FGF-2 induces proliferation of human periodontal

ligament cells and maintains differentiation potentials

of STRO-1
+
/CD146
+
periodontal ligament cells
Tatsuhiro Hidaka, Toshiyuki Nagasawa, Kaname Shirai, Takashi Kado, Yasushi Furuichi *
Division of Periodontology and Endodontology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido,
1757 Kanazawa, Ishikari-Tobetsu, Hokkaido, Japan
1. Introduction
Periodontitis is an infectious disease that causes pathologic
alterations in tooth-supporting tissues, potentially leading to
tooth loss. The periodontal ligament (PDL) consists of dense
brous connective tissue located between the alveolar bone
and cementum.
1
PDL cells have potential to maintain or
reconstruct periodontal tissue in response to environmental
changes caused by e.g. periodontitis, injury and orthodontic
movements.
2,3
The PDL cells, in addition, have been reported
to play important roles in periodontal tissue regeneration.
4
Recent advances in stem cell biology and regenerative
medicine have presented opportunities for tissue engineering,
in which cells, growth factors and scaffolds are indispensable.
5
The presence of somatic stem cells in the adult body has been
reported. Bone marrow derived mesenchymal stem cells, for
example, have multipotency, including differentiation into
osteoblasts and adipocytes.
6
Mesenchymal stem cells have been
found to residein dental pulp, boneand fat.
79
Seo et al. reported,
for the rst time, that mesenchymal stem cells reside in the
PDL.
10
In the aforementioned study, they isolated STRO-1
+
/
CD146
+
[melanoma cell adhesion molecule (MCAM), MUC18]
cells using magnetic activated cell sorting (MACS) and reported
the differentiation of these cells into adipocytes and osteoblasts
in vitro. They also implanted the STRO-1
+
/CD146
+
cells into
surgically created defects of the jaw in immunocompromised
mice and found theformationof PDL/cementum-likestructures,
a r c hi v e s o f or a l b i o l og y 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0
a r t i c l e i n f o
Article history:
Accepted 6 December 2011
Keywords:
Periodontal ligament
FGF-2
STRO-1
+
/CD146
+
stem cells
Cell sorting
Proliferation
Differentiation
a b s t r a c t
The presence of humanSTRO-1
+
/CD146
+
periodontal ligament (PDL) cells has been reported,
but obtaining a large amount of these cells is difcult. The purpose of this study was to
evaluate the percentages of STRO-1
+
/CD146
+
cells in PDL cells and determine the effects of
FGF-2 on the proliferation and multilineage differentiation potency of these cells. Human
PDL (HPDL) cells were individually prepared from 15 extracted teeth. HPDL cells were
cultured with or without FGF-2, and the percentages of STRO-1
+
/CD146
+
cells in each HPDL
cell culture was examined using FACSAria
TM
. The STRO-1
+
/CD146
+
cells were sorted with
FACSAria
TM
, and the mRNA expression and differentiation potency of the sorted cells were
subsequently examined. The numbers of the STRO-1
+
/CD146
+
cells in the FGF-2 cultures
were signicantly higher than those cultured in the absence of FGF-2. The sorted STRO-1
+
/
CD146
+
cells expressed mRNA of PDL markers and differentiated into adipocytes and
osteoblast-like cells. The present study shows that FGF-2 augmented the proliferation of
the STRO-1
+
/CD146
+
cells in the HPDL cultures whilst retaining adipogenic and osteogenic
differentiation potentials. Thus, it may be useful to culture HPDL cells with FGF-2 for the
application of the human STRO-1
+
/CD146
+
PDL cells in periodontal tissue regeneration.
# 2011 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +81 133 23 1211x3320; fax: +81 133 23 1414.
E-mail address: furuichi@hoku-iryo-u.ac.jp (Y. Furuichi).
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
00039969/$ see front matter # 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2011.12.003
indicating that STRO-1
+
/CD146
+
cells were indeed PDL stem cells
(PDLSCs). Transplantation of PDLSCs is thus regarded as a
promising periodontal regenerative therapy.
2,11
To date, there
have been few reports on the presence of PDLSCs around teeth
indicated for extraction.
10,12
However, the amount of the PDL
tissues recovered from such teeth has been limited, and it is,
therefore, difcult to harvest enough PDLSCs for periodontal
tissue regeneration. To obtain a sufcient amount of PDLSCs for
periodontal tissue regeneration, expansion of PDLSCs by cell
culture is essential. Tsutumi et al. reported that bone marrow
stem cells cultured with broblast growth factor-2 (FGF-2)
retained adipogenic and osteogenic potential throughout many
mitotic divisions.
13
However, the effect of FGF-2 on PDLSCs
remains to be elucidated.
The purpose of the present study was to determine the
percentage of human STRO-1
+
/CD146
+
PDL cells from
extracted teeth and to examine the effects of FGF-2 on the
growth and differentiation of the human STRO-1
+
/CD146
+
PDL
cells.
2. Materials and methods
2.1. Reagents
The following reagents were used in this study: Dulbeccos
Modied Eagles Medium (DMEM; SigmaAldrich, St Louis, MO,
USA), foetal bovine serum (FBS; Valley Biomedical, Inc.,
Winchester, VA, USA), L-glutamine (Invitrogen, Ltd., Paisley,
UK), kanamycin (SigmaAldrich), amphotericin B (Sigma
Aldrich), FGF-2 (Kaken Pharmaceutical, Tokyo, Japan), bovine
serum albumin (BSA; Wako Pure Chemical Industries, Osaka,
Japan), HEPES (SigmaAldrich), 1% normal human serum
(Millipore, Billerica, MA, USA), HBSS () (Wako), anti-human
STRO-1 IgM mouse monoclonal antibody (R&D Systems,
Minneapolis, MN, USA), FITC-labelled anti-human CD146
mouse IgG
1
monoclonal antibody (AbD Serotec, Kidlington,
Oxford, UK), Alexa Fluor
1
546-conjugated goat anti-mouse
IgM (Invitrogen), Alexa Fluor
1
633-conjugated goat anti-
mouse IgM (Invitrogen), Mouse monoclonal IgM (Santa Cruz
Biotechnology, Delaware, CA, USA), FITC-labelled mouse IgG
1
monoclonal antibody (AbD Serotec), MSCGM
TM
medium
(Lonza, Walkersville, MD, USA), Isogen (Nippongene Co.,
Toyama, Japan), Omniscript RT Kit (Qiagen, Hilden, Germany),
10 CL Buffer (Qiagen), Q solution (Qiagen), dNTP (Qiagen),
RNase free water (Qiagen), Taq DNA Polymerase (Qiagen),
agarose (SigmaAldrich), ethidium bromide (SigmaAldrich),
methylisobutylmethylxanthine (SigmaAldrich), hydrocorti-
sone(SigmaAldrich), indomethacin (SigmaAldrich), Oil-red-
O (SigmaAldrich), L-ascorbic acid (SigmaAldrich), b-glycer-
ophosphate (SigmaAldrich), dexamethasone (SigmaAldrich)
and Alizarin-Red S (SigmaAldrich).
2.2. Human PDL cells (HPDL cells)
15 HPDL tissues were collected from 10 extracted healthy third
molars, 3 anterior teeth, 1 premolar and 1 molar, from15
subjects (mean age 32.3 yrs, range 2061 yrs, 9 males and 6
females). The experimental protocol was approved by the
ethics committee of the Health Sciences University of
Hokkaido (Number 18, Approval date; 9 June 2007), and
informed consent was obtained from all subjects.
HPDL tissue was removed from the middle third of the root
by using a sterile scalpel. The tissue fragments obtained were
rinsed two times with phosphate buffered saline (PBS) and
cultured on type I collagen-coated plastic culture dishes with
biopsy medium (DMEM supplemented with 10% FBS, 2 mM L-
glutamine, 200 mg/ml kanamycin, penicillin/streptomycin
100 mg/ml and 5 mg/ml amphotericin B) at 37 8C with 5%
CO
2
. The cells that grew from the tissues were cultured in
growth medium (DMEM supplemented with 10% FBS, 2 mML-
glutamine and 200 mg/ml kanamycin) at 37 8C with 5% CO
2
and
used for analyses according to the methods reported by
Nagatomo et al.
14
15 HPDL cell populations from 15 HPDL
tissues were denoted as HPDL1-15. Until otherwise stated, the
cells used in this study were maintained in humidied
incubators at 37 8C with 5% CO
2
.
2.3. Flow cytometry analysis
The 15 HPDL cell populations were individually cultured in
growth medium, and the culture medium was exchanged every
23 days. In subconuent cultures, the portion of the each cell
population was subjected to ow cytometry analyses (Fig. 1A,
left). The rest of each cell population was subsequently cultured
for another 10 days after conuency either in the presence or
absence of 20 ng/ml FGF-2, and each cell population was
subjected to ow cytometry analyses (Fig. 1A, right). The single
cell suspensions of the 15 HPDL cell cultures were obtained
through a 70 mm cell strainer (Becton Dickinson). Samples were
centrifuged at 400 g for 10 min and re-suspended in blocking
buffer (HBSS, 20 mM HEPES, 1% normal human serum, 1% BSA,
5% FBS) for 20 min on ice and stained with anti-human STRO-1
mouse IgM monoclonal antibody for 1 h on ice. FITC-labelled
anti-human CD146 mouse IgG
1
monoclonal antibody was also
added and incubated for 1 h on ice. After washing 3 times, Alexa
Fluor
1
633-conjugated goat anti-mouse IgM was added and
incubated for 1 h on ice. The expressions of STRO-1 and CD146
on HPDL cells were analysed, and the STRO-1
+
/CD146
+
cells
were sorted using FACSAria
TM
(Becton Dickinson, Franklin
Lakes, NJ, USA). The data was analysed with FACS DiVa
TM
6.0
(Becton Dickinson). The auto-uorescence of HPDL cells was
increased by the reaction with isotype control antibody, and
gating for the STRO-1 and CD146 positive cells was determined
on the highest uorescence of isotype control antibodies (FITC-
labelled mouse IgG
1
monoclonal antibody and mouse mono-
clonal IgM, followed by Alexa Fluor
1
633-cnjugated goat anti-
mouse IgM antibody).
2.4. Cell sorting
To examine marker gene expression and differentiation
potency of STRO-1
+
/CD146
+
cells in the periodontal ligament
cells cultured with and without FGF-2, the STRO-1
+
/CD146
+
cells were isolated from HPDL2 (donor; 26 yrs of age, male,
healthy third molar) using FACSAria
TM
. The STRO-1
+
/CD146
+
cells obtained from the HPDL2 cells cultured with FGF-2 were
denoted as HPDLSC2f cells and without FGF-2 were HPDLSC2
cells. HPDLSC2 and HPDLSC2f cells were subcultured and
subjected to the below experiments (Fig. 1B).
a r c hi v e s o f or a l b i ol o gy 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0 831
2.5. Human bone marrow mesenchymal stem cells
(hBMMSC)
hBMMSC cells were purchased from Lonza and seeded in
MSCGM
TM
medium. The cells were subcultured and subjected
to the below experiments (Fig. 1B).
2.6. Immunocytochemistry
The cells were plated into each well of type I collagen coated
8-chamber slides (Becton Dickinson) at a density of
1 10
5
cells/well, and subcultured. The cells were xed in
4% paraformaldehyde for 30 min and permeabilized with
0.2% Triton X-100 in PBS. After background inhibition in 2%
BSA in PBS, cells were labelled with anti-human STRO-1 IgM
mouse monoclonal antibody and incubated at room tem-
perature for 2 h. Following washing of the primary antibody
with 0.2% Triton X-100 in PBS, the cells were incubated with
FITC-labelled anti-human CD146 mouse IgG
1
monoclonal
antibody for 2 h at room temperature. After washing 3
times, the cells were incubated with Alexa Fluor
1
546-conjugated goat anti-mouse IgM antibody for 2 h at
Fig. 1 Experimental protocol. (A) The 15 HPDL cell populations were individually cultured in growth medium, and the
culture medium was exchanged every 23 days. In subconfluent cultures, the portion of the each cell population was
subjected to flow cytometry analyses (left). The rest of each cell population was subsequently cultured for another 10 days
after confluency either in the presence or absence of 20 ng/ml FGF-2, and each cell population was subjected to flow
cytometry analyses (right). (B) STRO-1
+
/CD146
+
cells obtained from HPDL2 cells cultured with FGF-2 were called HPDLSC2f
and without FGF-2 were HPDLSC2. HPDL2, HPDLSC2, HPDLSC2f and hBMMSC cells at subconfluency were subjected to
immunocytochemistry and RT-PCR. HPDLSC2, HPDLSC2f and hBMMSC cells were subcultured and subjected to adipogenic
and osteogenic induction. Adipogenic differentiation potency of the cells was analysed using RT-PCR and Oil-red-O
staining. Osteogenic differentiation potency of the cells was analysed using RT-PCR and Alizarin-Red staining.
a r c hi v e s o f o r a l b i o l og y 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0 832
room temperature. The cells were washed three times with
0.2% Triton X-100 in PBS. The signals were subsequently
detected using a confocal laser scanning microscope (Nikon
TE2000-E, Nikon Corp., Tokyo, Japan).
2.7. Reverse transcription-polymerase chain reaction (RT-
PCR) analysis
Total RNA was extracted from the cells using Isogen according to
the manufacturers protocol. Complementary DNA (cDNA) was
synthesized with Omniscript reverse transcriptase (Qiagen)
using (dT)
15
primer (1 mM). Subsequent amplications were, for
the detection of HPDL cell cDNAs, performedusing the requisite
number of cycles, under the following conditions: 94 8C for 30 s,
annealing temperature optimized for each primer pair for 45 s
and 72 8C for 60 s. The target gene of each specic forward/
reverse primer (Table 1) was used: periodontal ligament-
associated protein-1 (PLAP-1),
15
periostin (accession number
NM 006475), S100A4,
16
scleraxis,
17
runt-related gene 2 (Runx2;
accession number NM 001024630), type I collagen (Col I),
17
osteocalcin (OCN),
17
peroxisome proliferator-activated receptor
g (PPARg),
17
lipoprotein lipase (LPL),
17
von Willebrand factor
(vWF; accession number NM000552), CD34 (accession number
NM 001025109), calponin (accession number AK223234), nestin
(accession number NM 006617), CD146 (accession number NM
006500), FGF receptor 1 (FGFR1)
17
and glyceraldehyde-3-phos-
phate dehydrogenase (GAPDH; accession number NM 002046).
The amplied products were separated by agarose gel electro-
phoresis and stained with ethidium bromide. PCR experiments
were performed using samples from at least three different cell
preparations, and the results were conrmed by triplicate PCR
experiments from the same cell samples.
2.8. Induction of adipogenic differentiation
HPDLSC2, HPDLSC2f and hBMMSC cells were cultured in 6-
well and 24-well culture plates (1 10
4
cells/cm
2
) with an
adipogenic induction medium (growth medium containing
0.5 mM methylisobutylmethylxanthine, 0.5 mM hydrocorti-
sone and 60 mM indomethacin). The cells of the control
groups were cultured with growth medium. After 20 days,
the mRNA expressions of adipogenic marker genes, includ-
ing PPARg and LPL in the cultured cells, were assayed using
RT-PCR. The cultured cells were xed with 10% formalin for
30 min, incubated with 60% propan-2-ol for 1 min and then
stained with 0.2% Oil-red-O in propan-2-ol for 10 min
(Fig. 1B).
2.9. Induction of osteogenic differentiation
HPDLSC2, HPDLSC2f and hBMMSC cells were cultured in 6-
well and 24-well culture plates (1 10
4
cells/cm
2
) with
osteogenic induction medium (growth medium including
50 mg/ml L-ascorbic acid, 10 mM b-glycerophosphate and
5 mM dexamethasone). The cells of the control groups were
cultured with growth medium. After 20 days, the mRNA
expressions of osteoblastic marker genes including Runx2,
Col I and OCN in the cultured cells were assayed using RT-
PCR. The cells were xed with 4% paraformaldehyde in PBS
(pH 7.2) at room temperature for 30 min. The monolayers
were washed with distilled water three times and treated
with 40 mM Alizarin-Red solution for 60 min. They were
washed again with distilled water at least three times andPBS
one time, and observed under a phase-contrast microscope
(Fig. 1B).
Table 1 Sequences of primers and conditions used for RT-PCR.
Target gene Primer sequence forward/reverse (5
0
3
0
) Annealing
temperature
( 8C)
Cycles Predicted
size (bp)
References
PLAP-1 5
0
-CGATACAAAGAACTACAAAGGCTGG-3
0
/5
0
-GCATTTCCCAGTATTT-
CACCG-3
0
60 30 291 15
Periostin 5
0
-GTCTTTGAGACGCTGGAAGG-3
0
/5
0
-CAAGATCCGTGAAGGTGGTT-3
0
58 28 205 NM 006475
S100 A4 5
0
-GGCCCTGGATGTGATGGTGT-3
0
/5
0
-TCCACCACCCTGTTGCTGTA-3
0
57 40 350 16
Scleraxis 5
0
-CTGGCCTCCAGCTACATCTC-3
0
/5
0
-CTTTCTCTGGTTGCTGAGGC-3
0
58 35 210 17
Runx2 5
0
-TCTGGCCTTCCACTCTCAGT-3
0
/5
0
-GACTGGCGGGGTGTAAGTAA-3
0
55 32 199 NM 0010246030
Col I 5
0
-AGAGCCTGGCCCTGTTGGTG-3
0
/5
0
-TGCCATCAGGACCAGGGCTG-3
0
61 27 308 17
OCN 5
0
-GGTGCAGCCTTTGTGTCCAAGC-3
0
/5
0
-GGCAAGGGGAAGAGGAAA-
GAAGG-3
0
59 28 284 17
PPARg 5
0
-CTCCTATTGACCCAGAAAGC-3
0
/5
0
-GTAGAGCTGAGTCTTCTCAG-3
0
55 28 346 17
LPL 5
0
-ATGGAGAGCAAAGCCCTGCTC-3
0
/5
0
-GTTAGGTCCAGCTGGATCGAG-3
0
60 35 563 17
vWF 5
0
-GTGACGGTGAATGGGAGACT-3
0
/5
0
-GTCATTGGCTCCGTTCTCAT-3
0
55 40 222 NM 000552
CD34 5
0
-AGGGGCAGGTAAACTCCTGT-3
0
/5
0
-CAAAGATTCCTGGGAGAAA-3
0
55 40 242 NM 001025109
Calponin 5
0
-AGGCTCCGTGAAGAAGATCA-3
0
/5
0
-CTCCCACGTTCACCTTGTTT-3
0
58 30 227 AK 223234
Nestin 5
0
-GTCCTCAGGGGAGGACTAGG-3
0
/5
0
-GCAAGAGATTCCCTTTGCAG-3
0
55 30 250 NM 006617
CD146 5
0
-CTGCTGAGTGACCACAGGA-3
0
/5
0
-CACCTGGCCTGTCTCTTCTC-3
0
55 30 115 NM 006500
FGFR1 5
0
-CCCTGGAAGAGAGGCCGGGCAGTGATGAC-3
0
/5
0
-GGTTTGCCTAAGAC-
CAGTCTGTCCCG-3
0
58 38 367 17
GAPDH 5
0
-CGACCACTTTGTCAAGCTCA-3
0
/5
0
-AGGGGTCTACATGGCAACTG-3
0
60 25 228 NM 002046
a r c hi v e s o f or a l b i ol o gy 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0 833
2.10. Statistical analysis
Statistical differences were evaluated using Students t-test
and analysis of variance (ANOVA) with Tukeys tests. A P value
of less than 0.05 was considered statistically signicant.
3. Results
3.1. The percentage of STRO-1
+
/CD146
+
cells in the HPDL
cells
The mean percentage of the STRO-1
+
cells, CD146
+
cells and
STRO-1
+
/CD146
+
cells in 15 HPDL cell populations cultured
without FGF-2 at subconuency were 0.85 .75%, 0.23 .13%
and 0.07 .08%, respectively (Table 2).
3.2. The effect of FGF-2 on STRO-1
+
/CD146
+
cells in the
HPDL cells
The mean percentage of the STRO-1
+
cells, CD146
+
cells and
STRO-1
+
/CD146
+
cells in 15 HPDL cell populations cultured
with FGF-2 10 days after conuency were 1.58 1.81%,
1.84 2.00% and 0.43 .64%, respectively. The corresponding
mean percentage of the cells cultured without FGF-2 was
0.13 0.14%, 0.07 0.07% and under the detection limit,
respectively. The percentages of the STRO-1
+
cells, CD146
+
cells and STRO-1
+
/CD146
+
cells in the HPDL cells cultured with
FGF-2 were signicantly higher (P < 0.05) than those without
FGF-2 (Table 3). The mean number of the STRO-1
+
/CD146
+
cells
cultured without FGF-2 were under the detection limit, and the
corresponding number of cells cultured with FGF-2 were
2076 3129 cells/culture dish (Table 3).
3.3. STRO-1
+
/CD146
+
cells in the HPDL cells
The STRO-1
+
/CD146
+
cells in the HPDL cells were examined by
immunocytochemistry. It was found that STRO-1
+
/CD146
+
cells resided in the proliferating colony in the HPDL2 cells
(Fig. 2ad). No uorescence was observed in the isotype control
staining of the HPDL2 cells (Fig. 2eh).
3.4. mRNA expression proles of the HPDL cells
RT-PCR analysis showed that the HPDL2 cells expressed PDL
markers, including PLAP-1, periostin, S100A4 and scleraxis
mRNA. The HPDL2 cells also expressed Runx2, Col I, OCN,
CD34, calponin, nestin, CD146 and FGFR1 mRNA. However,
vWF, PPARg and LPL mRNA expressions were not observed in
the HPDL2 cells (Fig. 3).
Table 2 The percentage of STRO-1
+
/CD146
+
cells in
HPDL cells.
Subconuent Percentage
STRO-1
+
cells 0.85 0.75
CD146
+
cells 0.23 0.13
STRO-1
+
/CD146
+
cells 0.07 0.08
HPDL cells were cultured in growth medium until subconuent,
and the percentages of STRO-1
+
and/or CD146
+
cells in HPDL cells
were analysed. The percentage of positive cells (%) is displayed as
mean standard deviation (SD) (n = 15).
Table 3 The effect of FGF-2 on STRO-1
+
/CD146
+
cells in
HPDL cells.
FGF-2 +
STRO-1
+
cells Percentage 0.13 0.14 1.58 1.81
*
Number (cells) 648 704 7721 8790
CD146
+
cells Percentage 0.07 0.07 1.84 2.00
*
Number (cells) 324 352 8954 9748
STRO-1
+
/CD146
+
cells Percentage UDL 0.43 0.64
Number (cells) UDL 2076 3129
The HPDL cells cultured with and without FGF-2 were subjected to
ow cytometry analysis 10 days after conuency. The percentages
of positive cells (%) and the numbers of positive cells (cells) are
displayed as mean standard deviation (SD) (n = 15).
*
P < 0.05 Students t-test (vs FGF-2(-)).
UDL: under the detection limit.
Fig. 2 Expressions of STRO-1 and/or CD146 in HPDL2 cells. HPDL2 cells were fluorescently labelled with antibodies for
STRO-1 (a; red) and CD146 (b; green). The nuclei were counterstained with DAPI (c and g; blue). The images were merged (d).
The STRO-1
+
/CD146
+
cells in the HPDL2 cultures were observed in the proliferating colony in the HPDL cells. No fluorescence
was observed in the isotype control staining in the HPDL cells (e, f and h). The signals were subsequently detected using a
confocal laser scanning microscope. Magnification: 200T; Bar: 50 mm.
a r c hi v e s o f o r a l b i o l og y 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0 834
3.5. Sorted STRO-1
+
/CD146
+
cells derived from the HPDL
cells using FACSAria
TM
STRO-1
+
/CD146
+
cells in the HPDL2 cells cultured with or
without FGF-2 were sorted, and the cells obtained were
denoted as HPDLSC2f and HPDLSC2 cells, respectively. The
HPDLSC2 and HPDLSC2f cells were cultured for 15 days and
examined using phase-contrast microscopy. Both HPDLSC2
and HPDLSC2f cells were observed in the proliferating colony
(Fig. 4A, a and c) and were spindle-shape (Fig. 4A, b and d). The
STRO-1
+
/CD146
+
cells in the HPDLSC2, HPDLSC2f and hBMMSC
(positive control cells) cultures were examined by immunocy-
tochemistry (Fig. 4B, d, h and l). Five microscopic appearances
were randomly chosen on a voluntary basis, the numbers of
the STRO-1
+
/CD146
+
cells and DAPI measured, and the
percentages of the STRO-1
+
/CD146
+
cells calculated (Fig. 4C).
The percentage of the STRO-1
+
/CD146
+
cells in the HPDLSC2
(72.82 12.23%), HPDLSC2f (85.00 13.69%) and hBMMSC
(78.10 23.53%) cultures were signicantly higher than those
in the HPDL 2 cultures (19.66 9.70%, Fig. 2).
3.6. mRNA expression proles of the HPDLSCs (sorted
STRO-1
+
/CD146
+
cells)
The RT-PCR analysis showed that the HPDLSC2, HPDLSC2f and
hBMMSC cells expressed PLAP-1, periostin, S100A4 and
scleraxis mRNA. However, the HPDLSC2 and HPDLSC2f cells
revealed higher expression of PLAP-1 mRNA than the hBMMSC
cells, and the hBMMSC cells higher expression of periostin
mRNA than the HPDLSC2 and HPDLSC2f cells. The HPDLSC2
cells expressed Col I and OCN mRNA, and HPDLSC2f cells
expressed OCN mRNA. However, Runx2 mRNA expression
was not observed in the HPDLSC2 or HPDLSC2f cells. The
hBMMSC cells expressed Runx2, Col I and OCN mRNA. PPARg
and LPL mRNA expressions were not observed in the HPDLSC2,
HPDLSC2f or hBMMSC cells. vWF mRNA expression was
observed in the hBMMSCs, but not in the HPDLSC2 or
HPDLSC2f cells. The HPDLSC2, HPDLSC2f and hBMMSCs cells
expressed CD34, calponin, nestin, CD146 and FGFR1 mRNA
(Fig. 5).
3.7. Adipogenic differentiation of the HPDLSCs (sorted
STRO-1
+
/CD146
+
cells)
The RT-PCR analysis showed that the HPDLSC2, HPDLSC2f and
hBMMSC cells expressed adipogenic markers following adipo-
genic induction. The HPDLSC2 and hBMMSC cells expressed
both PPARg and LPL mRNA, and the HPDLSC2f cells expressed
PPARg mRNA (Fig. 6A). Oil-red-O staining revealed the
accumulation of lipid vacuoles within the cytoplasm of the
HPDLSC2, HPDLSC2f and hBMMSC cells, following adipogenic
induction (Fig. 6B, df). These lipid vacuoles were not observed
in the HPDLSC2, HPDLSC2f or hBMMSC cells without adipo-
genic induction (Fig. 6B, ac).
3.8. Osteogenic differentiation of the HPDLSCs (sorted
STRO-1
+
/CD146
+
cells)
The HPDLSC2 cells did not express Col I mRNA without
osteogenic induction, but expressed with osteogenic induc-
tion. Runx2 and OCN mRNA expressions were also detected in
the HPDLSC2 cells with osteogenic induction. The HPDLSC2f
and hBMMSC cells expressed Runx2, Col I and OCN mRNA
with osteogenic induction (Fig. 7A). Mineralized nodule
formations visualized with Alizarin-Red staining were ob-
served in the HPDLSC2, HPDLSC2f and hBMMSC cells in the
osteogenic induction medium (Fig. 7B, df), but not in the
corresponding control medium (Fig. 7B, ac).
4. Discussion
In the present study, we investigated the effect of FGF-2 on
STRO-1
+
/CD146
+
cells detected in PDL cell cultures obtained
from 15 different subjects/teeth. We found that (i) the number
of the STRO-1
+
/CD146
+
cells in the HPDL cell cultures with FGF-
2 reached nearly 15-fold higher cell numbers than those
without FGF-2 (Table 3); (ii) the sorted STRO-1
+
/CD146
+
cells
from the representative HPDL cells expressed PDL marker
genes, including PLAP-1 and scleraxis (Fig. 5); (iii) the
percentage of the STRO-1
+
/CD146
+
cells was comparable to
that of the hBMMSCs (Fig. 4C), and (iv) the sorted STRO-1
+
/
CD146
+
cells had the potential to differentiate into osteoblast-
like cells and adipocytes (Figs. 6 and 7).
Previous studies showed that bone marrow, dental pulp,
and PDL cells with multiple differential potencies expressed a
cell surface marker of stem cells such as STRO-1, CD44, CD105,
or CD146 (see review Huang et al.).
18
However, because
mesenchymal progenitor cells often express stem cell markers
at various levels, utilization of multiple stem cell markers for
the isolation of highly puried stem cells was recommended.
Fig. 3 mRNA expression profiles of HPDL2 cells. As shown
are the expressions of PDL markers: PLAP-1, periostin,
S100A4 and scleraxis; adipogenic markers: PPARg and
LPL; osteoblastic markers: Runx2, Col I and OCN; vascular
endothelial cell markers: vWF and CD34; vascular smooth
muscle cell marker: calponin; neural stem cell marker:
nestin; mesenchymal stem cell marker: CD146, and FGF
receptor 1 mRNA. GAPDH was used as an internal control.
a r c hi v e s o f or a l b i ol o gy 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0 835
Human STRO-1
BRIGHT
/VCAM-1
+
(vascular cellular adhesion
molecule 1, CD106) cells isolated from bone marrow stromal
cells using MACS were shown to possess multi-differentiation
potentials as highly puried stem cells.
19
Seo et al. trans-
planted human STRO-1
+
/CD146
+
cells into periodontal defects
of immunodecient rats and observed the capacity of the
STRO-1
+
/CD146
+
cells to generate cementum/PDL-like struc-
tures.
10
Gay et al. examined the expression of STRO-1 on HPDL
tissue, and found that about 27% of HPDL cells were positive,
and 3% were strongly positive.
20
Xu et al. reported that 2.6%
(max = 7.13%; min = 0.13%; n = 6) of HPDL cells were STRO-1
+
/
CD146
+
cells in average. The percentage of STRO-1
+
/CD146
+
cells in the HPDL cells in the present study was relatively lower
compared with these reports.
21
Zheng et al. investigated the
percentage of STRO-1 or CD146 positive HPDL cells from young
donors (15 yrs of old in average) or from the aged donors (54 yrs
of old in average), and found that the mean percentage of
STRO-1
+
or CD146
+
HPDL cells in the young donors was higher
(18.5 2.6% or 26.8 2.4%, respectively) than those in the aged
donors (3.15 1.3%, 4.3 1.8%, respectively).
22
The mean age
of the donors was 32.3 yrs in the present study, suggesting that
the relatively old donors inuenced the lower percentage of
STRO-1
+
/CD146
+
cells in the HPDL cell cultures without FGF-2
in the present study.
The concentration of FGF-2 introduced in the present study
was decided to be 20 ng/ml based on the results reported by
Takayama et al.
23
In the aforementioned study, it was reported
that FGF-2 enhanced the proliferation of periodontal ligament
cells in a dose-dependent manner, and that the response
reached plateau at the concentration of 10 ng/ml, and
decreased at >100 ng/ml, whilst alkaline phosphatase activi-
ties of the periodontal ligament cells were completely
inhibited when the cells were cultured with more than
10 ng/ml of FGF-2. We found that the number of the STRO-
1
+
/CD146
+
cells in the HPDL cell cultures with 20 ng/ml FGF-2
Fig. 4 Sorted STRO-1
+
/CD146
+
cells derived from HPDL2 cells using FACSAria
TM
. The STRO-1
+
/CD146
+
stem cells obtained
from the HPDL2 cells cultured with FGF-2 were called HPDLSC2f cells and without FGF-2 were HPDLSC2 cells. (A) The
proliferating colony of the HPDLSC2 and HPDLSC2f cells are shown. (a and b) Lower magnification: 40T; Bar: 500 mm (c and
d) Higher magnification 200T: Bar: 30 mm (B) Expression of STRO-1 and/or CD146 in HPDLSC2, HPDLSC2f and hBMMSC cells
were examined by immunocytochemistry. The cells were fluorescently labelled with antibodies for STRO-1 (a: HPDLSC2, e:
HPDLSC2f, i: hBMMSC; red) and CD146 (b: HPDLSC2, f: HPDLSC2f, j: hBMMSC; green). The nuclei were counterstained with
DAPI (c, g and k; blue). The images were merged (d: HPDLSC2, h: HPDLSC2f, l: hBMMSC). (C) The percentage of STRO-1
+
/
CD146
+
cells in the HPDLSC2, HPDLSC2f and hBMMSC cells are shown. *P < 0.05 ANOVA with Tukeys test; significantly
different from HPDL2 cells.
a r c hi v e s o f o r a l b i o l og y 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0 836
Fig. 5 The mRNA expression profiles of HPDLSC2, HPDLSC2f and hBMMSC cells were analysed. As shown are the
expressions of the PDL markers: PLAP-1, periostin, S100A4 and scleraxis; adipogenic markers: PPARg and LPL; osteoblastic
markers: Runx2, Col I and OCN; vascular endothelial cell markers: vWF and CD34; vascular smooth muscle cell marker:
calponin; neural stem cell marker: nestin; mesenchymal stem cell marker: CD146, and FGF receptor 1 mRNA. GAPDH was
used as internal control.
Fig. 6 Adipogenic differentiation of the HPDLSCs. (A) Expression of PPARg and LPL mRNA in HPDLSC2, HPDLSC2f and
hBMMSC cells are shown. (B) Oil-red-O staining of HPDLSC2, HPDLSC2f and hBMMSC cells in adipogenic induction groups
(d: HPDLSC2, e: HPDLSC2f, f: hBMMSC) and control groups (a: HPDLSC2, b: HPDLSC2f, c: hBMMSC). Cont: control groups,
Adipo: adipogenic induction groups. Magnification: 200T; Bar: 15 mm.
a r c hi v e s o f or a l b i ol o gy 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0 837
reached nearly 15-fold higher cell numbers than those without
FGF-2. This result in turn supports the proposal by Takayama
et al. that topical application of FGF-2 may increase the
number of multi-potent immature cells which play critical
roles in periodontal tissue regeneration.
23
In the present study, FGF-2 increased the ratio and number
of the STRO-1
+
/CD146
+
cells in the HPDL cells, the sorted
STRO-1
+
/CD146
+
cells revealed multiple differentiation capac-
ities, and the number of the STRO-1
+
/CD146
+
cells in the
cultures without FGF-2 was under the detection limit. Baddoo
et al. reported that FGF-2 reversibly inhibited the differentia-
tion of mesenchymal stem cells into adipocytes, chondro-
cytes, and osteoblasts in vitro.
24
The result of the present study
suggested that the stem cells including STRO-1
+
/CD146
+
cells
cultured without FGF-2 might have differentiated into other
cell populations, and the percentage of STRO-1
+
/CD146
+
cells
gradually decreased during culture in the absence of FGF-2.
Relationships between growth factors and bone marrow
stromal cells have been reported by several investigators.
13,2527
Gronthos and Simons reported that growth factors in the serum
were essential for the proliferation of bone marrow stem cells,
and that epidermal growth factor (EGF) and platelet-derived
growth factor (PDGF) can support the colony growth without
serum.
25
Martin et al. examined the effect of various growth
factors, including EGF, PDGF, FGF-2, insulin-like growth factor
and transforming growth factor-beta, on the growth of bone
marrow stem cells and found that FGF-2 was most effective in
promoting the growth of bone marrow stem cells in vitro.
26
Walsh et al. reported that treatment of bone marrow stromal
cells with FGF-2 for 28 days enhanced the growth of STRO-1
+
cells 2.3 times (max = 3.9, min = 1.0) higher than those without
FGF-2.
27
Tsutsumi et al. reported that mesenchymal stem cells
expanded in vitro with FGF-2, but not those cultured without
FGF-2, exhibited a multiple differentiation capacity.
13
These
reports, in addition to our study, suggest an essential role of
FGF-2 in self-renewal of not only HPDL stem cells, but also
mesenchymal stem cells, including bone marrow stromal cells.
The HPDLSC2 and HPDLSC2f cells possessed adipogenic
differentiation potency. The HPDLSC2 or HPDLSC2f cells
expressed PPARg and LPL mRNA in the adipogenic
induction medium (Fig. 6) and formed mineralized nodules
following osteogenic induction (Fig. 7). These results suggest
Fig. 7 Osteogenic differentiation of HPDLSCs. (A) The expressions of osteoblastic markers mRNA in osteogenic induction
groups and control groups are shown. (B) Alizarin-Red staining of HPDLSC2, HPDLSC2f and hBMMSC cells in the osteogenic
induction groups (d: HPDLSC2, e: HPDLSC2f, f: hBMMSC) and control groups (a: HPDLSC2, b: HPDLSC2f, c: hBMMSC). Cont:
control groups, Osteo: osteogenic induction groups. Magnification: 100T; Bar: 40 mm.
a r c hi v e s o f o r a l b i o l og y 5 7 ( 2 0 1 2 ) 8 3 0 8 4 0 838
that HPDLSC2 and HPDLSC2f cells were able to differentiate
into not only adipocytes but also into osteoblast-like cells.
We investigated the mRNA expression proles of
HPDLSCs, including the markers for PDL, bone, fat, vessel,
mesenchymal and neural stem cells. HPDLSCs expressed
CD146 mRNA, FGF-2 receptor mRNA and mRNA of PDL
markers including PLAP-1, periostin, S100A4 and scleraxis
(Fig. 5). HPDLSCs strongly expressed PLAP-1, periostin
mRNA, and weakly expressed S100A4 and scleraxis mRNA.
The hBMMSCs strongly expressed periostin mRNA, and
weakly expressed S100A4, scleraxis and PLAP-1 mRNA. In
support of our ndings, Seo et al. also reported the
expression of scleraxis mRNA in PDLSCs. The present study
demonstrated for the rst time the expression of PLAP-1 in
HPDLSCs. PLAP-1 is a member of a small leucine-rich
proteoglycan family.
28
PLAP-1 is expressed in HPDL and has
been reported to suppress bone morphogenetic protein-2
activity.
15
PLAP-1 has been reported to suppress the
cytodifferentiation of PDL cells into mineralized-tissue-
forming cells, suggesting that PLAP-1 maintains PDL space
through the suppression of mineralization.
29,30
The previous
studies reported that human STRO-1
+
/CD146
+
PDL cells
expressed mRNA of alkaline phosphatase, bone sialoprotein
and OCN.
18
In addition to these molecules, HPDLSCs
expressed mRNA of osteoblastic markers, including Col I
and OCN in this study. HPDLSCs also expressed vascular
endothelial cell marker CD34 mRNA, as well as vascular
smooth muscle cell marker calponin mRNA in this study.
We have previously reported that a swine PDLSC-like cell
line (TesPDL3) expressed vWF, vascular endothelial-cad-
herin, CD31, calponin, a-smooth muscle actin, smoothelin,
Osterix and Runx2 mRNA, and constructed tube-like
structures in response to FGF-2 stimulation, suggesting
that FGF-2 stimulated the differentiation of PDLSC-like cells
into vascular cell lineages.
31
Transplantation of human PDLSCs into periodontal defects
has been reported to be useful for periodontal tissue
regeneration.
2,4
However, the number of the STRO-1
+
/
CD146
+
cells in extracted teeth are limited. In this study, we
found that FGF-2 augmented the number of STRO-1
+
/CD146
+
cells in PDL cells isolated from extracted teeth. A large number
of the STRO-1
+
/CD146
+
cells may be obtained by cell sorting
after expanding HPDL cells with FGF-2. The present study
suggests that stimulation with FGF-2 might be a useful
strategy to obtain sufcient cell numbers of the STRO-1
+
/
CD146
+
cells for periodontal tissue regeneration.
Acknowledgement
The present study was partly supported by High-Tech
Research Center Project for Private Universities: matching
fund subsidy from Ministry of Education, Culture, Sports,
Science and Technology-Japan.
Funding: None.
Competing interests: There are no conicts.
Ethical approval: The experimental protocol was approved
by the ethics committee of the Health Sciences University of
Hokkaido, and informed consent was obtained from all
subjects.
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