Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors
Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors
Zhi Zhou 0
Kazuhisa Kohda 0
Keiji Ibata 0
Jun Kohyama 0
Wado Akamatsu 0
Michisuke Yuzaki 0
Hirotaka James Okano
Erika Sasaki 0
Hideyuki Okano 0
0 Department of Physiology, Keio University School of Medicine , 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 , Japan
Background: The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets. Results: Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state. Conclusions: Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets.
Common marmoset; Direct reprogramming; Induced neuronal cells; Transcription factor; Regenerative medicine; Disease modeling; Cell-fate plasticity; Transdifferentiation
The common marmoset (Callithrix jacchus) is a New
World primate that has recently attracted considerable
attention as a non-human primate model for
biomedical research . Specific features of the common
marmoset are its small size, ease of handling, high fertility,
early sexual maturity, its similarity of physiological
properties with humans, drug metabolism, and
neurophysiological functions . Thus far, transgenic mice
modeling human neurodegenerative diseases have
contributed to disease research and drug development.
However, none of them have succeeded in faithfully
recapitulating the full spectrum of disease pathologies
observed in humans [2,3]. In a recent report by our group,
transgenic marmosets with germline transmission were
successfully generated for the first time by lentiviral
vector-mediated gene transfer . For these reasons,
our novel transgenic non-human primate models may
be suitable for studying human diseases, particularly
those that are neurodegenerative, such as Alzheimers
and Parkinsons disease. These in vivo models are
expected to faithfully recapitulate pathophysiology of
human diseases, and thus provide for the missing link
between mouse and human disease research with
subsequent drug development. However, results of studies
from these models may require an extended period
because of the longer lifespan of common marmosets
compared with mice . Moreover, detailed in vitro
analyses using primary neuronal cultures of the affected
area of the common marmoset transgenic models are
Recent studies using human neuronal cells derived
from either pluripotent stem cells or somatic cells have
succeeded in modeling human neurological disorders
in vitro [6,7]. These results prompted us to develop a
convenient and rapid method for obtaining common
marmoset neuronal cells from accessible somatic cells.
Therefore, we focused on somatic cell reprogramming
technology, including induced pluripotent stem (iPS)
cell and direct conversion technology [8,9]. However,
few studies have succeeded in generating common
marmoset iPS cells from neonatal skin fibroblasts, fetal liver
cells, and adult bone marrow-derived cells [10-12].
Moreover, only a few protocols exist for obtaining functional
common marmoset neuronal cells from pluripotent stem
cells . Furthermore, little attention has been given to
the direct conversion technology of common marmoset
dermal fibroblasts into neuronal cells thus far. Therefore,
in the present study, we aimed to generate common
marmoset neuronal cells directly from dermal fibroblasts. Our
results provide the first line of evidence for the generation
of electrophysiologically functional neuronal cells from
common marmoset somatic cells by defined neuronal
Results and discussion
Validation of the lentivirus-mediated overexpression of
neuronal transcription factors
Recently, generation of induced neuronal (iN) cells was
reported using mouse and human dermal cells [9,14]. In
the present study, we used a set of neuronal
transcription factors for human iN cells on common marmoset
embryonic skin fibroblasts (cjF) isolated from embryonic
day 91 (E91) embryos to determine whether common
marmoset somatic cells could be converted into
neuronal cells. We first confirmed transgene expression in
the mouse fibroblast cell line, NIH3T3, by infecting
these cells with lentiviral vectors coding the neuronal
transcription factors: ASCL1, BRN2, MYT1L, and
NEUROD1 , under the control of tetracycline response
element (TRE) together with reverse tetracycline
transactivator (rtTA)-expressing vector. Upregulation of transgenes
in NIH3T3 cells after doxycycline (dox) treatment was
confirmed by immunocytochemistry (Additional file 1).
Generation of neuron-like cells from common marmoset
To address whether cjFs, which were immunonegative for
the neural progenitor marker, SRY (sex determining region
Y)-box 2 (Sox2) (data not shown), can be converted into
cjiN cells, cjFs were infected with these lentiviral vectors at
0 day in vitro (div) (Figure 1A). Synapsin reporter-positive
mouse iN cells have been shown to be more functionally
mature than negative cells . Therefore, the reporter
lentivirus, which expresses fluorescent protein (enhanced
green fluorescent protein or DsRed) under the control of
the human synapsin I promoter, was used in the present
study to monitor neuronal conversion of cjFs [16-18]
(Figure 1A). When cjFs were treated with dox at 1 div to
induce neuronal conversion (Figure 1B), synapsin
reporterpositive cells with typical neuronal morphologies were
observed (Figure 2A, B). However, cjFs without dox
treatment did not generate synapsin reporter-positive cells
(Figure 2A). Notably, the morphology of synapsin
reporterpositive cells resembled fibroblasts at 9 div and then changed
into neuronal ones during reprogramming (Figure 2A, B).
The reprogramming efficiency was monitored by the
number of synapsin reporter-positive cells with neuronal
morphology, and depended on the concentration of dox
yielding 0.3 0.1, 21.8 0.8, or 32.6 2.6 cjiN cells/cm2 at
16 div when treated with 0, 1, or 2 g/mL dox,
respectively (Figure 2C) (P*** < 0.001, P## < 0.01, n = 4). However,
the overall induction efficiency was much lower (<1%)
than those in previous studies using mouse and human
fibroblasts [9,14]. This discrepancy was probably due to the
low infection efficiency of lentivirus in cjFs compared with
those in mouse and human fibroblasts. Previous studies
have also shown that polycistronic vector, micro RNAs
and small molecules can facilitate iN cell induction
Figure 1 Schematic overview of the experimental procedure for generation of common marmoset induced neuronal cells from
common marmoset embryonic skin fibroblasts. (A) Common marmoset embryonic skin fibroblasts (cjFs) were infected with drug-inducible
lentiviral vectors coding a set of induced neuronal (iN) cell factors: ASCL1, BRN2, MYT1L, and NEUROD1. Synapsin reporter lentivirus expressing
green fluorescent protein (GFP) or DsRed under the human synapsin I promoter was used to monitor neuronal induction. The lentiviral vector
that stably drives rtTA expression under the EF1 promoter was transduced to induce the expression of iN-factors. (B) Time course for common
marmoset induced neuronal (cjiN) cell induction. Lentivirus infection was conducted at 0 day in vitro (div) in fibroblast medium containing 10%
fetal bovine serum, and cells were exposed to doxycycline (dox) at 1 div for cjiN induction. The culture medium was then replaced at 3 div with
dox-containing neural meium composed of N2B27 medium, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF). For calcium
imaging, the cAMP analog, 8-(4-Chlorophenylthio) adenosine 3, 5-cyclic monophosphate (8-CPT; 100 M) , was added to promote neuronal
maturation and survival.
[7,19,20]. Therefore, the induction conditions of cjiN cells
may be optimized in the future study to enhance the
conversion efficiency. Nevertheless, our results show that the
four-factor set of neuronal transcription factors is
sufficient to convert cjFs to cjiN cells.
The duration of dox treatment was critical for neuronal
Next, we investigated the required duration of exogenous
neuronal transcription factor expression to drive neuronal
transdifferentiation. Therefore, we examined the exposure
time of dox and the cjiN induction efficiency (Figure 2D).
Treatment with dox from 13 div promoted the
conversion of cjFs into synapsin reporter-positive cells with
neuronal morphology (Figure 2E). Similarly, a previous study
has shown that the endogenous neuronal transcriptional
factor network is activated 48 h after dox treatment .
Our findings also revealed that the induction efficiency at
23 div depended on the exposure time of dox (Figure 2F).
The treatment of cjFs with dox from 111 div followed by
a culture without dox treatment from 1123 div generated
112.1 28.1 cjiN cells/cm2 (Figure 2F). However, cells
cultured with dox from 13 div followed by a culture without
dox treatment from 323 div generated only 10.6 5.4
cjiN cells/cm2 (Figure 2F). The efficiency of the former
group was significantly (P*** < 0.001, n = 4) higher (more
than 10-fold) compared with the latter group (Figure 2F).
These results indicate that a longer expression of
exogenous neuronal transcriptional factors is required for
efficient lineage conversion of somatic cells.
cjiN cells expressed a subset of neuronal genes and
To characterize cjiN cells as neuronal cells, we
performed reverse transcription-polymerase chain reaction
(RT-PCR) analysis using bulk RNA samples, including
the remaining synapsin reporter-negative cells to explore
the expression of neuronal genes (see Table 1 for the
primer sets used). We detected an upregulation of neuronal
genes (Figure 3), which included cytoskeletal markers
Figure 2 Generation of iN cells. (A, B) Transgene-dependent conversion of common marmoset fibroblasts into neuronal cells, in which
synapsin reporter activation and morphological changes were dependent on the exogenous transcription factor and time. Cells became synapsin
reporter-positive accompanied by a gradual morphological changes into neuronal cells, both effects of which were not found in cells cultured in
neural medium without dox at 21 div. (B) Magnified images of Figure 1A showing synapsin reporter-positive cells with morphological changes
from fibroblasts to neuronal cells. (C) Counts of synapsin reporter-positive cells with neuronal morphology showed a dox-dependent increase in
their number (positive cells with fibroblast morphology were excluded from the counts). P*** < 0.001, P## < 0.01 (one-way ANOVA followed by
Tukeys test). (D-F) The effect of sustained dox treatment on the production of cjiN cells. (D) Cells were treated with dox at 1 div until 3, 5, 7, 9 or
11 div and then maintained without dox until 23 div. (E) Synapsin reporter-positive cells with neuronal morphology were observed in all
treatment group. (F) Cell counts revealed that a longer treatment time of dox increased the number of cjiN cells. Although dox treatment from
13 div sufficiently promoted cjiNs, a longer treatment time increased their number. P*** < 0.001 (one-way ANOVA followed by Tukeys test).
Scale bar; 200 m.
(MAP2, DCX and TUBB3), synaptic vesicle markers (SYN1
and VGLUT1), and cation channel-related genes (SCN1A,
GRIN1 and GRIA1) in cjiN cells at 21 div (Figure 3).
However, expression of these neuronal genes was not detected
in cjFs at 0 div (Figure 3). These results were in line with
the concentration-dependent induction efficiency of dox
in Figure 2C. We also detected endogenous expression of
BRN2 and NEUROD1 in cjiN cells at 21 div (Figure 3),
indicating that ectopic expression of neuronal transcription
factors activated the endogenous neuronal program. This
effect may have caused neuronal transdifferentiation from
somatic cells [9,14,21]. Thus, ectopic neuronal differentiation
Table 1 Primer sets used in RT-PCR analysis
signals are likely to work together with the endogenous
neuronal program to efficiently convert non-neuronal
cells into neuronal cells .
To further characterize the property of cjiN cells, we
performed immunocytochemistry for the neuronal markers.
The results showed that most synapsin reporter-positive
cells also expressed (>88%) the pan-neuronal marker,
microtubule-associated protein 2 (MAP2) (data not shown).
This result indicated that the synapsin reporter using the
sequence of human synapsin I promoter was also
functional in common marmoset cells, and was thus a reliable
reporter for neuronal conversion, as previously reported
[15,16]. In cjiN cells at 43 div, MAP2-negative cells were
found to be negative for glial fibrillary acidic protein and
only weakly positive for -smooth muscle actin (data not
shown), raising the possibility that they were partially
reprogrammed cells. The MAP2- and synapsin
reporterpositive cells also showed immunoreactivity for the synaptic
Figure 3 Neuronal marker gene expression in cjiN cells. Dox
treatment upregulated cytoskeletal (MAP2, DCX, and TUBB3) and
synaptic vesicular (SYN1 and VGLUT1) marker genes, and cation
channel-related genes (SCN1A, GRIN1 and GRIA1). The results also
showed the activation of endogenous BRN2 and NEUROD1 genes.
vesicle marker, synaptophysin, at 38 div (Figure 4A).
However, immunostaining for synaptophysin and PSD95
revealed that these cells were unlikely to make synapse
structures at 38 div (data not shown). Although the cjiN
cells expressed neuronal marker genes and proteins,
they did not appear to be mature enough to make
synaptic contacts themselves. However, a future study may
facilitate synaptic formation by improving induction
efficiency and by co-culturing with astrocytes [9,14].
The majority of cjiN cells were glutamatergic
To characterize the neurotransmitter phenotype of iN
cells, we examined the expression of neurotransmitters
in MAP2-positive cjiN cells. Immunostaining at 38 div
for vesicular glutamate transporter 1 (vGlut1) and gamma
aminobutyric acid (GABA) revealed the presence of both
excitatory glutamatergic and inhibitory GABAergic
neuronal cells, respectively (Figure 4B). Counts of
vGlut1 and GABA-positive cjiN cells showed a
significantly (P*** < 0.001, n = 4) greater number of
vGlut1positive cells (39.2 1.9/cm2) than GABA-positive cells
(16.4 2.8 cells/cm2) (Figure 4C). These results are in
accordance with previous studies showing that the
majority of iN cells are excitatory cells [7,14,22]. Our
findings thus indicate that iN induction may be feasible in
the future for the in vitro analysis of the transgenic
common marmoset model of Alzheimers disease, in
which forebrain excitatory neurons are expected to be
affected. Moreover, our cjiN cell induction protocol is
likely advantageous over the previously reported
neuronal differentiation protocol which used common
marmoset embryonic stem (ES) cells and iPS cells, because
the ES/iPS cell-derived neural precursor cells showed
caudal identity . Thus far, several groups have
succeeded in generating reprogrammed neuronal cells
with specific neuronal subtypes, such as dopaminergic
neurons and motor neurons [23-26], which implicate the
cell fate plasticity of terminally differentiated somatic cells.
Figure 4 Immunocytochemistry of cjiN cells. (A) Synapsin reporter-positive-cjiN cells expressed microtubule-associated protein 2 (MAP2) and
synaptophysin. (B) cjiN cells were vesicular glutamate transporter 1 (vGlut1)- and gamma-aminobutyric acid (GABA)-positive, thereby displaying a
heterogeneous pool of excitatory and inhibitory neurons, respectively. (C) A significantly greater number of vGlut1-positive cells was generated
compared with GABA-positive cells. P*** < 0.001 (Students t-test). Scale bar; 50 m.
Our success in reprogramming common marmoset
somatic cells into excitatory and inhibitory neuronal cells using
defined iN factors may therefore provide great promise in
the future for generating specific subtypes of neuronal
cells with specific sets of neuronal transcription factors.
cjiN cells were functional as matured neurons
To further confirm the successful conversion of cjFs into
functional neuronal cells, we performed calcium imaging
analysis. cjiN cells cultured with dox were incubated
with the calcium indicator, Fluo-4 AM , followed by
response recordings. The intracellular calcium level
([Ca2+]i) in cjiN cells at 15 div was increased in cjiN cells
perfused with 80 mM of KCl, which was then decreased
by washout (Additional file 2). Furthermore, electrical
field stimulation on cjiN cells at 28 div increased [Ca2+]i,
which was reversibly blocked by the voltage-gated
sodium channel blocker, tetrodotoxin (0.2 M) (Figure 5),
suggesting that the increase in [Ca2+]i was likely evoked
by action potentials through voltage-gated sodium
channels. These results showed that cjiN cells derived from
cjFs were functionally comparable to common marmoset
ES cell-derived neuronal cells , and thus strongly
suggest that reprogramming of common marmoset somatic
cells generates functional neuronal cells. Moreover,
electrophysiological analyses of cjiN cells at 2942 div
revealed that action potentials were elicited by
membrane depolarization in current-clamp mode in 17 out
of 21 cjiN cells (81.0%) (Figure 6A and Additional file 3).
Among these 17 cjiN cells, 7 cjiN cells (41.2%) generated
a single action potential and the remaining 10 cjiN cells
(58.8%) generated repetitive action potentials (Figure 6A
and Additional file 3). In the voltage-clamp mode, both
fast-activating and inactivating sodium currents and
outward currents were observed (Figure 6B). The resting
membrane potentials of cjiN cells ranged between -26
and -55 mV, with a mean SEM of -38.1 2.0 mV
(Figure 6C and Additional file 3). This mean value was
significantly (P*** < 0.001) lower than that of cjFs (-16.4
0.6 mV) with a range between -13.1 and -19.0 mV
(Figure 6C and Additional file 3). Overall, our patch
clamp recordings showed that cjiN cells are functional
cjiN cells were directly converted from cjFs without
passing through a proliferative state
To determine whether cjiN cells were directly converted
from cjFs without passing through a proliferative neural
Figure 5 Increase in the intracellular calcium level upon electrical field stimulation. (A) cjiN cells (synapsin reporter-positive with a neuronal
morphology). (B, C) Intracellular calcium level ([Ca2+]i) in cjiN cells was measured by the intensity of Fluo-4 fluorescence. Electrical field stimulation
(40 Hz for 5 seconds) induces a robust elevation of [Ca2+]i in some synapsin reporter-positive cells. The increase in [Ca2+]i was reversibly suppressed by
the specific voltage-gated sodium channel blocker, tetrodotoxin. Scale bar; 50 m.
Figure 6 Neuronal electrophysiological property of cjiN cells. (A) cjiN cells elicited repetitive action potential by membrane depolarization in
current-clamp mode. (B) cjiN cells showed both fast-activating and inactivating sodium currents and outward currents in voltage-clamp mode.
(C) cjiN cells exhibited significantly lower resting membrane potentials than cjFs. P*** < 0.001 (Mann-Whitney U-test).
progenitor-like cell state, 5-bromodeoxyuridine (BrdU;
10 M) was added to the media at 1, 3 or 5 div until 24
div (Figure 7A), and the percentage of double-positive
cells for BrdU and MAP2 among the MAP2
singlepositive cells was determined (Figure 7B, C). The result
showed that while 85.3 5.4% of MAP2-positive cells
incorporated BrdU when treated from 124 div, only 30.1
9.0% (P** < 0.01) and 1.5 1.5% (P*** < 0.001) of
MAP2positive cells incorporated BrdU when treated from 324
and 524 div, respectively (Figure 7C). This result
indicates that most of the cells that were destined to be cjiN
cells became postmitotic at the early phase of the
reprogramming process, suggesting that cjiN induction is a
direct process unless cells pass through a proliferative neural
progenitor-like cell state, from which neuronal cells can
be differentiated. In the present study, however, no
Sox2positive cells were found during 25 div, while
MAP2positive cells were present at 21 div (data not shown),
indicating that the induction of neural progenitor-like
cells is unlikely during 1-5 div. Therefore, these results
show that cjiN cells are directly converted from cjFs
without passing through proliferative neural progenitor
In the present study, we established an in vitro method to
convert common marmoset somatic cells into functional
neuronal (i.e. cjiN) cells. The majority of the cjiN cells were
Figure 7 Cells destined to be cjiN cells became postmitotic at the early phase of the reprogramming process. (A) Time course for the
BrdU incorporation experiment. BrdU (10 M) was added into the culture medium at 1, 3 or 5 div until cells were fixed at 24 div. (B, C)
Immunocytochemistry against MAP2 and BrdU revealed that when cells were treated with BrdU at 1 div until 24 div, 85.2 5.4% of cjiN cells
were BrdU + MAP2+, while when treated at 3 or 5 div until 24 div, only 30.1 9.0% or 1.5 1.5% of cjiN cells were BrdU + MAP2+, respectively,
showing that the cjFs became postmitotic soon after transgene expression. P*** < 0.001 P** < 0.01. Scale bar; 50 m.
vGlut1-positive excitatory neuronal cells and expressed the
neuronal marker genes: TUBB3, DCX, MAP2, SYN1,
VGLUT1, SCN1A, GRIN1 and GRIA1. Importantly,
cjFderived cjiN cells exhibited functional neuronal properties
and responded to exogenous stimulation. Overall, these
findings suggest that direct conversion technology may be
beneficial in rapid and robust screening of neuronal
phenotypes of transgenic common marmoset models of
human diseases and analyzing underlying molecular
mechanisms of diseases.
Common marmoset embryonic fibroblasts, NIH3T3 cells
and 293T cells were cultured in Dulbeccos modified
Eagles medium supplemented with 10% fetal bovine
serum, 100 U/mL of penicillin and 100 g/mL of
streptomycin (10% FP medium) at 37C with 5% CO2 incubation.
Molecular cloning and lentivirus production
cDNA entry clone of human ASCL1 [GenBank: NM_004316]
was purchased from DNAFORM (clone ID: 100006383,
Japan). cDNAs of human BRN2 [GenBank: NM_0056
04.3], MYT1L [GenBank: NM_015025.2] and NEUROD1
[GenBank: NM_002500.4] were cloned into
pENTR-DTOPO vector (Invitrogen, USA). Then cDNAs were
inserted into a self-inactivation human immunodeficiency
virus-1-based lentivirus construct, CSIV-TRE-RfA
(CSIVTRE-RfA-CMV-KT was kindly provided by Dr. Hiroyuki
Miyoshi (RIKEN BRC, Japan), and then modified by Dr.
Takuji Maeda (Nagoya University, Japan)), by LR reaction
(Invitrogen, USA). Similarly, reverse tetracycline
transactivator (rtTA) gene was inserted into
CSII-EF1-RfATK-HygR construct . The human synapsin I reporter
constructs, pCSC-hSynI-GFP  and
pHIV7-hSynIDsRed , were kindly provided by Dr. Fred H. Gage,
Salk Institute, USA, and Dr. Alysson R. Muotri, University
of California, USA, respectively. Besides,
CSIV-hSynI-GFPIRES2-NeoR and CSIV-hSynI-DsRed-IRES2-NeoR were
constructed in-house. These reporters were constructed
using CSIV-TRE-RfA-CMV-KT, pCSC-hSynI-GFP,
pHIV7hSynI-DsRed and pIRESneo3 (Clontech, USA) with
PCRand restriction enzyme-based method. For lentivirus
production, 293T cells were transfected with lentivirus
plasmid, pCAG-HIVgp and pCMV-VSV-G-RSV-Rev 
(kindly provided by Dr. Hiroyuki Miyoshi, RIKEN BRC,
Japan). After 16-20 h, supernatant was replaced by fresh
media followed by 48-72 h incubation. The virus containing
media were then collected and 0.45 m-filterated followed
by ultracentrifugation. The concentrated virus was
suspended in PBS and used in subsequent experiments.
Induction of common marmoset iN cells
Common marmoset embryonic fibroblasts were seeded
directly on culture ware at 1 104 cells/cm2. Twenty-four
hours later, the cells were infected with lentivirus in 10%
FP media containing polybrene (8 g/mL) (Sigma-Aldrich,
USA). After 1620 h in media containing lentivirus, the
cells were switched into fresh 10% FP medium containing
doxycycline (dox) (2 g/mL) to drive transgene
expression. Procedures for experiments determining the
sufficient concentration and duration of dox are shown in
the main text. After 48 h in 10% FP media with dox, the
media was replaced with dox-containing neural media
composed of N2B27 media , brain-derived
neurotrophic factor (BDNF) (10 ng/mL, R&D systems, USA)
and neurotrophin-3 (NT-3) (10 ng/mL, R&D systems,
USA). For calcium imaging, 8-(4-Chlorophenylthio)
adenosine 3, 5-cyclic monophosphate (8-CPT, 100 M,
Sigma-Aldrich, USA), one of the cAMP analogs ,
was also supplemented to promote neuronal
maturation. The media was changed every 23 days during
culture period. BrdU incorporation assay was performed
as previously described  incubating cells with BrdU
(10 , BD, USA) until cells were fixed.
Cells were fixed with 4% paraformaldehyde for 15
minutes at room temperature and then processed for
immunocytochemistry . Samples were rinsed with PBS
three times. Then, samples were incubated at 4C
overnight with the primary antibodies diluted in PBS
containing 5% of fetal bovine serum and 0.3% Triton X-100.
The primary antibodies used were as follows;
Synaptophysin (1:50000, Millipore, USA), MAP2 (1:1000,
SigmaAldrich, USA), MAP2 (1:500, Millipore, USA), vGlut1
(1:2000, Synaptic systems, Germany), GABA (1:1000,
Sigma-Aldrich, USA), PSD95 (1:500, Millipore, USA),
SMA (1:500, Sigma-Aldrich, USA), Sox2 (1:500, R&D,
USA), Ascl1 (1:200, BD, USA), Brn2 (1:200, Santa Cruz,
USA), Myt1L (1:500, Abcam, England) and NeuroD1 (1:500,
Santa Cruz, USA). After three washes with PBS, samples
were incubated with secondary antibodies conjugated with
Alexa-488, Alexa-555 and Alexa-647 (Invitrogen, USA).
Nuclei were counterstained with 4,
6-diamidino-2-phenylindole (DAPI; 1:1000, Dojindo, Japan). After washing with
PBS, samples were mounted on slides with FluorSave
reagent (Calbiochem, Germany) and examined under a
universal fluorescence microscope (Axioplan 2; Carl Zeiss,
Germany). For anti-BrdU staining (1:500, Abcam, England),
cells were treated with 1 N HCl in PBS for 30 minutes at
37C and then rinsed with PBS three times before primary
RNA isolation and reverse transcription-polymerase chain
Total RNA was isolated with RNeasy Micro Kit with
DNase I treatment (QIAGEN, Germany) and was used
to synthesize cDNA with ReverTraAce qPCR RT Kit
(TOYOBO, Japan) according to the manufacturers
instruction. RT-PCR was conducted using Ex Taq HS
(TAKARA, Japan) according to the manufacturers
instruction. Common marmoset ES (cjES) cells and cjES-derived
neurons were used as control . The primer sets used
are listed in Table 1.
Calcium imaging and electrical stimulation
Calcium imaging analyses were performed as described
previously . To load the calcium imaging dye, cells
were incubated with 1 M Fluo-4 AM (Invitrogen, USA)
in imaging solution consisting of 117 mM NaCl, 2.5 mM
KCl, 2 mM CaCl2, 2 mM MgSO4, 25 mM HEPES and
30 mM D-(+)-glucose, (pH 7.4), at 37C for 20 minutes,
followed by washing for 30 minutes in imaging solution.
Coverslips were placed on a custom-made field
stimulation chamber and mounted on the stage of a Nikon
Eclipse microscope with a 20 (NA 0.45) objective. Cells
were perfused at 2 ml/minute with the imaging solution
at room temperature with or without 0.2 M
tetrodotoxin (TTX; Alomone Labs Ltd., Israel). Images were
acquired at 2 Hz (500 millisecond exposure time) with
a cooled CCD camera (Andor iXon, DU897).
Extracellular field stimulation was performed with two parallel
platinum wires at 25 V/cm. Each stimulation was a
train of 500 microsecond pulses at 40 Hz for 5 seconds.
Images were analyzed with ImageJ software (NIH,
Electrophysiological recordings were performed as
described previously . Synapsin reporter-positive cjiN
cells were identified under an inverted microscope
(Diaphot-TMD 200; Nikon, Japan) and whole cell patch clamp
recordings were done using Axopatch 200B (Axon
Instruments, USA) at room temperature. The extracellular
solution composed of 117 mM NaCl, 2.5 mM KCl, 2 mM
CaCl2, 2 mM MgCl2, 15 mM D-Glucose and 20 mM
HEPES (pH 7.4 adjusted with NaOH, 304 mOsm) was
continuously perfused during recordings. Patch pipettes
had a resistance of 5-6 M filled with the intracellular
solution containing 130 mM K-gluconate, 1 mM CaCl2,
1 mM MgCl2, 10 mM EGTA, 10 mM Sucrose and 20 mM
HEPES (pH adjusted with KOH, 305 mOsm). In
voltage-clamp recordings, iN cells were held at -70 mV
and voltage steps (10 mV, 300 msec) were applied to elicit
voltage-activated currents. Action potentials were evoked
by injecting step currents (20-40 pA, 500 msec) in the
current-clamp mode. Data were digitized at 10 kHz
with a 2 kHz low-pass filter. Liquid junction potential
All data were expressed as means SEM. The statistical
significance of differences was analyzed by Students t-test,
Mann-Whitney U-test or one-way ANOVA followed by
Tukeys test using Graph Pad Prism5 software. Differences
of P < 0.05 were considered statistically significant.
Additional file 1: Doxycycline-dependent transgene induction in
NIH3T3 cells. Immunocytochemistry against Ascl1, Brn2, Myt1L and
NeuroD1 in a mouse fibroblast cell line, NIH3T3 cells, that were lentivirally
transduced with iN factors and treated with doxycycline from 14 div
revealed doxycycline-dependent transgene expressions. Scale bar;
Additional file 2: KCl perfusion increased the intracellular calcium
level. (A) cjiN cells (synapsin reporter-positive with a neuronal
morphology). (B, C) Intracellular calcium level ([Ca2+]i) in cjiN cells was
measured by the intensity of Fluo-4 fluorescence. KCl (80 mM) perfusion
caused a robust elevation of [Ca2+]i in some synapsin reporter-positive
cells. This increase was reversibly suppressed by washout. Scale bar;
Additional file 3: Electrophysiological parameters in cjiN cells at
cjiN cells: Common marmoset induced neuronal cells; cjF: Common
marmoset embryonic fibroblast; RT-PCR: Reverse transcription-polymerase
chain reaction; dox: Doxycycline; rtTA: Reverse tetracycline transactivator.
ZZ conceived the concept of this study, designed the experiments,
performed experiments and analyzed data. KI, KK and MY performed calcium
imaging assays and electrophysiology, and ZZ, KI, KK and MY analyzed data.
JK, WA, HJO and HO coordinated the study. ES prepared and provided
common marmoset embryonic fibroblasts. HO provided financial support for
the experiments. ZZ, JK and HO wrote the paper. All authors read and
approved the final manuscript.
We are grateful to memebers of Okano laboratory, Keio Unversity school of
medicine for helpful advice and discussions. Dr. Hiroyuki Miyoshi, RIKEN
BRC, Japan, kindly provided us with lentiviral vectors. Dr. Takuji Maeda,
Nagoya University, Japan, kindly provided us with modified lentiviral vector.
Dr. Fred H. Gage, Salk Institute, USA, and Dr. Alysson R. Muotri, University of
California, USA, kindly provided us with synapsin reporter lentiviral vectors.
This work is supported in part by a Grants-in Aid to Keio University from the
Global COE Program of the Ministry of education, Culture, Sports, Science and
Technology, Japan, Keio University Grant-in-Aid for Encouragement of Young
Medical Scientists, and Funding Program for World-leading Innovative R&D on
Science and Technology (FIRST) program of the Cabinet Office, Government of
Japan and the Japan Society for the Promotion of Science (JSPS).
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