SCN1A in SMEI and mild febrile seizures patients showed heterozygous mutation
on 4243 T to A (left panel) and 5768 A to G (right panel), respectively. (B) Morphology of one iPSC line (Passage 5) derived from SMEI patient with F1415I mutation.
Scale bar: 200 mm. (C) Karyotype analysis of the SMEI patient-derived iPSC line (Passage 6). (D) Immunofluorescent staining of the SMEI patient-derived iPSC line
(Passage 6) with pluripotency markers for OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81 (scale bar: 100 mm). (E) Scatter plot analysis of microarray data shows the
differentially expressed genes between fibroblasts, SMEI patient iPSCs (Passage 12) and human ES cells (Passage 11). Red or green color indicates differentially up- or
down-regulated genes, respectively. (F) Teratoma formation for the F1415I SMEI patient-derived iPSC (Passage 8) line shows tissues from three germ layers. Scale
bar: 50 mm.
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The inactivation of sodium channels was further examined in
differentiated neurons derived from the F1415I patient iPSCs,
Q1923R patient iPSCs and control iPSCs. The V50 value for inactivation in control cells was 259 + 0.35 mV (n ¼ 17). In contrast, significant depolarizing shifts in the inactivation curves
were detected in F1415I iPSC-derived neurons (V50:
236.63 + 0.72 mV, n ¼23), and relative minor impairment
of inactivation of sodium channels was observed in Q1923R
mild febrile seizures patient iPSC-derived neurons (V50 ¼
247.61 + 0.60 mV, n ¼ 19) (Fig. 3E). Comparison of I– V
curves in neurons derived from the two iPSC lines of the same
patient showed no difference in the sodium channel activities
(Supplementary Material, Fig. S7B – D).
Neurons derived from SMEI and mild febrile seizures iPSCs
displayed alterations in action potential characteristics
In consistent with the sodium channel activity data, action potentials evoked following depolarizing current injection of SMEI
and mild febrile seizures iPSC-derived neurons had apparently
larger amplitude and higher frequencies (Fig. 4A). Further analysis on the amplitude of the action potentials showed significant
enlargement in the F1415I neuron, whereas the Q1923R showed
similar amplitude as wild-type cells (Fig. 4B). Furthermore, we
examined the numbers of action potentials evoked by injection
of currents. F1415I neurons showed significant more action
potentials than the wild-type cells, and Q1923R neurons
showed slightly more action potentials than the control cells
(Fig. 4C). These results indicate that, in accordance with the
enlarged and sustained activated sodium currents, neurons of
the SMEI patient showed larger and more action potentials,
whereas mild febrile seizures neurons showed less severe phenotype. Taken together, the results demonstrated that SMEI patient
iPSC-derived glutamatergic neurons showed hyperexcitability.
Neurons differentiated from SMEI and mild febrile seizures
patient iPSCs showed epileptiform discharges
We next examined whether the SMEI and mild febrile seizures
iPSC-derived neurons can recapitulate the epileptic phenotype.
Spontaneous action potentials showed higher frequency in the
F1415I and Q1923R neurons than wild-type with different
extent, and typical epileptic firing was detected in a portion of
iPSC-derived neurons obtained from F1415I and Q1923R
patients (Fig. 5). The frequencies of action potential of F1415I
were 5 Hz, which is significantly higher than the wild-type
cells of 2 Hz, and the Q1923R neurons showed moderate frequencies of 3– 4 Hz (Fig. 5A and B).
Besides the higher frequencies observed, epileptic firing characterized by action potentials with amplitudes of 50 mV and
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Figure 2. Differentiation of SMEI iPSCs into functional neurons. (A) Immunofluorescent staining of neurons differentiated from the F1415I SMEI patient iPSCs
shows expression of the neuronal markers TUJ-1 and MAP2. Scale bar: 200 mm (left panel). Immunofluorescent staining of individual neurons differentiated
from the F1415I SMEI patient iPSCs. Scale bar: 100 mm (right panel). (B) Whole-cell currents recorded following depolarizing voltage steps (280 to 60 mV) in
neurons differentiated from the SMEI iPSCs (left panel). Action potentials recorded after injection of current steps (250 to 90 pA) from neurons differentiated
from the SMEI iPSCs (right panel). (C) Sodium currents and action potentials were eliminated by TTX (1 mM). Whole-cell currents were recorded following depolarizing voltage steps (280 to 60 mV) in neurons differentiated from mild febrile seizures iPSCs. Action potentials evoked following injection of current steps (250 to
90 pA) recorded from neurons differentiated from mild febrile seizures iPSCs. (D) SMEI iPSC-derived neurons express GAD67, VGLUT1, SYNAPSIN as shown by
immunocytochemistry. Scale bars: GAD67, 20 mm; SYNAPSIN, 50 mm; VGLUT1, 50 mm. (E) Whole-cell neuronal recording in which spontaneously miniature
excitatory post-synaptic currents (miniature EPSCs) were detected in neurons derived from SMEI iPSCs.
Human Molecular Genetics, 2013
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firing frequencies of .5 Hz was identified in some neurons of
F1415I and Q1923R (Fig. 5). These action potentials showed
paroxysmal depolarization shifts (PDSs) featured with elongated repolarization times, deep cuneiform waveforms and
enlarged wave widths (Fig. 5C). These observations demonstrate that SMEI and mild febrile seizures patient iPSC-derived
neurons recapitulated the abnormal sodium currents, action
potentials and epileptiform firing.
and alleviated delayed inactivation of sodium channel
(Fig. 6A). Accordingly, the stimulated action potentials by injection of current steps reduced in frequency and amplitude
(Fig. 6B). Moreover, the epileptic form of the spontaneous
action potentials disappeared upon phenytoin treatment
(Fig. 6C). These results indicate that the iPSC-derived neurons
can respond to antiepileptic drug and can be potentially
applied in drug screening in the future.
Treatment of SMEI patient neurons with phenytoin can
alleviate the hyperexcitability
Direct conversion of SMEI and mild febrile seizures patient
fibroblasts to functional glutamatergic neurons
We next sought to investigate whether the patient iPSCdifferentiated neurons can respond to the drug used for treating
epilepsy. When treated with phenytoin (100 mM), a traditional
antiepileptic drug, in perfusion system of patch clamp, the
SMEI iPSC-derived neurons showed reduced sodium currents
To generate iNs from fibroblasts directly, fibroblasts from the two
patients and the unaffected control were infected with lentiviruses
carrying six transcription factors including ASCL1, BRN2,
MYT1Ll, NEUROD1, OLIG2 and ZIC1 (26) in the presence of
neural survival factors BDNF, GDNF and NT3. One month after
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Figure 3. Sodium channel of neurons differentiated from SMEI iPSCs shows enlarged magnitude and delayed inactivation. (A) The iPSC-derived neurons showed
90% neurons positive for glutamatergic neuron marker VGLUT1 and ,10% neurons expressed GABAergic neuron marker GAD67. Scale bars: 50 mm.
(B) Recorded neurons differentiated from iPSCs, marked by biocytin, are positive for VGLUT1. Scale bar: 50 mm. (C) Current–voltage relationships of whole-cell
sodium currents from iPSC-derived neurons from the Q1923R (mild febrile seizures), F1415I (SMEI) and unaffected control. F1415I showed significant enlarged
magnitude and slowed inactivation of sodium channels. Q1923R showed relatively minor enlarged magnitude and slowed inactivation of sodium channels. (D) A
plot showing normalized current– voltage relationships of sodium channels from iPSC-derived neurons obtained from F1415I and Q1923R patients and the unaffected
control. Currents were normalized to the peak current amplitude. (E) Voltage dependence of inactivation of sodium currents from iPSC-derived neurons obtained from
F1415I and Q1923R patients and the unaffected control. The protocol is depicted. Currents were normalized to the peak current amplitude.
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Human Molecular Genetics, 2013
induction, ,1% of the cells showed a neuron-like morphology
(Supplementary Material, Fig. S8A). No significant differences
in the kinetics or the efficiency of conversion from fibroblasts to
neurons were detected. The neuron-like cells expressed panneuronal markers such as TUJ-1, MAP2 and NEUN. Approximately half of the iNs were positive for the mature glutamatergic
neuron marker VGLUT1, and very few iNs expressed the
mature GABAergic neuron marker GAD67 (Fig. 7A).
We further assessed the physiological properties of the iNs
with patch-clamp recordings at days 25– 35 of culture. We
found that majority of iNs displayed typical neuronal Na+ and
K+ channels properties (Fig. 7B). Moreover, single and multiple
action potentials could be evoked with a series of depolarizing
current injections in most iNs (Fig. 7C). However, the resting
membrane potential of the iNs was around 230 mV, indicating
an immature state (Supplementary Material, Fig. S8B). Owing to
the extremely low conversion efficiency (,1%, Supplementary
Material, Fig. S8A), the miniature EPSCs were not detected in
the iNs. The analysis of properties of evoked action potentials
and spontaneous action potentials was prevented by the immature state of the iNs. These results demonstrate that we can directly convert SMEI patient and mild febrile seizures patient
fibroblasts into neurons possessing basic function.
SMEI and mild febrile seizures patient converted neurons
showed delayed inactivation of sodium channels
Physiological properties of the converted neurons from SMEI
and mild febrile seizures patients and the unaffected control
were studied under identical conditions using whole-cell patch-
clamp recordings. Figure 8A illustrated the representative
whole-cell currents from iN cells converted from the patient
cell lines and the unaffected control. In general, the F1415I
SMEI iNs exhibited rapid activation which was similar to
control iNs. However, F1415I iNs had impaired inactivation
properties (Fig. 8A and B). Sodium channels of iNs from the
F1415I patient displayed delayed inactivation compared with
the control, whereas neurons from the Q1923R mild febrile
seizures patient displayed no obvious differences from the
control. In all cases, sodium channels were confirmed to be
TTX-sensitive (Fig. 8C).
We further examined the electrophysiological properties of
sodium channels in neurons converted from SMEI patient fibroblasts. Current – voltage relationships and voltage dependence of
activation and inactivation of the differentiated neurons were
examined. We observed obvious delays in the inactivation of
sodium channels for F1415I iNs, so we decided to test the
current – voltage relationship first. I – V curves from F1415I
sodium channels showed reverse potential shifts to depolarization when compared with the control. Similar to the results
obtained in the iPSC-derived neurons, current– voltage relationships of sodium channels showed delayed inactivation in iNs
converted from F1415I fibroblasts (Fig. 8D).
The inactivation of sodium channels was examined in converted neurons from F1415I and Q1923R patients as well as
the control. In F1415I patient neurons, inactivation curves
were shifted in the depolarized direction. In Q1923R patient
neurons, this shift was less noticeable (Fig. 8E). The V50
value for inactivation in control iNs was 258.65 + 0.48 mV
(n ¼ 21). Significant depolarization shifts in the inactivation
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Figure 4. Action potential characteristics of neurons derived from SMEI iPSCs. (A) The mode of evoked action potentials in iPSC-derived neurons from F1415I and
Q1923R patients and the unaffected control. Action potentials of F1415I patient showed higher frequencies and larger amplitude patterns compared with unaffected
control. Action potentials of Q1923R patient showed slightly higher frequencies and larger amplitude patterns compared with unaffected control. (B) The histogram
shows average amplitude of evoked action potentials (AP) in iPSC-derived neurons from F1415I and Q1923R patients and the unaffected control. Numbers 1 and 2
represent individual F1415I, Q1923R and WT iPSC lines, respectively. The action potentials of F1415I and WT iPSC-derived neurons had significant differences,
which were examined by Student’s t-test (∗∗∗ P , 0.001). (C) The numbers of evoked action potentials (AP) elicited by depolarizing current steps in iPSC-derived
neurons from F1415I and Q1923R patients and the unaffected control.
Human Molecular Genetics, 2013
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curves were detected for F1415I iNs (V50: 251.02 + 2.05 mV,
n ¼ 23) and Q1923R (V50: 255.37 + 0.99 mV, n ¼ 25). These
results indicate a delayed inactivation of voltage-gated sodium
channels in F1415I and Q1923R iNs, which is similar to the
results obtained with iPSC-derived neurons. Collectively,
these results demonstrate that delayed inactivation of sodium
channels can be detected in directly converted neurons from
SMEI patient fibroblasts, and relative minor impairment of
sodium channels was observed in directly converted neurons
from mild febrile seizures patient fibroblasts.
Comparison between the two methods in obtaining patientspecific neurons revealed that the percentage of neurons differentiated from the iPSCs was .80%, whereas the percentage of
iNs was ,1% (Supplementary Material, Fig. S8A). Furthermore, the neurons derived from iPSCs possessed resting membrane potentials around 255 mV, which is comparable with
neurons in brain slices, whereas the resting membrane potentials
of iNs is around 230 mV, indicating an immature state (Supplementary Material, Fig. S8B). The results demonstrated that although the derivation of neurons from iPSCs takes longer (3
months from human fibroblasts) than direct conversion from
fibroblasts (1 month), neurons derived from iPSCs exhibit
higher efficiency and better maturation.
DISCUSSION
The pathophysiology of seizures is believed to be an alteration in
the normal balance of inhibition and excitation. A hyperexcitable state of neuronal network which causes seizures can result
from increased excitatory synaptic neurotransmission and/or
decreased inhibitory neurotransmission. The major excitatory
neurotransmitter is glutamate and the major inhibitory neurotransmitter is GABA. Hyperexcitability of glutamatergic
neurons and/or hypoexcitability of GABAergic neurons results
in the hyperexcitability of the neuronal network (30).
Several approaches have been developed to investigate the
pathological mechanism of SMEI. Expression of mutated
genes in cell lines provided an in vitro tool to study the ion
channel properties (31,32). Investigations with the cell lines
transfected with the mutated SCN1A found delayed inactivation
of sodium currents. However, although the transfected cell lines
possessed ion channels on their cell membranes, they were not
capable of recapitulating neuronal phenotypes and functions.
Knockout mouse models have been widely used to model epilepsy in vivo (5). The mouse model possessed the neuronal
circuit that is disrupted in SMEI and reflected the disease phenotype in some aspects. Knockout mouse model showed reduced
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Figure 5. Spontaneous action potential characteristics of SMEI iPSC-derived neurons. (A) Spontaneous firing of iPSC-derived neurons recorded in current clamp
mode from Q1923R, F1415I patients and the unaffected control. Spontaneous action potentials from Q1923R and F1415I cells showed higher frequencies and epileptiform patterns. (B) The frequency of spontaneous action potentials (AP) in iPSC-derived neurons from F1415I and Q1923R patients and the unaffected control. The
frequency of F1415I and Q1923R iPSC-derived neurons showed significant differences versus the unaffected control (∗∗∗ P , 0.001). (C) Spontaneous action potentials of neurons derived from SMEI iPSC recorded in current clamp mode show PDS.
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Human Molecular Genetics, 2013
sodium currents in the GABAergic neurons. However, mouse
models are limited because of discrepancies between mice and
humans in terms of disease progression and the underlying
disease mechanisms. To date, the precise mechanism of SMEI
remains controversial and elusive.
The development of iPSCs provides a revolutionized approach to investigate the pathological mechanism of many diseases, especially neuronal diseases. We demonstrated the
modeling of SMEI, using functional neurons obtained using
iPSC technology and technology for direct conversion of fibroblasts to neurons. Further analysis on the sodium channels of
the neurons revealed delayed inactivation in iPSC-derived
neurons from a patient with an F1415I mutation. Moreover,
examination of the spontaneous firing of iPSC-derived neurons
from patients showed action potentials typical of epilepsy. The
abnormal firing observed in epileptic neurons could be reproduced by in vitro modeling of the neurons differentiated from
the patient iPSCs.
At present, two models have been proposed to interpret the
pathological mechanism of SMEI. In the first model, loss of
function in the SCN1A genes causes reduced sodium current
in inhibitory GABAergic neurons, leading to insufficient inhibition, hyperexcitability in the neuronal network and epilepsy (5).
The second model proposes a gain-of-function mutation in
which persistent activation of sodium currents leads to hyperexcitability in neurons, causing epilepsy (6). Based on the diversity
of the mutations in SCN1A in SMEI patients, different
MATERIALS AND METHODS
Mutation detection
Genomic DNAs of fibroblasts from patients and patient-specificiPSCs were extracted with the Tissue and Cells Genomic DNA
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Figure 6. The hyperexcitability of the SMEI patient iPSCs could be alleviated by
phenytoin. (A) The whole-cell sodium currents of SMEI iPSC-derived neurons
before and after the treatment of phenytoin. (B) The action potentials evoked
by injected current step at 10 pA of SMEI iPSC-derived neurons before and
after the treatment of phenytoin. (C) Spontaneous action potentials of SMEI
iPSC-derived neurons showed PDS, which was eliminated by treatment of
phenytoin.
pathological mechanisms might be responsible for a common
epileptic phenotype. An explanation for inconsistencies in previous studies is that various mutations change the conformation of
the SCN1A protein in various ways, resulting in diverse functional consequences. Here, we observed a gain-of-function
effect for both F1415I and Q1923R mutations reflected by the
epileptiform discharges from iPSC-derived neurons. A change
in electrophysiological behavior of sodium channels was the
major cause of the epileptic phenotype with the F1415I mutation
but was relatively minor for the Q1923R mutation. The results
are in accordance with the different locations of the mutation
on SCN1A. The mutation lies in the linker domain between S5
and S6 in SMEI patient, which is critical for the formation of
pore, and lies in the C terminal in mild febrile seizures patient,
which is considered to be less important (Supplementary Material, Fig. S1) (33). Our results indicate that at least a portion of
SMEI patients may have persistent sodium channel activation
that causes neurons to become hyperexcitable. Moreover, the severity of impairment of sodium channels in the two patients is
consistent with the severity of the symptoms.
Phenytoin is considered to inhibit seizure by decreasing highfrequency repetitive firing of action potentials through enhancing
sodium channel inactivation (34). Here, we applied phenytoin to
the SMEI iPSC-derived neurons. Reduced sodium currents and
action potentials were detected and also the epileptic firing was
eliminated. The results demonstrated that the neurons derived
from iPSCs can respond to antiepileptic drug and thus could be
used for drug screening.
Both iPSC-derived neurons and directly converted neurons
have been utilized in disease modeling. In this study, we used
both approaches to generate functional neurons from one
SMEI patient and one mild febrile seizures patient, and consistent phenotypic and electrophysiological results were obtained.
The technology for iPSC generation and neuron differentiation
is well established and has high efficiency (.80%) for neuron
differentiation. Compared with iPSC technology, the efficiency
for generating directly converted neurons remains very low
(,1%). We also found that the iNs in this experiment possessed
higher resting membrane potential, indicating the immature state
of the neuron. However, this technology can significantly
shorten the period of time required for generating functional
neurons from fibroblasts.
In conclusion, we obtained functional neurons from the differentiation of SMEI patient-specific iPSCs and from the direct
conversion of mild febrile seizures and SMEI patient fibroblasts.
The iPSC-derived neurons revealed mild-to-severe hyperexcitability in the glutamatergic neurons of mild febrile seizures
and SMEI patients; and also recapitulated the typical epileptic
phenotype; and could respond to an antiepileptic drug. These
neurons reveal that SMEI and mild febrile seizures arise due to
the altered firing of glutamagergic neurons, more importantly,
can potentially be applied for screening appropriated drugs for
personalized clinical treatment of SMEI.
Human Molecular Genetics, 2013
9
Extraction Kit (Biomed) following the instructions provided by
the producer. Each exon of the SCN1A was cloned from the
genomic DNA with PCR. Phusion DNA polymerase (Thermo
scientific) was utilized in the amplification reaction following
the instructions of the producer. Sequences of primers of each
exon were described previously (2). Amplification products
were sequenced with Sanger method (conducted in Invitrogen
Co.). The numbering of the mutation was started from the
ATG codon of the coding sequence of SCN1A.
SMEI and mild febrile seizures patient-specific iPSC line
derivation
Fibroblasts collected from SMEI patients containing the F1415I
mutation and mild febrile seizures patients containing Q1923R
mutation were utilized for iPSC induction. Plasmids (pMIG)
containing human OSKM were obtained from Addgene and
were transiently co-transfected with package plasmids into
293 T cells, using the Vigofect transfection reagent (Vigorous).
Viral supernatants were harvested after 48 h, filtered through a
0.45 mm low protein-binding cellulose acetate filter (Millipore)
and concentrated by centrifugation. A total of 5 × 104 fibroblasts
from each patient were incubated with virus for 24 h and then
seeded onto irradiated mouse embryonic fibroblasts (MEFs)
feeder cells and cultured for 5 days. iPSC culture medium was
substituted after 7 days. iPSC colonies were manually picked
and mechanically dissociated for the next passage.
iPSC maintenance
SMEI patient-specific iPSCs and control human ES cell lines
were cultured on irradiated ICR MEF feeders, using the following culture medium: DMEM/F12 (1:1) medium (Invitrogen)
containing 20% knockout serum replacement (Invitrogen),
1 mM non-essential amino acids (Invitrogen), 3 mM L-glutamine
(Invitrogen), 0.1 mM b-mercaptoethanol (Sigma-Aldrich),
100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen) and
8 ng/ml bFGF (R&D Systems). iPSCs and hESCs were either
passaged mechanically or passaged with 1 mg/ml collagenase
IV (Sigma-Aldrich).
Karyotype analysis
The iPSCs were incubated in culture medium containing
0.25 mg/ml colcemid (Invitrogen) for 4 h and were then harvested and incubated in hypotonic solution containing 0.4%
sodium citrate and 0.4% kalium chloratum (1:1, v/v) at 378C
for 5 min, before fixation with three washes of a mixture of
methanol:acetic acid (3:1, v/v). Slides were aged for 8 – 12 h at
658C and digested with 0.8% trypsin for 15– 30 s. Slides were
then stained with Giemsa for 10 min and air-dried. Twenty metaphase chromosome karyoschisis images were examined, and
5 G-band images were analyzed.
Neural differentiation of SMEI patient-specific iPSCs
SMEI and mild febrile seizures patient-specific iPSCs and
hESCs were passaged with collagenase, and cell clumps were
cultured on low attachment plates (Costar) in iPSC culture
medium without bFGF. The medium was replaced every
2 days and gradually changed to EB culture medium (DMEM
supplemented with 20% fetal bovine serum, 1× non-essential
amino acid, 50 mM b-mercaptoethanol, 100 U/ml penicillin
and 100 mg/ml streptomycin) by substituting 25% more of the
ES medium with EB medium each time the medium was
replaced. After 10 days, the EBs in suspension were collected
and plated on PDL/laminin-coated plates in neural rosettes
medium (DMEM/F12 supplemented with 1× N2, 100 U/ml
penicillin and 100 mg/ml streptomycin) with 25 ng/ml bFGF
(Peprotech). After 10 – 12 days, rosettes were manually picked
Downloaded from http://hmg.oxfordjournals.org/ by guest on July 4, 2013
Figure 7. Direct conversion of SMEI and mild febrile seizures patient fibroblasts into functional neurons. (A) Immunofluorescent staining of neurons converted from
the SMEI patient and mild febrile seizures patient fibroblasts lines show the expression of neuronal markers including TUJ-1, MAP2, NEUN, VGlUT1 and GAD67.
Scale bar: 50 mm. (B) Whole-cell currents recorded following depolarizing voltage steps (280 to 60 mV) in neurons converted from mild febrile seizures patient
fibroblasts. (C) Action potentials evoked following injection of current steps recorded from neurons converted from mild febrile seizures patient fibroblasts.
10
Human Molecular Genetics, 2013
and plated on PDL/laminin-coated plates in neural rosettes
medium. The first passage was performed with 0.05% trypsin/
EDTA (Invitrogen), and the following passages were performed
with Accutase (Sigma). Neuronal differentiation was induced by
changing the neural rosettes medium to neuron culture medium
consisting of Neurobasal (Invitrogen) supplemented with 1×
B27 (Invitrogen), 10 ng/ml BDNF (Peprotech), 10 ng/ml
GDNF (Peprotech), valproic acid (10 mM, Sigma), insulin-like
growth factor 1 (IGF-1, 10 ng/ml, Peprotech), cAMP (1 mM,
Sigma), 10 ng/ml NT3 (Peprotech), 100 U/ml penicillin and
100 mg/ml streptomycin. The neuron medium was refreshed
every 2 to 3 days. The cells were cultured in neuron medium
on human astrocytes layer (Sciencell) for more than 1 month
to obtain full maturation.
Direct conversion of SMEI patient fibroblasts to neurons
SMEI patient fibroblasts were cultured in fibroblast culture
medium (DMEM with 10% FBS, 1× glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin). Plasmids (pFUW containing ASCL1, BRN2, MYT1L, NEUROD1, OLIG2, ZIC1 and
rtTA; obtained from Addgene) were transiently co-transfected
with package plasmids into 293 T cells, using Vigofect transfection reagent (Vigorous). Viral supernatants were harvested after
48 h, filtered through 0.45 mm low protein-binding cellulose
acetate filters (Millipore) and concentrated by centrifugation.
A total of 5 × 104 fibroblasts from each patient were incubated
with virus for 6 –8 h. After virus infection, cells were cultured
in fibroblast culture medium with 1 mg/ml doxycycline for
Downloaded from http://hmg.oxfordjournals.org/ by guest on July 4, 2013
Figure 8. Delayed inactivation of sodium channels in directly converted neurons from F1415I and Q1923R patients. (A) Representative sodium currents from neurons
induced from F1415I, Q1923R and unaffected control cells. Whole-cell currents were recorded following depolarizing voltage steps (280 to 60 mV). (B) Typical
sodium currents from neurons induced from F1415I, Q1923R and unaffected control cells. The currents were normalized. (C) Sodium currents could be eliminated in
the iNs (directly converted neurons from fibroblasts) with TTX (1 mM). (D) Current– voltage relationships of whole-cell currents in directly converted neurons
obtained from F1415I, Q1923R and unaffected control cells. (iN, directly converted neurons from fibroblasts). (E) Voltage dependence of inactivation of sodium
currents from iNs obtained from F1415I, Q1923R and unaffected control cells. Currents were normalized to the peak current amplitude. (iN, directly converted
neurons from fibroblasts).
Human Molecular Genetics, 2013
2 days. The medium was then changed to neuron culture medium
with 1 mg/ml doxycycline, and the resulting neurons were maintained in culture on human astrocytes layer for more than
1 month.
Immunofluorescent staining
Teratoma formation
Pluripotent cells were harvested by collagenase IV treatment,
collected in tubes and centrifuged, and the pellets were suspended in DMEM/F12. Cells from one 60 mm dish were injected
subcutaneously into the dorsal flank of a recipient NOD/SCID
mouse (Jackson Laboratory). Paraffin sections of formalin-fixed
teratoma specimens were prepared 9 weeks after injection.
Analysis of H&E-stained tissue sections was performed for
each specimen.
Electrophysiology
Electrophysiological experiments were performed using directly converted iNs and iPSC-derived neurons. Whole-cell patchclamp recordings in either voltage or current clamp mode were
conducted to measure voltage-activated sodium/potassium currents and action potentials. An EPC-10 (HEKA) amplifier was
used for recording the electrophysiological signals. Data were
acquired using the Patchmaster software (HEKA) and analyzed
using Igor pro (Wavemetrics). Glass micropipettes (2.0 –
4.0 MV) containing a solution of 130 mM K+-gluconate,
20 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 4 mM Mg2ATP,
0.3 mM Na2GTP, 10 mM Na2+-phosphocreatine (at pH 7.3,
290– 310 mOsm) and 1% biocytin (Invitrogen) were used for
patch-clamp recordings. The bath solution contained 140 mM
NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES
and 10 mM glucose (at pH 7.4). All electrophysiological experiments were performed at room temperature.
Microarray analysis
Total RNA was extracted from SMEI patient fibroblasts, and
SMEI iPSCs and H9 hESCs after induction using Trizol
reagent (Invitrogen). Affymetrix GeneChipw Human Genome
U133 plus 2.0 (Affymetrix, Inc.) was used, and all experiments
were performed at Beijing Capitalbio Corporation. Data were
analyzed using the GCOS 1.4 software provided by Affymetrix.
Signal values of probes presented in two samples were plotted in
a scatter graph. Pearson’s correlation coefficient (R) between
samples was calculated using Excel. The microarray data sets
have been deposited in NCBI’s Gene Expression Omnibus
under accession number GSE46472.
AUTHORS’ CONTRIBUTIONS
J.J. and Y.Y.: conception and design, collection and/or assembly
of data, data analysis and interpretation and manuscript writing.
Y.S., J.C., R.G., Y.F, H.Y. and W.L.: provision of study material
and data analysis and interpretation. X.-F.S. and S.G.: conception and design, financial support, data analysis and interpretation, manuscript writing and final approval of the manuscript.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG online.
ACKNOWLEDGEMENTS
We are grateful to Dr Li Yingji from ICE Bioscience Co. for his
assistance on electrophysiological experiments. We are grateful
to our colleagues in our laboratory for their help with experiments and in the preparation of this manuscript.
Conflict of Interest statement. None declared.
FUNDING
This project was supported by the Ministry of Science and Technology (grants 2010CB944900 and 2011CB964800 to S.G.) and
NSFC-Guangdong Joint Fund (U1132005 to X.-F. S.).
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