Praveen-Characterization of Human Dental Pulp-Derived Cell

8/9/2019 Praveen-Characterization of Human Dental Pulp-Derived Cell

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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



AIM

To establish and characterize different types of

fibroblastic cell lines derived from dental pulp

tissue



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)



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



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




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




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




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




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




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




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



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



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



RESULTS

All established cells showed positive staining

for these antibodies, indicating that these

cell lines had fibroblastic lineages



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



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)



RESULTS

These genes were purified, sub-cloned and

sequenced

The obtained sequences were databasesearched



RESULTS

Table 2 shows the results of the search



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



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



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



RESULTS

To examine the expression of Thy-1, immuno-

histochemical staining was conducted using

anti-Thy-1 antibody



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



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



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



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



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



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



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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Praveen-Characterization of Human Dental Pulp-Derived Cell