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Characterization of human dental
pulp-derived cell
lines
H. Suguro1,2, M. Asano3,4, Y. Kaneko3, D. Omagari3, B. Ogiso1,2, I. Moro5 & K.Komiyama3,4
1Department of Endodontics, Dental Research Center, Nihon University School of Dentistry; 2Division of Advanced Dental
Treatment, Dental Research Center, Nihon University School of Dentistry;3Department of Pathology, Dental Research Center,
Nihon University School of Dentistry; 4Division of Immunology and Pathobiology,
Dental Research Center, Nihon UniversitySchool of Dentistry; and 5Division Advanced Research Institute for the Sciences and
Humanities, Nihon University, Chiyoda-ku,
Tokyo, Japan
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AIM
To establish and characterize different types of
fibroblastic cell lines derived from dental pulp
tissue
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DEFINITION
TRANSFECTION
It is the process of introducing nucleic acids into cellsby non-viral methods
Transfection typically involves opening transient poresor "holes" in the cell membrane, to allow the uptake of material
Genetic material (such as supercoiled plasmid DNA or
siRNA constructs), or even proteins such as antibodies,may be transfected
In this study SV40 large T antigen was transfected withLIPOFECTAMINE (proprietary transfection reagents)
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DEFINITION
SV40 large T antigen (Simian Vacuolating Virus
40 TAg) is a hexamer protein that is an
oncogene derived from the polyomavirus
SV40 which is capable of transforming a
variety of cell types
The transforming activity of TAg is due in large
part to its perturbation of the retinoblastoma
and p53 tumour suppressor proteins
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MATERIALS AND METHODS
Cells
Human dental pulp cells were isolated from a mandibularright third molar extracted with the donors informed
consent (21-year-old female) Immediately after extraction, the tooth was rinsed in
phosphate-buffered saline (PBS), and the crown and rootportions were separated
The pulp tissue was obtained and minced in Dulbeccos
modified Eagles medium (DMEM) with 10% foetal calf serum (FCS), l-glutamine (50 lg/mL and 1%penicillin/streptomycin Solution with scissors on a 10-cmculture dish
The cells were maintained at 37oC in a 5% CO2 incubator
until they reached confluence
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MATERIALS AND METHODS
Immortalization of dental pulp-derived cells
The confluent cells were trypsinized and freshly plated on a 10-cmculture dish on the day before transfection
The cells were then transfected with 1 lg of SV40-large T antigen-encoded plasmid (pSV40) using the Lipofectamine Plus transfectionmethod
The transfection media were then applied to the cells andincubated for 24 h at 37 C in a 5% CO2 incubator
The transfection media were replaced with fresh 10% FCS-DMEM
and further cultured for 24 h The cells were replated and cultured with 10% FCSDMEM
supplemented with 400 lg/mL of geneticin (G418) to selecttransfectants
After 7 days, the surviving cells were collected with a cloning ring
Each cell was grown and sub-cloned by limited dilution
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MATERIALS AND METHODS
Detection of differentially expressed genes
To identify the genes expressed differentially
amongst established cell lines, total RNA was
extracted from each cell line and subjected to
Gene Fishing differentially expressed gene (DEG)
reactions Aliquots of the differential PCR were examined on
a 2% agarose gel stained with ethidium bromide
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MATERIALS AND METHODS
Cloning and sequencing
The differentially expressed bands were extracted
from the gel and directly cloned The cloned plasmids were sequenced with ABI
PRISM 3100 Genetic Analyzer
The obtained sequences were checked against
the database to identify the corresponding genes
Gene expression level of each gene wascompared between nine cell clones by RT-PCRusing sequence specific primers
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MATERIALS AND METHODS
Immunofluorescence and Immunohistochemical
staining
After all the initial preparation was done, the cells
were incubated with fluorescein-5-isothiocyanate
(FITC)-conjugated goat anti-mouse antibody or
(FITC)-conjugated goat anti-rabbit antibody
The cells were washed and mounted in Aqua-Polymount
The images were viewed and photographed with
a confocal laser microscope
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MATERIALS AND METHODS
Immunofluorescence and Immunohistochemicalstaining
For immunohistochemical study, the dental pulp
tissues from the extracted teeth were carefullyremoved and fixed in 4% paraformaldehyde inPBS
The fixed tissues were then dehydrated and
embedded in paraffin according to standardhistological procedures
Then 6- µm sections were cut, mounted andstored at room temperature
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MATERIALS AND METHODS
Immunofluorescence and Immunohistochemicalstaining
The sections after processing were then incubated with
a 100· dilution of polyclonal rabbit anti-human Thy-1antibody for 1 h
After washing in PBS three times, the sections wereincubated with a 1000· dilution of peroxidase-
conjugated goat anti-rabbit IgG (H + L) for 1 h The sections were thoroughly washed with PBS and
were counterstained with Mayers haematoxylin
The images were viewed and photographed using alight microscope
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RESULTS
The outgrowing cells created a sheet-likestructure on the culture dish
At this time point, cells were replated and
transfected with SV40 large T antigen After selection of SV40 large T antigen
transfectants with 400 lg/mL of G418, more than30 cell lines were observed
Although some cells ceased replication and diedafter approximately 20 passages, nine differentcell lines were maintained and to date these cellshave been growing actively for more than 2 years
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RESULTS
To determine the cell lineages of establishedcell lines, immunofluorescent cell staining wasfirst performed
Anti-fibroblast, anti-vimentin and anti-collagen type I and type III antibodies wereused
Vimentin is a member of the intermediatefilament family of proteins which exists as adynamic structure in the fibroblast
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RESULTS
All established cells showed positive staining
for these antibodies, indicating that these
cell lines had fibroblastic lineages
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RESULTS
Detection of differentially expressed genes
Although the established cell lines had
fibroblastic characteristics, their geneexpression patterns could have been different
To identify the genes which are not expressed
in dental pulp tissue, a differential display
using a Gene Fishing DEG kit was performed
using 20 arbitrary PCR primers
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RESULTS
Results of the first screening, revealed sevendifferent genes (indicated by arrows)
The DEGs were observed when arbitrary primers17 (indicated on the top) were used (each gene
was designated as DEG 1, 2, 3, 7, 9, 10 and 11respectively)
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RESULTS
These genes were purified, sub-cloned and
sequenced
The obtained sequences were databasesearched
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RESULTS
Table 2 shows the results of the search
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RESULTS
Sub-cloning of product NO. 7 yielded two
different clones designated as DEG 7A and 7B
A total of eight different genes were obtained,two of which (DEG 9 and 10) did not show any
similarity to other known sequences
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RESULTS
To confirm the obtained results, sequence-
specific primers for DEG 1, 2, 3, 7A, 7B and 11
was designed and RT-PCR was performed
Results showed that all the cell lines showed
variable expression patterns for these DEGs
and overall expression patterns were
consistent with the data obtained
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RESULTS
Detection of Thy-1+ cells in dental pulp tissue
Amongst the identified DEGs, the DEG 11 gene
was focused upon as the DNA sequenceanalysis revealed that this gene was identical
to Thy-1
Thy-1 is known as a T-cell marker, but it was
not known whether it existed in dental pulp
tissue
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RESULTS
To examine the expression of Thy-1, immuno-
histochemical staining was conducted using
anti-Thy-1 antibody
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RESULTS
Thy-1+ cells were sparsely observed in thepulp tissue
These positive reactions were specific,
because no positive staining was detected inthe negative controls
Not all cells with fibroblastic shape had Thy-1
antigen on their surface This report is the first one to confirm presence
of Thy-1 in the pulp tissue
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DISCUSSION
Hence the goal of this study to establish differentcell lines derived from human dental pulp tissuehas been achieved
This has also been established by Galler et al.(2006) who obtained only one clone whichachieved continuous growth
Galler used electroporation to introduce the SV40large T antigen but in this study Lipofectaminemethod was, which is much less harmful to thecells and hence nine different clones wereobtained
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DISCUSSION
Hence the goal of this study to establish differentcell lines derived from human dental pulp tissuehas been achieved
This has been established earlier by Galler et al.(2006) who obtained only one clone whichachieved continuous growth
Galler used electroporation to introduce the SV40large T antigen but in this study Lipofectamine
method was used and 9 different clones wereobtained, hence efficiency of cell immortalizationmay vary with the transfection method used
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DISCUSSION
All of the established cell lines stained positive
for fibroblast-specific antigen, vimentin and
collagen type I and type III, indicating that
they originated as pulp fibroblasts
Characterization of these cells has been
contributing to the understanding of the
dental pulp tissue
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DISCUSSION
Further understanding of the characteristics of
fibroblast subpopulations in dental pulp tissue
might prove advantageous in future and aid in
regeneration therapy of the pulp
Amongst the genes identified, the focus was
on Thy-1, which was thought to be a T-cell-
specific antigen and was not originallyexpected to be detected
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DISCUSSION Ovarian cancer cells, endothelial cells, neurons &
haematopoietic cells are known to express Thy-1on their surfaces
Thy-1 contributes to many biological functions,
such as cell adhesion, cell signalling, apoptosis orproliferation
But, since this is the first report to demonstratethe expression of Thy-1 in dental pulp tissue,further experiments would be needed to identifythe function of Thy-1 on dental pulp-derivedfibroblasts
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DISCUSSION
Nine fibroblastic cell clones were established,however, not all of these clones were positivefor Thy-1
Clones A7, A10 and A18 were negative for Thy-1 and DEG 2, 3, 7A and 7B, which shows thereare at least two different sub-populations of fibroblasts in dental pulp tissue
The functional differences of these two groupsare not known; hence further study isrequired to study the precise function OFthese sub-populations
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THE END