Download Protein Interacting with Never in Mitosis A

Regenerative Endodontics
Protein Interacting with Never in Mitosis A-1
Induces Glutamatergic and GABAergic Neuronal
Differentiation in Human Dental Pulp Stem Cells
Young-Ah Cho, DDS, PhD,* Duck-Su Kim, DMD, PhD,† Miyeoun Song, PhD,*
Won-Jung Bae, MSD,* Soojung Lee, DMD, PhD,‡ and Eun-Cheol Kim, DDS, PhD*
Abstract
Introduction: The purpose of this study was to investigate the role of protein interacting with never in mitosis
A-1 (PIN1) in the neuronal or glial differentiation of human dental pulp stem cells (hDPSCs) and whether PIN1
can regulate determination of neuronal sub-phenotype.
Methods: After magnetic-activated cell sorting to separate CD34+/c-kit+/STRO-1+ hDPSCs, cells were cultured
in neurogenic medium. Differentiation was measured as
Nissl staining and marker protein or mRNA expression
by reverse transcriptase polymerase chain reaction,
immunofluorescence, and flow cytometric analysis.
Results: PIN1 mRNA levels were upregulated in a
time-dependent fashion during neurogenic differentiation. The PIN1 inhibitor juglone suppressed neuronal
differentiation but promoted glial differentiation as
assessed by the number of Nissl-positive cells and
mRNA expression of neuronal markers (nestin, bIIItubulin, and NeuN) and a glial marker (glial fibrillary
acidic protein). Conversely, overexpression of PIN1 by
infection with adenovirus-PIN1 increased neuronal differentiation but decreased glial differentiation. Moreover, PIN1 overexpression increased the percentage of
glutamatergic and GABAergic cells but decreased that
of dopaminergic cells among total NeuN-positive
hDPSCs. Conclusions: This is the first study to demonstrate that PIN1 overexpression induced glutamatergic
and GABAergic neuronal differentiation but suppressed
glial differentiation of hDPSCs, suggesting that enhancing PIN expression is important to obtain human glutamatergic and GABAergic neurons from hDPSCs. (J Endod
2016;42:1055–1061)
Key Words
Glial cells, human dental pulp stem cells, neural differentiation, neuron, PIN1
H
uman dental pulp
Significance
stem cells (hDPSCs)
PIN1 overexpression induced glutamatergic and
that originate from adult
GABAergic neuronal differentiation but suppressed
tooth pulp tissue are neuglial differentiation of human dental pulp stem cells,
ral crest–derived adult
which may serve as useful sources of neuro- and
stem cells capable of
gliogenesis in degenerative disorders of the CNS.
differentiating into a variety of cell lineages such
as odontoblasts, osteoblasts, adipocytes, and hepatocytes (1–3). In addition, hDPSCs
were able to differentiate into glial or neuronal cells on the basis of cellular
morphology, expression of the neural progenitor marker nestin, and expression of
the glial marker glial fibrillary acidic protein (GFAP) (4). Furthermore, in vivo studies
demonstrated that grafted dental pulp cells (DPCs) and stem cells from human exfoliated deciduous teeth (SHED) into the central nervous system (CNS) survived for several
months and expressed neuronal markers (5, 6). Culture-expanded hDPSCs exposed to
neurogenic medium (NM) differentiated into functionally active mature neurons both
in vitro and in vivo (7). In addition, engrafted hDPSCs integrated into the rat brain
showed neuronal properties not only by expressing neuron-specific markers but
also by exhibiting voltage-dependent sodium and potassium channels (8). The neural
progenitor marker nestin is more highly expressed in undifferentiated hDPSCs than in
human adipose stem cells or human skin-derived mesenchymal stem cells (MSCs) (9).
Human neurodegenerative diseases are caused by chronic and progressive loss of
specific types of neurons: cerebral cortex glutamatergic and basal forebrain cholinergic
neurons in Alzheimer’s disease (AD), midbrain dopaminergic neurons in Parkinson’s
disease (PD), and striatal GABAergic neurons in Huntington’s disease. An alternative
treatment approach in neurodegenerative disease is transplantation of easily expandable cells that have the capacity to generate those specific neuron types that are lost
in the different disorders (10). However, fetal or embryonic cell transplantation has
significant ethical, technical, and practical limitations. Recently, hDPSCs have been proposed as promising stem cells for nerve regeneration because of their close embryonic
origin and ease of harvest (1, 4).
DPCs from both rats and humans have the phenotypic characteristics of embryonic
dopaminergic neurons and protect dopaminergic neurons against the neurotoxin
6-hydroxy-dopamine in vitro (6). Differentiation of SHED into dopaminergic
neuron-like cells in vitro has been reported (11). Neurogenic-differentiated murine
DPSCs expressed markers for cholinergic, GABAergic, and glutamatergic neurons, indicating a mixture of CNS and peripheral nervous system cell types (12). Although hDPSCs
From the *Department of Oral and Maxillofacial Pathology and Research Center for Tooth and Periodontal Tissue Regeneration (MRC), †Department of Conservative
Dentistry, and ‡Department of Oral Physiology, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea.
Address requests for reprints to Dr Eun-Cheol Kim, Department of Oral and Maxillofacial Pathology and Research Center for Tooth and Periodontal Tissue Regeneration (MRC), School of Dentistry, Kyung Hee University, Seoul 130-701, Republic of Korea. E-mail address: [email protected]
0099-2399/$ - see front matter
Copyright ª 2016 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2016.04.004
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PIN1 and Neurogenic Differentiation in Dental Pulp Stem Cells
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exposed to either dopaminergic or motor NM undergo neuronal differentiation (13), regulatory controls for neuronal differentiation toward
specific neurons or glial cells remain to be elucidated in hDPSCs.
Protein phosphorylation of certain serine or threonine residues is a
central signaling mechanism in diverse cellular processes (14). Protein
interacting with never in mitosis A-1 (PIN1) is involved in cis-trans isomerization of phosphorylated serine/threonine-proline bonds in phosphoproteins, which regulates numerous key signaling molecules involved in
cell growth and differentiation (15, 16). We recently demonstrated that
PIN1 inhibition can promote odontogenic differentiation of hDPSCs but
inhibits adipogenesis (17).
PIN1 has been shown to be involved in neurodegenerative disorders such as AD, PD, and amyotrophic lateral sclerosis (18, 19).
PIN1-deficient mice display both tau-related and Abeta-related pathologies and neurodegeneration in an age-dependent manner, resembling AD (20, 21). In contrast, PIN1 overexpression in postnatal
neurons effectively suppresses tau-related pathology and neurodegeneration in a mouse model of AD (22). Furthermore, PIN1 depletion
suppressed neuronal differentiation, whereas PIN1 overexpression
enhanced it without any effects on gliogenesis in neural progenitor
cells (NPCs) (23).
Although defective neuronal differentiation in PIN1 knockout
NPCs was rescued in vitro by overexpression of b-catenin (23), little
is known about the role of PIN1 in the neuronal differentiation of
hDPSCs. In addition, PIN1 is expressed in most neurons in the brain
but is present at an especially low level in those neurons most vulnerable to neurodegeneration in AD (20). Moreover, the major challenge
for the development of neuronal replacement therapies for brain
diseases is the identification of reliable sources of easily expandable
cells with the capacity to generate those specific neuron types that are
lost in the different neurodegenerative disorders (10, 20). Therefore,
the aim of the present study was to investigate the role of PIN1 on
neuronal differentiation toward specific neurons or glial cells of
hDPSCs.
Materials and Methods
Cell Culture of hDPSCs
Human dental pulp tissue was obtained from the healthy premolars of young adults undergoing routine extractions at the dental hospital of Kyung Hee University (Seoul, Korea) who provided informed
consent. Human dental pulp tissue was isolated, and human dental
pulp cells (hDPCs) were separated enzymatically as described previously (1). Primary hDPCs were grown in a-MEM (Invitrogen, Carlsbad,
CA) supplemented with 10% fetal bovine serum. The hDPSC CD34+/
c-kit+/STRO-1+ cell population was sorted from the primary hDPCs
by a magnetic activating cell sorting method and was isolated and
cultured as described previously (3, 24).
Neurogenic Induction of hDPSCs
The hDPSCs were cultured in NM (Sigma-Aldrich, St Louis, MO)
supplemented with 1.55 mg/mL glucose, 0.073 mg/mL L-glutamine,
1.69 mg/mL sodium bisulfite, N-2 supplement (R&D Systems Inc, Minneapolis, MN), 20 ng/mL EGF (R&D Systems), and 20 ng/mL FGF (R&D
Systems) at 37 C in a 5% humidified CO2 atmosphere for 2 weeks, as
described previously for DPSCs (25).
Preparation of Recombinant PIN1 Adenovirus
An adenovirus encoding PIN1 (Ad-PIN1) (provided by Professor
Byung-Hyun Park, Jeonbuk National University, Korea) was created
by using the ViraPower Adenovirus Expression System (Invitrogen)
according to the manufacturer’s instructions.
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Cho et al.
Nissl Staining
Formalin-fixed cells were washed with phosphate-buffered saline
and stained in 0.1% Nissl staining solution (0.12 g cresyl violet acetate
with 120 mL distilled water and 0.2 mL glacial acetic acid) at room temperature for 30 minutes. After staining, the specimens were carefully
washed in distilled water, dehydrated in alcohols, and mounted in
balsam at different time points.
RNA Isolation and Reverse Transcriptase
Polymerase Chain Reaction
Total RNA of cells was extracted by using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions and then reversetranscribed by using AccuPower RT pre-mix (Bioneer, Daejeon, Korea).
cDNA was amplified by using AccuPower PCR PreMix (Bioneer) in a
DNA thermal cycler. Primer sequences for genes were reported previously (24). Polymerase chain reaction (PCR) products were subjected
to electrophoresis on 1.2% agarose gels stained with ethidium bromide.
Immunofluorescence
Cells were fixed in 4% paraformaldehyde and incubated with AntiNeuN (Merck Millipore, Darmstadt, Germany), GFAP (Merck Millipore), or nestin (Santa Cruz Biotechnology, Santa Cruz, CA) primary
antibodies overnight at 4 C. Cells were then washed in phosphatebuffered saline and incubated with anti-mouse Alexa Fluor 488 or
anti-rabbit Alexa Fluor 594 conjugated secondary antibodies (Molecular Probes Inc, Eugene, OR) for 1 hour at 4 C. Slides were imaged on a
confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan).
Cells stained with a secondary antibody without the primary antibody
served as negative control.
Flow Cytometric Analysis
Cells were fixed in ice-cold 50% methanol and stained by using antivesicular glutamate transporter-1 (VGluT1) (Abcam, Cambridge, UK),
gamma-aminobutyric acid (GABA) (Abcam), and tyrosine hydroxylase
(TH) (Abcam) primary antibodies. Secondary antibodies used were
Alexa Fluor 488. Cells stained only with a single secondary antibody
were used as negative controls. Ten thousand events were acquired by
using a BD FACSVerse flow cytometer (BD Biosciences, San Jose, CA),
and data were analyzed by using FACSuite software (BD Biosciences).
Statistical Analysis
The Student t test was used to detect significant differences between groups after determining that the data were normally distributed
and exhibited equal variances. Values in all figures are presented as the
means standard deviations. P value < .05 was considered statistically
significant.
Results
Characterization of Neural Differentiation of hDPSCs
To characterize neurogenic differentiation of hDPSCS, cells were
treated with NM up to 14 days, and then their morphology was analyzed.
The shape of hDPSCs cultured in NM changed from an elongated spindleshape to rounded or formed nest cells at 7 days, but diverse cellular morphologies including elongated bipolar cells, star-shaped cells forming a
network, and round cells with numerous processes at 14 days, consistent with neural cell phenotypes, were observed (Fig. 1A).
To further evaluate the neuronal phenotype of hDPSCs, the expression of mRNA and protein levels of the glial cell-specific marker GFAP
and neural progenitor marker nestin, early neuronal marker bIIItubulin, and late neuronal marker NeuN were assessed by reverse
transcriptase (RT)-PCR (Fig. 1B) and confocal microscopy (Fig. 1C).
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Regenerative Endodontics
B
Undifferentiated
A
PIN1
Nestin
20 μm
20 μm
β III-tubulin
7 days
NeuN
GFAP
β-actin
50 μm
20 μm
20 μm
0
1
3
7
14
(days)
14 days
NM
50 μm
Nestin
DAPI
Merge
PIN1
NeuN
20 μm
Merge
βIII-tubulin
NeuN
DAPI
7 days
Merge
DAPI
Merge
GFAP
DAPI
14 days
1 day
C
20 μm
Figure 1. Characterization of neuronal differentiation in hDPSCs. Cells were treated with NM for 14 days. (A) Representative morphologic changes during neuronal
predifferentiation of hDPSCs. Control undifferentiated hDPSCs had a spindle-shaped fibroblast-like morphology (arrowheads). After 7 days of differentiation, cells
committed toward the neural lineage became shortened, rounded, and dendritic. After 14 days of differentiation, a heterogeneous population of cells was observed,
including elongated cells, star-shaped cells (arrow), and round cells with processes. (B) mRNA expression of neuronal or glial markers and PIN1 during neuronal
induction of hDPSCs. mRNA levels were assessed by RT-PCR. (C) Representative double or triple immunofluorescence images of undifferentiated and differentiated
hDPSCs. Scale bar = 25 mm. These data are representative of 3 independent experiments.
Transcript levels of nestin increased early, with highest expression 1 day
after NM treatment, after which levels decreased. Expression of bIIItubulin mRNA increased until day 7, with a significant reduction on
day 14. Expression of mRNA levels of NeuN increased in a timedependent manner after exposure to NM until 14 days, showing a
similar expression pattern to glial cell marker GFAP and PIN1 mRNA
(Fig. 1B). Confocal microscopic images revealed that nestin protein
was present in undifferentiated hDPSCs for 1 day, whereas PIN1,
bIII-tubulin, NeuN, and GFAP proteins were expressed in differentiated
cells for 7 or 14 days (Fig. 1C). PIN1 and NeuN were co-expressed in the
nucleus of differentiated hDPCs (Fig. 1C).
Effects of PIN1 Inhibition and Overexpression
on Neural Differentiation of hDPSCs
To investigate the role of PIN1 in neurogenic differentiation of
hDPSCs, PIN1 expression was inhibited by the PIN1 inhibitor juglone
and overexpressed by Ad-PIN1 in NM-stimulated hDPSCs. Juglone
concentration-dependently decreased the number of Nissl bodies, a
mature neuron marker (Fig. 2A), and downregulated mRNA expression of nestin, bIII-tubulin, and NeuN (Fig. 2C). To examine whether
inhibition of PIN1 transcripts by juglone is nonspecific cytotoxic effects in higher doses, cell viability was analyzed by MTT assay. Juglone
did not significantly affect NM-induced cell viability (Fig. 2D). Treatment of hDPSCs with Ad-PIN1 enhanced NM-induced upregulation of
nestin, bIII-tubulin, and NeuN but blocked GFAP expression
(Fig. 2G). Although the number of Nissl body–stained cells did not
JOE — Volume 42, Number 7, July 2016
change significantly (Fig. 2F), flow cytometry analysis revealed that
the number of NeuN-positive cells was significantly higher in the
Ad-PIN1–treated cells than in negative control and cells transfected
with control adenovirus expressing b-galactosidase (Ad-LacZ)
(Fig. 2H). In contrast, GFAP expression was enhanced by juglone in
a concentration-dependent manner (Fig. 2C) but attenuated by
Ad-PIN1 (Fig. 2G).
Because the NM-induced increase in GFAP mRNA level was inhibited by Ad-PIN1, immunofluorescence staining and flow cytometry
for GFAP were performed to confirm the expression of this glial marker
protein. GFAP-positive cells were rarely detected in either the control or
Ad-PIN1 groups (Fig. 3A). The number of GFAP-positive cells decreased
significantly in response to treatment with Ad-PIN1 on the basis of flow
cytometric analysis (Fig. 3B).
Effects of PIN1 Overexpression on the Ability
of hDPSCs to Differentiate into Neurons
with Different Neurochemical Phenotypes
To determine the neuronal population types obtained, the expression of the glutamatergic marker VGluT1, GABAergic marker GABA, and
dopaminergic marker TH were assessed by confocal microscopy
(Fig. 4A). Immunoreactivity for VGluT1, GABA, and TH was observed
in both NM-treated control cells and Ad-PIN1–infected cells
(Fig. 4A). The percentage of double positive cells (VGluT1+ NeuN+,
GABA+ NeuN+, or TH+ NeuN+) was evaluated by flow cytometry. As
shown in Figure 4B, PIN1 overexpression significantly increased the
PIN1 and Neurogenic Differentiation in Dental Pulp Stem Cells
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A
14 days
B
Nissl body
total cells (%)
NM
Juglone (μM)
-
+
-
+
0.1
+
1
+
2
#
40
30
20
10
0
NM
Juglone (μM)
-
+
-
+
0.1
C
+
1
D
PIN1
PIN1
7 days
β -actin
80
60
40
20
PIN1
0
NM
Juglone (μM)
NeuN
14 days
GFAP
β-actin
NM
Juglone (μM)
% of Control
β -actin
β ΙΙΙ-tubulin
120
100
1 day
Nestin
+
2
-
+
-
+
0.1
+
1
-
+
-
+
0.1
+
1
+
2
+
2
Figure 2. Effects of PIN1 inhibition and overexpression on neuronal and glial differentiation of hDPSCs. Cells were treated with PIN1 inhibitor juglone and NM for
14 days (A–D)(continued).
percentages of glutamatergic and GABAergic cells but decreased the
proportion of dopaminergic cells.
Discussion
hDPSCs can undergo differentiation to neural progenitors such as
glial or neuronal cells in vitro (4, 6). Because of the clinical advantages
of hDPSCs as an alternative source of stem cells, we investigated whether
glial or specific neural phenotypes can be derived from CD34+/c-kit+/
STRO-1+ hDPSCs by modulation of PIN1 expression.
hDPSCs constitutively express the neural precusor marker nestin,
which is an intermediate filament that is expressed in neural stem or
NPCs (4, 6). bIII tubulin is the only phosphorylated tubulin and is
considered to be an early/intermediate neuronal-specific marker
expressed during neuronal differentiation of hDPSCs (7). NeuN is a
mature neuronal marker and has been observed in more than half of neuronally committed hDPSCs (8). Furthermore, GFAP has been shown to be
a marker of glial cells among hDPSCs (4). To neuronally differentiate
hDPSCs, we exposed hDPSCs to NM, which has previously been used to
induce DPSCs (25) and MSCs (26) to become neurogenic cells. Culture
in NM resulted in morphologic changes of the majority of the cells toward
a neuron-like morphology, and co-expression of neurogenic or glial cell
markers was found in hDPSCs, consistent with previous studies (25, 27).
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Our findings confirm that hDPSCs have the potential to differentiate
into neuronal and glial cell types. Moreover, we demonstrated peak
upregulation of nestin at 1 day (proliferation stage), bIII-tubulin at
7 days (early differentiation stage), and NeuN and GFAP at 14 days (late
differentiation stage) in NM-induced differentiated hDPSCs. The sequential
expression of these markers according to neural differentiation stage is
similar to that reported in a previous study of the differentiation of human
adult skin-derived neuronal precursors into mature neurons (28).
PIN1 level was found to be strongly increased during neuronal
differentiation of NPCs (23). However, the expression pattern of
PIN1 in differentiating cells may vary depending on the types of stem
cells and the lineages of cellular differentiation (17). The present
study showed that PIN1 mRNA level was time-dependently elevated
throughout the process of neurogenic differentiation of hDPSCs, suggesting the functional involvement of PIN1 in both early and late developmental stages of neurogenesis from hDPSCs. Consistent with these
results, time-dependent PIN1 upregulation was observed during adipogenic differentiation of hDPSCs (17) and NPC differentiation (23).
Stem cells can differentiate first into neuron-glia progenitors and
later into glial cells and functional neurons (29). However, the signaling
mechanisms by which neuronal or glial cell fates are decided in hDPSCs
are not well-understood. We found that differentiation of hDPSCs into
neurons was inhibited by the PIN1 inhibitor juglone but promoted by
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Regenerative Endodontics
E
7 days
NM
Ad-PIN1
-
+
-
+
+
+
+
Ad-LACZ
n.s.
Nissl body
total cells (%)
F
20
10
0
NM
Ad-PIN1
G
-
+
-
+
+
+
+
Ad-LACZ
H
PIN1
Nestin
NeuN+/ totalcells (%)
1 day
β -actin
PIN1
βΙΙΙ-tubulin
NeuN
7 days
GFAP
β -actin
NM
-
+
-
+
+
Ad-PIN1
-
-
+
+
AdLACZ
#
80
75
70
65
60
Control
Ad-PIN1
Ad-LACZ
Figure 2. (Continued). Cells were pretreated with Ad-PIN1 for 5 hours and post-treated with NM for 7 days (E–H). Adenoviral vector containing LACZ (Ad-LACZ)
served as a control. Cell viability was evaluated by MTT assay (D). Differentiation was assessed by (A and B) Nissl staining, (C and G) RT-PCR, and (H) flow
cytometric analysis. Quantification of % of Nissl-positive cells in each group (B and F). (H) Histogram indicates percentage of NeuN-positive cells among all hDPSCs.
Data were obtained from 3 independent experiments. #P < .05 versus NM alone control.
PIN1 overexpression, as evidenced by the formation of Nissl bodies and
upregulation of neural marker genes such as nestin, bIII-tubulin, and
NeuN. These results are consistent with previous data that the number of
bIII-tubulin–positive neurons increased in response to PIN1 overexpression but decreased in PIN1 knockout NPCs (23).
Merge
B
Ad-PIN1
Control
GFAP
Control
49± 3.3%
FITC(GFAP)
DAPI
Merge
Ad-PIN1
29± 6.4%
FITC(GFAP)
GFAP
Ad-LACZ
DAPI
Merge
Ad-LACZ
53± 1.0%
FITC(GFAP)
GFAP+/ total cells (%)
A
Concurrently, NM-induced glial differentiation was promoted by a
PIN1 inhibitor but inhibited by PIN1 overexpression, which was confirmed
by GFAP expression as determined by RT-PCR, immuofluorescence, and
flow cytometric analysis. Thus, our results indicate that PIN1 overexpression promotes neurogenic differentiation and inhibits glial differentiation,
GFAP
DAPI
#
60
50
40
30
20
10
0
1
Control
2
3
Ad-PIN1
Ad-LACZ
Figure 3. Effects of PIN1 overexpression on glial marker GFAP expression in hDPSCs. Expression was assessed by double immunofluorescence (A) and flow
cytometric analysis (B). Cells were pretreated with Ad-PIN1 for 5 hours and post-treated with NM for 7 days. Representative images revealed GFAP-positive cells
with relatively plump cytoplasm and multiple dendrites (scale bar = 25 mm). Histogram indicates percentage of GFAP-positive cells among total hDPSCs. Data were
obtained from 3 independent experiments. #P < .05 versus control. FITC, fluorescein isothiocyanate.
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PIN1 and Neurogenic Differentiation in Dental Pulp Stem Cells
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A
Control
Ad-LACZ
Ad-PIN1
Merge
VGluT1
NeuN
Merge
VGluT1
NeuN
Merge
VGluT1
NeuN
Merge
GABA
NeuN
Merge
GABA
NeuN
Merge
GABA
NeuN
Merge
TH
NeuN
Merge
TH
NeuN
Merge
TH
NeuN
B
Glutamatergic neuron
GABA+NeuN+/
NeuN+ cells (%)
FITC(NeuN)
30
0
Control Ad-PIN1 Ad-LACZ
80
60
40
20
0
Control Ad-PIN1 Ad-LACZ
Dopaminergic neuron
FITC(NeuN)
TH+NeuN+/
NeuN+cells (%)
PE (TH)
PE (TH)
60
GABAergic neuron
PE (GABA)
FITC(NeuN)
FITC(NeuN)
90
FITC(NeuN)
PE (TH)
PE (GABA)
PE (GABA)
FITC(NeuN)
FITC(NeuN)
VGluT1+NeuN+/
NeuN+ cells (%)
PE (VGluT-1)
PE (VGluT-1)
PE (VGluT-1)
FITC(NeuN)
FITC(NeuN)
Ad-LACZ
Ad-PIN1
Control
90
60
30
0
Control Ad-PIN1 Ad-LACZ
Figure 4. Effects of PIN1 overexpression on neuronal phenotype of differentiating hDPSCs. Cells were pretreated with Ad-PIN1 for 5 hours and post-treated with
NM for 7 days. Neuronal phenotype was examined by double immunofluorescence (A) and flow cytometric analysis (B). Histogram indicates percentage of glutamatergic, GABAergic, and dopaminergic cells as percentage of NeuN-positive hDPSCs. Data were obtained from 3 independent experiments. Scale bar = 50 mm.
#P < .05 versus control. FITC, fluorescein isothiocyanate.
whereas PIN1 inhibition has the opposite effects in hDPSCs. However, the
number of GFAP+ cells did not change in PIN1 knockout NPCs, which suggests that PIN1 knockout might specifically impair neuronal but not glial
differentiation (23). Further analysis of the mechanism of regulation of
neural and glial cell fates in hDPSCs is necessary.
NPCs can generate heterogeneous progeny; clones of mature neurons can synthesize multiple neurotransmitters such as GABA, glutamate,
dopamine, or some combination (30). The differentiation of SHED into
dopaminergic neuron-like cells in vitro has been reported (11). Glutamatergic and GABAergic axons are abundant in the dental pulp and
appear to play a role in mediating pulpal pain during inflammation
and injury (31, 32). In addition, murine DPSC-derived neural cells expressed markers for GABAergic and glutamatergic neuron but did not
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Cho et al.
express dopaminergic neuron marker (12). To the best of our knowledge, this study presents the first evidence of glutamatergic, GABAergic,
and dopaminergic neuronal potential in hDPSCs. Moreover, overexpression of PIN1 increased differentiation of hDPSCs to glutamatergic and
GABAergic neurons but decreased the percentage of dopaminergic neurons among the total NeuN-positive cell population. Because Nurr1, a
transcription factor, is essential for the development of midbrain
dopaminergic neurons and interacts with PIN1 (33), PIN1 overexpression-suppressed dopaminergic differentiation might be related to the
interaction between Nurr1 and PIN1. Further attempts at elucidating the
mechanisms by which PIN1 regulates the development of these specific
types of neurons would be the basis for sophisticated cell replacement
therapy in neurodegenerative diseases as well as in dental pulp lesions.
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Trigeminal nucleus caudalis (Vc), relaying nociceptive somatosensory information derived from the face, mouth, and dental pulp,
contains glutamatergic and GABAergic projection neurons and interneurons that exert crucial modulatory effects on primary sensory
afferent inputs from this region (34). Long-term consequences of peripheral injury and inflammation lead to neuroplastic changes (central
sensitization) in trigeminal nucleus that play a central role in acute and
chronic pain. Therefore, therapeutic targets for pain management and
development of therapeutic procedures should be focused on manipulating peripheral inputs and central processes within this region (Vc).
Neuropathic pain pathogenesis and maintenance involve interactions
among neurons, inflammatory immune cells, glial cells, and a wide
cascade of proinflammatory and anti-inflammatory cytokines (35, 36).
Recently, many researchers have tried to relieve neuropathic pain
by using stem cells with various origins, not only treating pain but also
repairing the damaged nervous system by replacing injured or lost
neural cells in experimental animal model (37). Intravenous neural
stem cell delivery in sciatic nerve injury model was reported to induce
a significant reduction of neuropathic symptoms, proinflammatory
cytokines, and spinal cord Fos expression in addition to increase of activated anti-inflammatory cytokines, improvement of nerve morphology,
and altering microenvironment within the spinal cord (38). MSCs are
also being studied as a therapeutic agent in the treatment of neuropathic
orofacial pain or atypical odontalgia besides CNS disorders (39).
Neuropathic pain results from inflammatory nerve damage and is maintained by glial activation in the spinal cord after peripheral nerve injury
(40). Because PIN1 upregulation can promote neurogenesis but inhibit
glial cell differentiation of hDPSCs, the transplantation of PIN1modulated hDPSCs might be an alternative treatment option in neuropathic orofacial pain by regenerating injured peripheral nerves without
excessive glial proliferation.
In conclusion, the present study is the first to elucidate the dual
functions of PIN1: activation of glutamatergic and GABAergic neuronal
differentiation and suppression of glial differentiation of hDPSCs. Our
findings suggest that PIN-modulated hDPSCs may serve as useful sources
of neurogenesis and gliogenesis in degenerative disorders of the CNS.
Acknowledgments
Young-Ah Cho and Duck-Su Kim contributed equally to this
study.
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Science, ICT and Future Planning
(no. 2015054225 and no. 2012R1A5A2051384).
The authors deny any conflicts of interest related to this study.
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