Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10229
RESEARCH ARTICLE
Proliferation rate of stem cells derived from human dental pulp and identification of differentially expressed genes Muhammad Fawwaz Abdullah1, Siti Fadilah Abdullah1, Nor Shamsuria Omar1, Zuliani Mahmood1, Siti Noor Fazliah Mohd Noor2, Thirumulu Ponnuraj Kannan1,3* and Khairani Idah Mokhtar1 1 School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia 2 Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Pulau Pinang, Malaysia 3 Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
Abstract Stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs) obtained from the dental pulp of human extracted tooth were cultured and characterized to confirm that these were mesenchymal stem cells. The proliferation rate was assessed using AlamarBlue1 cell assay. The differentially expressed genes in SHED and DPSCs were identified using the GeneFishingTM technique. The proliferation rate of SHED (P < 0.05) was significantly higher than DPSCs while SHED had a lower multiplication rate and shorter population doubling time (0.01429, 60.57 h) than DPSCs (0.00286, 472.43 h). Two bands were highly expressed in SHED and three bands in DPSCs. Sequencing analysis showed these to be TIMP metallopeptidase inhibitor 1 (TIMP1), and ribosomal protein s8, (RPS8) in SHED and collagen, type I, alpha 1, (COL1A1), follistatin-like 1 (FSTL1), lectin, galactoside-binding, soluble, 1, (LGALS1) in DPSCs. TIMP1 is involved in degradation of the extracellular matrix, cell proliferation and anti-apoptotic function and RPS8 is involved as a rate-limiting factor in translational regulation; COL1A1 is involved in the resistance and elasticity of the tissues; FSTL1 is an autoantigen associated with rheumatoid arthritis; LGALS1 is involved in cell growth, differentiation, adhesion, RNA processing, apoptosis and malignant transformation. This, along with further protein expression analysis, holds promise in tissue engineering and regenerative medicine. Keywords: DEGs; DPSCs; SHED; stem cells; proliferation rate Introduction Stem cells are defined as clonogenic cells capable of both selfrenewal and multi-lineage differentiation. Post-natal stem cells have been isolated from various tissues, including bone marrow, neural tissue, skin, retina and dental epithelium (Harada et al., 1999; Fuchs and Segre, 2000; Bianco et al., 2001; Blau et al., 2001). Gronthos et al. (2000) identified dental pulp stem cells (DPSCs) while Miura et al. (2003) identified stem cells from human exfoliated deciduous teeth (SHED). SHED and DPSCs from permanent teeth are rich in mesenchymal stem cells (MSCs). These adult stem cells have high proliferation and are able to differentiate into a variety of cell types including neural cells, adipocytes and odontoblasts (Miura et al., 2003). It has become important to assess the cell growth and the proliferation of cells in any cell culture study as it helps the researcher to optimise the culture conditions for the cells, to determine the growth factors as well as the
activity of cytokines. It also enables scientists to develop new therapeutic agents and to assess their efficacy, to evaluate the cytostatic potential of anti-cancer drugs, and also to assess the cell mediated toxicity. Measurements of viability are a help in assessing the death or life of cancerous cells, the rejection of implanted organs, or the effectiveness of a drug candidate. This assay aids in determining the optimal growth conditions of cell populations maintained in culture and collectively, they provide us with information on whether the cells under experimentation is healthy, dividing or in apoptosis (May, 2014). In addition, differences in proliferation rate between SHED and DPSCs will suggest the presence of other molecular interactions that might regulate the behaviour of the cells. The identification of differentially expressed genes (DEGs) observed in these stem cells (SCs) will enable further understanding regarding the fundamental aspects of stem cell biological activity. Identification of the signalling molecules and pathways associated in controlling the growth of SHED
Corresponding author: e-mail: [email protected]
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and DPSCs can then be applied in tissue regeneration. Hence, the gene expression profile and functional pathway of SHED and DPSCs need to be explored to determine their biological functional activity which can be used in determining the usefulness of these stem cells for cell-based regeneration therapy. The GeneFishingTM technique using annealing control primer (ACP) system allows the identification of differentially expressed mRNA transcripts (Kim et al., 2004). Hence, this study employs the GeneFishingTM technique to identify the DEGs in SHED and DPSCs which will throw light on the differences in the proliferation rate of stem cells from deciduous and permanent teeth.
Cell Technologies, Canada), 2 mM L-glutamine (GIBCOTM, Invitrogen, Japan), 100 U/mL penicillin and 100 mg/mL streptomycin (Gronthos et al., 2000). The culture was incubated at 378C in air plus 5% CO2. The stem cell lineage was confirmed using immunocytochemistry staining to detect human mesenchymal stem cell. Characterisation was performed by immunoperoxidase secondary detection system with primary antibodies mouse monoclonal antihuman endoglin/CD105 and mouse monoclonal antihuman CD166 (Chemicon, USA).
Materials and methods
The samples were plated in 96 multiwell plate at 1,000 cells in 100 mL aMEM, supplemented with 20% (v/v) FBS, 100 mM L-ascorbic acid, 1% (v/v) penicillin-streptomycin and 20 mM L-glutamine. Culture medium (100 mL) without cells was used as background control. AlamarBlue1 cell viability reagent (10 mL) (Invitrogen, USA) was added into each well and incubated for 24 h. After incubation, the absorbance was measured using ELISA Reader (570 and 600 nm) (Tecan, DKSH, Germany). The absorbance was measured every day from Day 1–10, to observe the growth pattern for SHED and DPSCs. The absorbance of alamarBlue was converted to percentage of reduction using the formula provided by Willard et al. (1965) as below.
Ethical approval Ethical approval for conducting this study was obtained from the Research and Ethics Committee (Human) of Universiti Sains Malaysia, Health Campus vide reference USMKK/PPP/ JEPeM(211.3[12]) dated 7 April, 2009 and revised on 22 June 2010.
Sample collection A total of eight teeth, four from patients aged between four and 7 years with at least one carious teeth class 1 (for SHED analysis) and 4 from patients aged 10–40 years (for DPSC analysis) were collected in this study. Prior to the collection of teeth, informed consent was obtained from the adults and in the case of children below 18 years of age, from the parent. The dental pulp was extracted and cultured (Fazliah et al., 2010). RNA isolation and differential display analyses were obtained from the confluent cells from passage 4.
Dissection of tissue and isolation of stem cells from dental pulp Within 24 h of collection, the teeth were cut at the enamelcementum junction using a hard material cutter. Cut teeth were briefly immersed in 75% ethanol followed by soaking in phosphate buffer saline (PBS). The pulp was separated from a remnant crown and digested in a solution of 3 mg/mL collagenase type I (Worthing Biochem, USA) overnight at 378C. Single-cell suspensions were obtained by passing the cells through a 70 mm strainer (BD FalconTM, USA).
Culture and characterisation of stem cells from human dental pulp Single-cell suspensions were cultured in alpha modified Eagle’s medium (aMEM), (BioWhittakerTM, USA) supplemented with 20% fetal bovine serum (FBS) (GIBCOTM, Invitrogen, USA), 100 mM L-ascorbic acid 2-phosphate (Stem Cell Biol Int 38 (2014) 582–590 ß 2013 International Federation for Cell Biology
Determination of proliferation rate by alamarBlue assay
%Reduction ¼
ðeOXÞl2 Al1 ðeOXÞl1Al2 ðeREDÞl1 A0 l2 ðeREDÞl2A0 l1
where, (eox)l2 ¼ molar extinction coefficient of alamarBlue oxidized from (BLUE) at 600 nm ¼ 117,216 (eox)l1 ¼ molar extinction coefficient of alamarBlue oxidized from (BLUE) at 570 nm ¼ 80,586 (eRED)l1 ¼ molar extinction coefficient of alamarBlue oxidized from (RED) at 570 nm ¼ 155,677 (eRED)l2 ¼ molar extinction coefficient of alamarBlue oxidized from (RED) at 600 nm ¼ 14,652 Al1 ¼ observed absorbance reading for test well at 570 nm Al2 ¼ observed absorbance reading for test well at 600 nm A0 l1 ¼ observed absorbance reading for negative well at 570 nm A0 l2 ¼ observed absorbance reading for negative well at 600 nm Determination of cell proliferation rate Cell multiplication rate (r) and population doubling time (PDT) were calculated according to the estimated cell number in the growth curve of SHED and DPSCs using the formula provided by Davis (2002) and analysed using Mann–Whitney test. Seven replicates of cultured SHED and DPSCs were measured for r and PDT. Multiplication rate (r) 583
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is the number of generations that occur per unit time and is usually expressed as population doublings per 24 h. Population doubling time (PDT) is the time, expressed in hours, taken for cell number to double, and is the reciprocal of the multiplication rate (i.e. 1/r) (Davis, 2002).
median proliferation rates were different between SHED and DPSCs. The proliferation rate of SHED was significantly higher than DPSCs (Table 1).
total time elapsed PDT ¼ number of generations
Two genes in SHED and three genes in DPSCs were highly expressed (Figure 2A). The sequencing and BLAST results showed that those genes were TIMP metallopeptidase inhibitor 1 (TIMP1), (A09) (with 82% query coverage and 93% identity), and ribosomal protein s8, (RPS8), (A16) (with 90% query coverage and 99% identity) while genes which were highly expressed in DPSCs were collagen, type I, alpha 1, (COL1A1), (A20) (97% query coverage and 99% identity), follistatin-like 1 (FSTL1), (A17) (96% query coverage and 100% identity), lectin, galactoside-binding, soluble, 1, (LGALS1), (A16) (with 75% query coverage and 96% identity). To confirm the efficacy of the ACP system, the DEGs were confirmed by RT-PCR using gene specific primer pairs. The primers (Table 2) designed using Primer3 Input program version 0.4.0 (http://frodo.wi.mit.edu/) and ordered from 1st BASE (M) SdnBhd Company, Malaysia were used for RT-PCR. The Arbitrary A09, A16, A17 and A20 were subjected to confirmation using RT-PCR (Figure 2A). The RT-PCR confirmed the ACP observation (Figure 2B). Analysis of relative intensity of gene expression also showed that SHED expressed significantly higher amounts of TIMP1 and RPS8 compared to DPSCs while DPSCs expressed significantly higher amounts of FSTL1 and LGALS1 compared to SHED, but not COL1A1 (Figure 3).
Reverse transcriptase-polymerase chain reaction (RT-PCR) – GeneFishing technique first-strand cDNA synthesis The cDNA synthesis was done according to the protocol provided (Epicentre1 Biotechnologies, USA). ACP – GeneFishing PCR Synthesis of second-strand cDNAwas done following the GeneFishingTM DEG Premix kit protocol (Seegene, Korea). The PCR products were electrophoresed on 2% agarose gel and visualised under UV light. DNA purification and sequencing The gels with differentially expressed bands were extracted using QIAquick1 gel extraction kit (Qiagen, USA) and the purified DNAs were sent for sequencing. The gene sequences were analysed using Basic Local Alignment Search Tool (BLAST) software. Confirmation with RT-PCR RT-PCR with specific gene primer pairs were used to confirm the differential expressions of DEGs. Human b-actin was used first as normalisation to the first-strand cDNA. Then, the normalised cDNA was used as a template. Results
Culture and characterisation of stem cells from human dental pulp The cells cultured in this study displayed the typical morphology of SHED and DPSCs (Figure 1A–F) and were grown to a confluence of 80 to 100%. The stem cells from dental pulp results showed the presence of brownish colour indicating positive reactivity for CD105 and CD166 primary antibodies, which characterised SHED and DPSCs (Figure 1G–L).
Proliferation rate of SHED and DPSCs The analysis results showed that SHED had lower r and shorter PDT (0.01429, 60.57 h) than DPSCs (0.00286, 472.43 h), respectively. At 5% level of significance, the 584
DEGs in SHED and DPSCs
Discussion Immunocytochemistry is used as junction methods for the identification of cells in tissues sections. Characterization of SHED and DPSCs relied on immunocytochemistry staining because of its stability, sensitivity, clear cytomorphological details, ease of use, cost-effectiveness and low technology needed (Leong, 1996). According to Dominici et al. (2006), the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed three minimal criteria to define human MSCs: they must be plastic-adherent when maintained in standard culture conditions, must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface molecules. Both SHED and DPSCs were plastic-adherent cells that were found positive for antibodies against human antigens CD105 (a component of the transforming growth factor (TGF) receptor) and CD166 (a member of the Ig superfamily), which are the mesenchymal stem cell markers and positive for negative control. This means that both SHED and DPSCs can be considered as human MSC. Cell Biol Int 38 (2014) 582–590 ß 2013 International Federation for Cell Biology
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Figure 1 Morphology of SHED and DPSCs. Based on stem cell cultures, observed on days 0, 7 and 15. Day 0–Digested pulp of both SHED (A) and DPSCs (D) showing fibroblasts, mesenchymal cells and macrophages floating inside the flask at this early stage; Day 7–Small rounded and spindle shaped cells seen attached to the flask (B, E); Day 15–Spindle-shaped cells showing 80% confluence (40) (C, F). Characterization of stem cells from dental pulp by immunocytochemistry staining using CD105 and CD166 antibodies were positive in SHED (G, K), DPSCs (H, L), hMSCs (I, M) (positive control-without any antibody) and hMSCs (J, N) (negative control) (100).
GeneFishingTM DEG Premix Kits consist of three steps; reverse transcription and two-stage PCR, which uses primers annealing specifically to the template thus amplifying only
Table 1 Comparison of multiplication rate (r) and population doubling time (PDT) between SHED and DPSCs using alamarBlue assay. Group r PDT
SHED DPSCs SHED DPSCs
Median (IqR) 0.0198 0.002 51 506
(0.006) (0.000) (21) (72)
Z-statistica
P-value
3.298
0.001
3.205
0.001
Mann–Whitney test (P < 0.05).
a
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genuine products. This system hence overcomes the difficulty in identifying the genes responsible for a specialized function during a certain biological stage as the gene is expressed at low levels, whereas most mRNA transcripts within a cell are abundantly expressed (Kim et al., 2004). Theoretically, in comparison with DPSCs, SHED provides a good source for stem cells and is enriched with extracellular matrix. SHED had discrete patterns of gene expression compared to DPSCs (Nakamura et al., 2009). TGF-b2 stimulates cell proliferation and collagen synthesis (Lee et al., 2006) and might regulate the biological activities in SHED. Nakamura et al. (2009) also demonstrated that SHED displayed increased expression of several growth factors such as FGF, TGF-b, connective tissue growth factor (CTGF), nerve growth factor (NGF) and bone morphogenetic protein 585
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Figure 2 GeneFishingTM DEG screening results. Based on the GeneFishing technique, five DEGs were detected. (A) The arrows show the genes which were highly expressed in SHED or DPSCs using arbitrary primers A09, A16, A17 and A20. Lanes 2, 4, 6, 8, 10, 12, 14: DEGs from DPSCs; Lanes 3, 5, 7, 9, 11, 13, 15: DEGs from SHED and Lane 1: DNA marker (100 bp). After gene sequencing, the efficacy analysis of five selected arbitrary ACP primers were confirmed by RT-PCR (B). Arbitrary A09 (TIMP1), A16 (LGALS1, RPS8), A17 (FSTL1) and A20 (COL1A1) were confirmed with RT-PCR to confirm the ACP observation. Lanes 2, 4, 6, 8, 10: Total DNA from DPSCs; Lanes 3, 5, 7, 9, 11: Total DNA from SHED; Lanes 12, 13:b-actin, a housekeeping gene and Lane 1: DNA marker (100 bp).
(BMP), which can promote the proliferation of legion types of cells. In addition, CTGF, whose expression was higher in SHED, is a matrix signalling molecule involved in TGF-binduced cell proliferation, matrix synthesis, angiogenesis, migration and osteoblast lineage differentiation of MSCs (Luo et al., 2004). These findings suggest that TGF-b and
CTGF might be involved in the regulation of biological functions of SHED. Based on sequencing analysis, the specific genes that differentially expressed in SHED and DPSCs were identified as TIMP1, RPS8, LGALS1, FSTL1 and COL1A1. They were identified from the results of the chromatograms of
Table 2 Primer pairs and product sizes for the differentially expressed genes and b-actin for reverse transcriptase-polymerase chain reaction designed using primer3 software. Gene
Arbitrary
Sequences
Size (bp)
COL1A1
A20
496
FSTL1
A17
LGALS1
A16
TIMP1
A09
RPS8
A16
F: 50 -ACAGTGATTGAATACAAAACCA-0 3 R: 50 -GTGGAGAAAGGAGCAGAAAG-0 3 F: 50 -TAGCATCTGTTAAGATCCAGTG-0 3 R: 50 -TCCACTCTTAGGAAGTAAATGG-0 3 F: 50 -TGGATACGAATTCAAGTTCC-0 3 R: 50 -TACTATGTGCCAAACTCTGTGT-0 3 F: 50 -CAACAGATGTATAAAGGGTTCC-0 3 R: 50 -CCTTCTGATAGACTGAAATTGG-0 3 F: 50 -GTTGTGCTAAGGATCACCTACT-0 3 R: 50 -GAGTTGAGAACAGGGACTTTAC-0 3 F: 50 -TGGCACCACACCTTCTACAATGAGC-30 R: 50 -GCACAGCTTCTCCTTAATGTCACGC-30
b-actin
586
—
391 347 270 350 395
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Figure 3 Analysis of relative intensity of gene expression for the differentially expressed genes in SHED and DPSCs. SHED expressed significantly higher amounts of TIMP1 and RPS8 compared to DPSCs while DPSCs expressed significantly higher amounts of FSTL1 and LGALS1 compared to SHED but not COL1A1.
sequencing analysis, using BioEdit software and further carrying out BLASTanalysis. BLASTanalysis also showed that all sequences produced significant alignment with highest query coverage and maximum identity. Interestingly, in our study, TIMP1 was one of the DEGs highly expressed in SHED. TIMP1 belongs to TIMPs that constitute a family of secreted proteins, whose primary function is inhibition of the degradative action of matrix metalloproteinases (MMPs) and regulation of ECM turnover and tissue remodelling. MMPs are zinc-dependent endopeptidases involved in only invasion and metastasis, but evidence suggests that certain MMPs also play a role in tumourigenesis (Chambers and Matrisian, 1997). ECM is a dynamic structure that not only provides a scaffold for organising tissue architecture, but also contributes signals that regulate cell function. TIMPs also are also involved in homeostasis of the ECM by regulating the activities of MMPs including proliferation, differentiation, apoptosis, tumour angiogenesis, tumourigenesis and metastasis (Visse and Nagase, 2003). However, the regulation of TIMP1 expression is incompletely understood. Hence, the increased cell proliferation of SHED in comparison to DPSCs could be attributed to the high expression of the TIMP1 gene. The role of TGF-b1 in liver fibrosis is in part related to impairment of ECM breakdown by stimulation of TIMP1. Flisiak et al. (2002) found that chronic viral hepatitis B and C resulted in a significant increase in plasma TIMP1 levels, but not TGF-b1. Yang et al. (2002) reported that there was correlation between the expression of TGF-b1 and MMP-1, but no correlation between TGF-b1 and TIMP1 in the expression of TGF-b1 MMP-1 and its inhibitor in human middle ear cholesteatoma. Cell Biol Int 38 (2014) 582–590 ß 2013 International Federation for Cell Biology
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Another DEG is COL1A1. Type I collagen is present in most organs, and synthesised only by a small number of discrete cell types, including fibroblasts, osteoblasts and odontoblasts, which is a major component of many ECMs, and its accumulation characterises most fibrotic processes. Cytokines including interleukin-1 (IL-1) and IL4, growth factors such as TGF-b and insulin-like growth factor-1 (IGF-1), vasoactive peptide such as endothelin-1, or other molecules such as lipid peroxidation products can stimulate type I collagen production by fibroblastic cells (Rossert et al., 1996). COL1A1 encodes the a1 chain of collagen type I. This gene is of particular interest because of its involvement in resistance and elasticity of the tissues. COL1A1 is elevated in tumour endothelium, compared with normal endothelium, suggesting that they play an important role in angiogenesis and formation of desmoplasia. Overexpression of COL1A1 can also be associated with invasive process in papillary thyroid cancer (PTC) (Lee et al., 2009). Many diseases, such as Ehler–Danlos syndrome, osteogenesis imperfect, chondrodysplasia, low bone mineral density (Kuivaniemi et al., 1997) and CDH (Skirving et al., 1984) have been associated with COL1A1. Our results show that COL1A1 was overexpressed in DPSCs compared to SHED, which is in contrast to the report of Wang et al. (2012). Col 1 contains the majority of the ECM and is important in maintaining the biological and structural integrity of the ECM architecture (Cen et al., 2008). Ascorbic acid can stimulate distinguishable expression of Col 1 in DPSCs because these cells had a structure that was enriched with ECM (Park et al., 2010). The expression of COL1A1 might be due to the addition of L-ascorbic acid 2-phosphate into the culture medium during the culturing process. L-ascorbic acid 2-phosphate can stimulate collagen accumulation (Hata and Senoo, 1989). The use of L-ascorbic acid 2-phosphate in the culture medium also increases the production of type I and type III collagen peptide in cultured rabbit keratinocytes (Saika et al., 1992). LGALS1 was also overexpressed in DPSCs compared to SHED. High levels of Gal-1, a b-galactoside binding secreted protein, correlate with aggressiveness of tumours and acquisition of a metastatic phenotype of tumours, resulting in a poor prognosis (Jung et al., 2007). Gal-1 also promotes proliferation and migration of glioblastoma cell lines in vitro (Camby et al., 2002), acts as pro-angiogenic factor in tumour angiogenesis (Le Mercier et al., 2008), promotes proliferation and migration of endothelial cells (Thijssen et al., 2006) and vascular smooth muscle cells (Moiseeva et al., 2000), leading to influences on the formation of new blood vessels. Crosstalk exists between tumour-derived suppressive factors; TGF-b triggers Gal-1 expression via (mothers against decapentaplegic homolog) SMAD3/SMAD4 dependent pathway (Daroqui et al., 2007) and in turn, Gal-1 induces IL-10 production in monocytes and T-cells (van der Leij et al., 2007). Moreover, 587
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Gal-1 is a key regulator of murine CD4þCD25þFoxp3 regulatory T-cells (Garin et al., 2007) which is essential in suppression of anti-cancer immunity (Orentas et al., 2006). Gal-1 also inhibits the immune response at the level of leukocyte infiltration; it decreases the adhesion and extravasation of lymphocytes and neutrophils through activate dendothelium (Cooper et al., 2008). In addition, Gal-1 expression in tumour cells or the surrounding stroma intimately regulates the function and vitality of infiltrating-Tcells (Gandhi et al., 2007). Gal-1 has many prospects for therapeutic applications. Gal-1 has critical importance in neuronal cell differentiation and survival in the central and the peripheral nervous systems, plus the establishment and maintenance of T-cell tolerance and homeostasis in vivo. Gal-1 expression or overexpression in tumours or the surrounding tissue can also be considered a sign of their malignant progression. The targeted overexpression (or delivery) of Gal-1 should be selected as a novel approach for the treatment of inflammation-related diseases, such as graft versus host disease (GVHD) (Baum et al., 2003), arthritis (Rabinovich et al., 1999), colitis (Santucci et al., 2003), and nephritis (Tsuchiyama et al., 2000). This gene could be also seen as a possible therapeutic aim in some neurodegenerative diseases (Kadoya and Horie, 2005) and muscular dystrophies (Goldring et al., 2002). Limiting the migration of cancer cells by depress-expressing the Gal-1 in the cancer cells restores a level of sensitivity to cell death, and also cytotoxic drugs (Lefranc et al., 2005). Hence, anti-Gal-1 compounds are needed to fight migrating cancer cells (NangiaMakker et al., 2002; Sorme et al., 2003; Ingrassia et al., 2006). We have shown the differences between SHED and DPSCs with regard to their characteristics, immunocytology, proliferation and gene expression. These results can be used to determine and measure their functional activities. The specific gene expression patterns of SHED and DPSCs, and the underlying differences that exist between them, might be useful in determining the functional roles of these relevant genes pertaining to its specific protein expression. This knowledge may have application in stem cell therapies, using stem cells obtained from human dental pulp as multipotent cell sources for genetic and tissue engineering technology. Conflict of interest statement The authors declare that they have no competing interests. Acknowledgements and funding We would like to express our gratitude to the clinicians, staff, patients and patients’ families for their cooperation in this study. This study was supported by the Universiti Sains Malaysia Short term grant (304/PPSG/6139051). 588
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in adamantinomatous craniopharyngiomas: their ability to regulate tumor adhesiveness to surrounding tissue and their contribution to prognosis. Neurosurgery 56(4): 763–76. Leong SY (1996) Principles and practice of medical laboratory science, vol. 1. New York: Churchill Livingstone. Luo Q, Kang Q, Si W, Jiang W, Park JK, Peng Y, Li X, Luu HH, Luo J, Montag AG, Haydon RC, Chuan T (2004) Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J Biol Chem 279(53): 55958– 68. May O (2014) Assay kits. Determining Cell Vitality. Cayman Chemical Company, Ann Arbor, Michigan, USA. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S (2003) SHED—stem cells from human exfoliated deciduous teeth. J Dent Res 82: B305. Moiseeva EP, Javed Q, Spring EL, de Bono DP (2000) Galectin 1 is involved in vascular smooth muscle cell proliferation. Cardiovasc Res 45(2): 493–502. Nakamura S, Yamada Y, Katagiri W, Sugito T, Ito K, Ueda M (2009) Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod 35(11): 1536–42. Nangia-Makker P, Conklin J, Hogan V, Raz A (2002) Carbohydrate-binding proteins in cancer, and their ligands as therapeutic agents. Trends Mol Med 8(4): 187–92. Orentas RJ, Kohler ME, Johnson BD (2006) Suppression of anticancer immunity by regulatory T cells: back to the future. Semin Cancer Biol 16(2): 137–49. Park HJ, Ock SM, Kim HJ, Lee YB, Choi JM, Cho CS, Lee JY, Cho BK, Cho DH (2010) Vitamin C attenuates ERK signalling to inhibit the regulation of collagen production by LL-37 in human dermal fibroblasts. Exp Dermatol 19(8): e258–64. Rabinovich GA, Daly G, Dreja H, Tailor H, Riera CM, Hirabayashi J, Chernajovsky Y (1999) Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis. J Exp Med 190(3): 385–98. Rossert JA, Chen SS, Eberspaecher H, Smith CN, de Crombrugghe B (1996) Identification of a minimal sequence of the mouse proalpha 1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts. Proc Natl Acad Sci USA 93(3): 1027–31. Saika S, Uenoyama K, Hiroi K, Ooshima A (1992) L-ascorbic acid 2-phosphate enhances the production of type I and type III collagen peptides in cultured rabbit keratocytes. Ophthalmic Res 24(2): 68–72. Santucci L, Fiorucci S, Rubinstein N, Mencarelli A, Palazzetti B, Federici B, Rabinovich GA, Morelli A (2003) Galectin-1 suppresses experimental colitis in mice. Gastroenterology 124(5): 1381–94. Skirving AP, Sims TJ, Bailey AJ (1984) Congenital dislocation of the hip: a possible inborn error of collagen metabolism. J Inherit Metab Dis 7(1): 27–31.
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Sorme P, Kahl-Knutsson B, Wellmar U, Magnusson BG, Leffler H, Nilsson UJ (2003) Design and synthesis of galectin inhibitors. Methods Enzymol 363: 157–69. Thijssen VLJL, Postel R, Brandwijk RJMG, Dings RPM, Nesmelova I, Satijn S, Verhofstad N, Nakabeppu Y, Baum LG, Bakkers J, Mayo KH, Poiner F, Griffioen AW (2006) Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy. Proc Natl Acad Sci USA 103(43): 15975–80. Tsuchiyama Y, Wada J, Zhang H, Morita Y, Hiragushi K, Hida K, Shikata K, Yamamura M, Kanwar YS, Makino H (2000) Efficacy of galectins in the amelioration of nephrotoxic serum nephritis in Wistar Kyoto rats. Kidney Int 58(5): 1941–52. van der Leij J, van den Berg A, Harms G, Eschbach H, Vos H, Zwiers P, van Weeghel R, Groen H, Poppema S, Visser L (2007) Strongly enhanced IL-10 production using stable galectin-1 homodimers. Mol Immunol 44(4): 506–13.
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Visse R, Nagase H (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 92(8): 827–39. Wang X, Sha XJ, Li GH, Yang FS, Ji K, Wen LY, Liu SY, Chen L, Ding Y, Xuan K (2012) Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch Oral Biol 57(9): 1231–40. Willard HH, Merritt LL, Dean JA (1965) Ultraviolet and visible absorption methods. In: Instrumental methods of analysis. New York: Van Nostrand, pp. 94–5. Yang X, Li X, Ma M, Zhang L, Zhang Q, Wang J, Wang B (2002) Expression of transforming growth factor-beta 1 matrix metalloproteinase-1 and its inhibitor in human middle ear cholesteatoma. Zhonghua Er Bi Yan Hou Ke Za Zhi 37(2): 121–23. Received 14 May 2013; accepted 10 December 2013. Final version published online 13 January 2014.
Cell Biol Int 38 (2014) 582–590 ß 2013 International Federation for Cell Biology
RESEARCH ARTICLE
Proliferation rate of stem cells derived from human dental pulp and identification of differentially expressed genes Muhammad Fawwaz Abdullah1, Siti Fadilah Abdullah1, Nor Shamsuria Omar1, Zuliani Mahmood1, Siti Noor Fazliah Mohd Noor2, Thirumulu Ponnuraj Kannan1,3* and Khairani Idah Mokhtar1 1 School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia 2 Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Pulau Pinang, Malaysia 3 Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
Abstract Stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs) obtained from the dental pulp of human extracted tooth were cultured and characterized to confirm that these were mesenchymal stem cells. The proliferation rate was assessed using AlamarBlue1 cell assay. The differentially expressed genes in SHED and DPSCs were identified using the GeneFishingTM technique. The proliferation rate of SHED (P < 0.05) was significantly higher than DPSCs while SHED had a lower multiplication rate and shorter population doubling time (0.01429, 60.57 h) than DPSCs (0.00286, 472.43 h). Two bands were highly expressed in SHED and three bands in DPSCs. Sequencing analysis showed these to be TIMP metallopeptidase inhibitor 1 (TIMP1), and ribosomal protein s8, (RPS8) in SHED and collagen, type I, alpha 1, (COL1A1), follistatin-like 1 (FSTL1), lectin, galactoside-binding, soluble, 1, (LGALS1) in DPSCs. TIMP1 is involved in degradation of the extracellular matrix, cell proliferation and anti-apoptotic function and RPS8 is involved as a rate-limiting factor in translational regulation; COL1A1 is involved in the resistance and elasticity of the tissues; FSTL1 is an autoantigen associated with rheumatoid arthritis; LGALS1 is involved in cell growth, differentiation, adhesion, RNA processing, apoptosis and malignant transformation. This, along with further protein expression analysis, holds promise in tissue engineering and regenerative medicine. Keywords: DEGs; DPSCs; SHED; stem cells; proliferation rate Introduction Stem cells are defined as clonogenic cells capable of both selfrenewal and multi-lineage differentiation. Post-natal stem cells have been isolated from various tissues, including bone marrow, neural tissue, skin, retina and dental epithelium (Harada et al., 1999; Fuchs and Segre, 2000; Bianco et al., 2001; Blau et al., 2001). Gronthos et al. (2000) identified dental pulp stem cells (DPSCs) while Miura et al. (2003) identified stem cells from human exfoliated deciduous teeth (SHED). SHED and DPSCs from permanent teeth are rich in mesenchymal stem cells (MSCs). These adult stem cells have high proliferation and are able to differentiate into a variety of cell types including neural cells, adipocytes and odontoblasts (Miura et al., 2003). It has become important to assess the cell growth and the proliferation of cells in any cell culture study as it helps the researcher to optimise the culture conditions for the cells, to determine the growth factors as well as the
activity of cytokines. It also enables scientists to develop new therapeutic agents and to assess their efficacy, to evaluate the cytostatic potential of anti-cancer drugs, and also to assess the cell mediated toxicity. Measurements of viability are a help in assessing the death or life of cancerous cells, the rejection of implanted organs, or the effectiveness of a drug candidate. This assay aids in determining the optimal growth conditions of cell populations maintained in culture and collectively, they provide us with information on whether the cells under experimentation is healthy, dividing or in apoptosis (May, 2014). In addition, differences in proliferation rate between SHED and DPSCs will suggest the presence of other molecular interactions that might regulate the behaviour of the cells. The identification of differentially expressed genes (DEGs) observed in these stem cells (SCs) will enable further understanding regarding the fundamental aspects of stem cell biological activity. Identification of the signalling molecules and pathways associated in controlling the growth of SHED
Corresponding author: e-mail: [email protected]
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and DPSCs can then be applied in tissue regeneration. Hence, the gene expression profile and functional pathway of SHED and DPSCs need to be explored to determine their biological functional activity which can be used in determining the usefulness of these stem cells for cell-based regeneration therapy. The GeneFishingTM technique using annealing control primer (ACP) system allows the identification of differentially expressed mRNA transcripts (Kim et al., 2004). Hence, this study employs the GeneFishingTM technique to identify the DEGs in SHED and DPSCs which will throw light on the differences in the proliferation rate of stem cells from deciduous and permanent teeth.
Cell Technologies, Canada), 2 mM L-glutamine (GIBCOTM, Invitrogen, Japan), 100 U/mL penicillin and 100 mg/mL streptomycin (Gronthos et al., 2000). The culture was incubated at 378C in air plus 5% CO2. The stem cell lineage was confirmed using immunocytochemistry staining to detect human mesenchymal stem cell. Characterisation was performed by immunoperoxidase secondary detection system with primary antibodies mouse monoclonal antihuman endoglin/CD105 and mouse monoclonal antihuman CD166 (Chemicon, USA).
Materials and methods
The samples were plated in 96 multiwell plate at 1,000 cells in 100 mL aMEM, supplemented with 20% (v/v) FBS, 100 mM L-ascorbic acid, 1% (v/v) penicillin-streptomycin and 20 mM L-glutamine. Culture medium (100 mL) without cells was used as background control. AlamarBlue1 cell viability reagent (10 mL) (Invitrogen, USA) was added into each well and incubated for 24 h. After incubation, the absorbance was measured using ELISA Reader (570 and 600 nm) (Tecan, DKSH, Germany). The absorbance was measured every day from Day 1–10, to observe the growth pattern for SHED and DPSCs. The absorbance of alamarBlue was converted to percentage of reduction using the formula provided by Willard et al. (1965) as below.
Ethical approval Ethical approval for conducting this study was obtained from the Research and Ethics Committee (Human) of Universiti Sains Malaysia, Health Campus vide reference USMKK/PPP/ JEPeM(211.3[12]) dated 7 April, 2009 and revised on 22 June 2010.
Sample collection A total of eight teeth, four from patients aged between four and 7 years with at least one carious teeth class 1 (for SHED analysis) and 4 from patients aged 10–40 years (for DPSC analysis) were collected in this study. Prior to the collection of teeth, informed consent was obtained from the adults and in the case of children below 18 years of age, from the parent. The dental pulp was extracted and cultured (Fazliah et al., 2010). RNA isolation and differential display analyses were obtained from the confluent cells from passage 4.
Dissection of tissue and isolation of stem cells from dental pulp Within 24 h of collection, the teeth were cut at the enamelcementum junction using a hard material cutter. Cut teeth were briefly immersed in 75% ethanol followed by soaking in phosphate buffer saline (PBS). The pulp was separated from a remnant crown and digested in a solution of 3 mg/mL collagenase type I (Worthing Biochem, USA) overnight at 378C. Single-cell suspensions were obtained by passing the cells through a 70 mm strainer (BD FalconTM, USA).
Culture and characterisation of stem cells from human dental pulp Single-cell suspensions were cultured in alpha modified Eagle’s medium (aMEM), (BioWhittakerTM, USA) supplemented with 20% fetal bovine serum (FBS) (GIBCOTM, Invitrogen, USA), 100 mM L-ascorbic acid 2-phosphate (Stem Cell Biol Int 38 (2014) 582–590 ß 2013 International Federation for Cell Biology
Determination of proliferation rate by alamarBlue assay
%Reduction ¼
ðeOXÞl2 Al1 ðeOXÞl1Al2 ðeREDÞl1 A0 l2 ðeREDÞl2A0 l1
where, (eox)l2 ¼ molar extinction coefficient of alamarBlue oxidized from (BLUE) at 600 nm ¼ 117,216 (eox)l1 ¼ molar extinction coefficient of alamarBlue oxidized from (BLUE) at 570 nm ¼ 80,586 (eRED)l1 ¼ molar extinction coefficient of alamarBlue oxidized from (RED) at 570 nm ¼ 155,677 (eRED)l2 ¼ molar extinction coefficient of alamarBlue oxidized from (RED) at 600 nm ¼ 14,652 Al1 ¼ observed absorbance reading for test well at 570 nm Al2 ¼ observed absorbance reading for test well at 600 nm A0 l1 ¼ observed absorbance reading for negative well at 570 nm A0 l2 ¼ observed absorbance reading for negative well at 600 nm Determination of cell proliferation rate Cell multiplication rate (r) and population doubling time (PDT) were calculated according to the estimated cell number in the growth curve of SHED and DPSCs using the formula provided by Davis (2002) and analysed using Mann–Whitney test. Seven replicates of cultured SHED and DPSCs were measured for r and PDT. Multiplication rate (r) 583
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is the number of generations that occur per unit time and is usually expressed as population doublings per 24 h. Population doubling time (PDT) is the time, expressed in hours, taken for cell number to double, and is the reciprocal of the multiplication rate (i.e. 1/r) (Davis, 2002).
median proliferation rates were different between SHED and DPSCs. The proliferation rate of SHED was significantly higher than DPSCs (Table 1).
total time elapsed PDT ¼ number of generations
Two genes in SHED and three genes in DPSCs were highly expressed (Figure 2A). The sequencing and BLAST results showed that those genes were TIMP metallopeptidase inhibitor 1 (TIMP1), (A09) (with 82% query coverage and 93% identity), and ribosomal protein s8, (RPS8), (A16) (with 90% query coverage and 99% identity) while genes which were highly expressed in DPSCs were collagen, type I, alpha 1, (COL1A1), (A20) (97% query coverage and 99% identity), follistatin-like 1 (FSTL1), (A17) (96% query coverage and 100% identity), lectin, galactoside-binding, soluble, 1, (LGALS1), (A16) (with 75% query coverage and 96% identity). To confirm the efficacy of the ACP system, the DEGs were confirmed by RT-PCR using gene specific primer pairs. The primers (Table 2) designed using Primer3 Input program version 0.4.0 (http://frodo.wi.mit.edu/) and ordered from 1st BASE (M) SdnBhd Company, Malaysia were used for RT-PCR. The Arbitrary A09, A16, A17 and A20 were subjected to confirmation using RT-PCR (Figure 2A). The RT-PCR confirmed the ACP observation (Figure 2B). Analysis of relative intensity of gene expression also showed that SHED expressed significantly higher amounts of TIMP1 and RPS8 compared to DPSCs while DPSCs expressed significantly higher amounts of FSTL1 and LGALS1 compared to SHED, but not COL1A1 (Figure 3).
Reverse transcriptase-polymerase chain reaction (RT-PCR) – GeneFishing technique first-strand cDNA synthesis The cDNA synthesis was done according to the protocol provided (Epicentre1 Biotechnologies, USA). ACP – GeneFishing PCR Synthesis of second-strand cDNAwas done following the GeneFishingTM DEG Premix kit protocol (Seegene, Korea). The PCR products were electrophoresed on 2% agarose gel and visualised under UV light. DNA purification and sequencing The gels with differentially expressed bands were extracted using QIAquick1 gel extraction kit (Qiagen, USA) and the purified DNAs were sent for sequencing. The gene sequences were analysed using Basic Local Alignment Search Tool (BLAST) software. Confirmation with RT-PCR RT-PCR with specific gene primer pairs were used to confirm the differential expressions of DEGs. Human b-actin was used first as normalisation to the first-strand cDNA. Then, the normalised cDNA was used as a template. Results
Culture and characterisation of stem cells from human dental pulp The cells cultured in this study displayed the typical morphology of SHED and DPSCs (Figure 1A–F) and were grown to a confluence of 80 to 100%. The stem cells from dental pulp results showed the presence of brownish colour indicating positive reactivity for CD105 and CD166 primary antibodies, which characterised SHED and DPSCs (Figure 1G–L).
Proliferation rate of SHED and DPSCs The analysis results showed that SHED had lower r and shorter PDT (0.01429, 60.57 h) than DPSCs (0.00286, 472.43 h), respectively. At 5% level of significance, the 584
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Discussion Immunocytochemistry is used as junction methods for the identification of cells in tissues sections. Characterization of SHED and DPSCs relied on immunocytochemistry staining because of its stability, sensitivity, clear cytomorphological details, ease of use, cost-effectiveness and low technology needed (Leong, 1996). According to Dominici et al. (2006), the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed three minimal criteria to define human MSCs: they must be plastic-adherent when maintained in standard culture conditions, must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface molecules. Both SHED and DPSCs were plastic-adherent cells that were found positive for antibodies against human antigens CD105 (a component of the transforming growth factor (TGF) receptor) and CD166 (a member of the Ig superfamily), which are the mesenchymal stem cell markers and positive for negative control. This means that both SHED and DPSCs can be considered as human MSC. Cell Biol Int 38 (2014) 582–590 ß 2013 International Federation for Cell Biology
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Figure 1 Morphology of SHED and DPSCs. Based on stem cell cultures, observed on days 0, 7 and 15. Day 0–Digested pulp of both SHED (A) and DPSCs (D) showing fibroblasts, mesenchymal cells and macrophages floating inside the flask at this early stage; Day 7–Small rounded and spindle shaped cells seen attached to the flask (B, E); Day 15–Spindle-shaped cells showing 80% confluence (40) (C, F). Characterization of stem cells from dental pulp by immunocytochemistry staining using CD105 and CD166 antibodies were positive in SHED (G, K), DPSCs (H, L), hMSCs (I, M) (positive control-without any antibody) and hMSCs (J, N) (negative control) (100).
GeneFishingTM DEG Premix Kits consist of three steps; reverse transcription and two-stage PCR, which uses primers annealing specifically to the template thus amplifying only
Table 1 Comparison of multiplication rate (r) and population doubling time (PDT) between SHED and DPSCs using alamarBlue assay. Group r PDT
SHED DPSCs SHED DPSCs
Median (IqR) 0.0198 0.002 51 506
(0.006) (0.000) (21) (72)
Z-statistica
P-value
3.298
0.001
3.205
0.001
Mann–Whitney test (P < 0.05).
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genuine products. This system hence overcomes the difficulty in identifying the genes responsible for a specialized function during a certain biological stage as the gene is expressed at low levels, whereas most mRNA transcripts within a cell are abundantly expressed (Kim et al., 2004). Theoretically, in comparison with DPSCs, SHED provides a good source for stem cells and is enriched with extracellular matrix. SHED had discrete patterns of gene expression compared to DPSCs (Nakamura et al., 2009). TGF-b2 stimulates cell proliferation and collagen synthesis (Lee et al., 2006) and might regulate the biological activities in SHED. Nakamura et al. (2009) also demonstrated that SHED displayed increased expression of several growth factors such as FGF, TGF-b, connective tissue growth factor (CTGF), nerve growth factor (NGF) and bone morphogenetic protein 585
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Figure 2 GeneFishingTM DEG screening results. Based on the GeneFishing technique, five DEGs were detected. (A) The arrows show the genes which were highly expressed in SHED or DPSCs using arbitrary primers A09, A16, A17 and A20. Lanes 2, 4, 6, 8, 10, 12, 14: DEGs from DPSCs; Lanes 3, 5, 7, 9, 11, 13, 15: DEGs from SHED and Lane 1: DNA marker (100 bp). After gene sequencing, the efficacy analysis of five selected arbitrary ACP primers were confirmed by RT-PCR (B). Arbitrary A09 (TIMP1), A16 (LGALS1, RPS8), A17 (FSTL1) and A20 (COL1A1) were confirmed with RT-PCR to confirm the ACP observation. Lanes 2, 4, 6, 8, 10: Total DNA from DPSCs; Lanes 3, 5, 7, 9, 11: Total DNA from SHED; Lanes 12, 13:b-actin, a housekeeping gene and Lane 1: DNA marker (100 bp).
(BMP), which can promote the proliferation of legion types of cells. In addition, CTGF, whose expression was higher in SHED, is a matrix signalling molecule involved in TGF-binduced cell proliferation, matrix synthesis, angiogenesis, migration and osteoblast lineage differentiation of MSCs (Luo et al., 2004). These findings suggest that TGF-b and
CTGF might be involved in the regulation of biological functions of SHED. Based on sequencing analysis, the specific genes that differentially expressed in SHED and DPSCs were identified as TIMP1, RPS8, LGALS1, FSTL1 and COL1A1. They were identified from the results of the chromatograms of
Table 2 Primer pairs and product sizes for the differentially expressed genes and b-actin for reverse transcriptase-polymerase chain reaction designed using primer3 software. Gene
Arbitrary
Sequences
Size (bp)
COL1A1
A20
496
FSTL1
A17
LGALS1
A16
TIMP1
A09
RPS8
A16
F: 50 -ACAGTGATTGAATACAAAACCA-0 3 R: 50 -GTGGAGAAAGGAGCAGAAAG-0 3 F: 50 -TAGCATCTGTTAAGATCCAGTG-0 3 R: 50 -TCCACTCTTAGGAAGTAAATGG-0 3 F: 50 -TGGATACGAATTCAAGTTCC-0 3 R: 50 -TACTATGTGCCAAACTCTGTGT-0 3 F: 50 -CAACAGATGTATAAAGGGTTCC-0 3 R: 50 -CCTTCTGATAGACTGAAATTGG-0 3 F: 50 -GTTGTGCTAAGGATCACCTACT-0 3 R: 50 -GAGTTGAGAACAGGGACTTTAC-0 3 F: 50 -TGGCACCACACCTTCTACAATGAGC-30 R: 50 -GCACAGCTTCTCCTTAATGTCACGC-30
b-actin
586
—
391 347 270 350 395
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Figure 3 Analysis of relative intensity of gene expression for the differentially expressed genes in SHED and DPSCs. SHED expressed significantly higher amounts of TIMP1 and RPS8 compared to DPSCs while DPSCs expressed significantly higher amounts of FSTL1 and LGALS1 compared to SHED but not COL1A1.
sequencing analysis, using BioEdit software and further carrying out BLASTanalysis. BLASTanalysis also showed that all sequences produced significant alignment with highest query coverage and maximum identity. Interestingly, in our study, TIMP1 was one of the DEGs highly expressed in SHED. TIMP1 belongs to TIMPs that constitute a family of secreted proteins, whose primary function is inhibition of the degradative action of matrix metalloproteinases (MMPs) and regulation of ECM turnover and tissue remodelling. MMPs are zinc-dependent endopeptidases involved in only invasion and metastasis, but evidence suggests that certain MMPs also play a role in tumourigenesis (Chambers and Matrisian, 1997). ECM is a dynamic structure that not only provides a scaffold for organising tissue architecture, but also contributes signals that regulate cell function. TIMPs also are also involved in homeostasis of the ECM by regulating the activities of MMPs including proliferation, differentiation, apoptosis, tumour angiogenesis, tumourigenesis and metastasis (Visse and Nagase, 2003). However, the regulation of TIMP1 expression is incompletely understood. Hence, the increased cell proliferation of SHED in comparison to DPSCs could be attributed to the high expression of the TIMP1 gene. The role of TGF-b1 in liver fibrosis is in part related to impairment of ECM breakdown by stimulation of TIMP1. Flisiak et al. (2002) found that chronic viral hepatitis B and C resulted in a significant increase in plasma TIMP1 levels, but not TGF-b1. Yang et al. (2002) reported that there was correlation between the expression of TGF-b1 and MMP-1, but no correlation between TGF-b1 and TIMP1 in the expression of TGF-b1 MMP-1 and its inhibitor in human middle ear cholesteatoma. Cell Biol Int 38 (2014) 582–590 ß 2013 International Federation for Cell Biology
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Another DEG is COL1A1. Type I collagen is present in most organs, and synthesised only by a small number of discrete cell types, including fibroblasts, osteoblasts and odontoblasts, which is a major component of many ECMs, and its accumulation characterises most fibrotic processes. Cytokines including interleukin-1 (IL-1) and IL4, growth factors such as TGF-b and insulin-like growth factor-1 (IGF-1), vasoactive peptide such as endothelin-1, or other molecules such as lipid peroxidation products can stimulate type I collagen production by fibroblastic cells (Rossert et al., 1996). COL1A1 encodes the a1 chain of collagen type I. This gene is of particular interest because of its involvement in resistance and elasticity of the tissues. COL1A1 is elevated in tumour endothelium, compared with normal endothelium, suggesting that they play an important role in angiogenesis and formation of desmoplasia. Overexpression of COL1A1 can also be associated with invasive process in papillary thyroid cancer (PTC) (Lee et al., 2009). Many diseases, such as Ehler–Danlos syndrome, osteogenesis imperfect, chondrodysplasia, low bone mineral density (Kuivaniemi et al., 1997) and CDH (Skirving et al., 1984) have been associated with COL1A1. Our results show that COL1A1 was overexpressed in DPSCs compared to SHED, which is in contrast to the report of Wang et al. (2012). Col 1 contains the majority of the ECM and is important in maintaining the biological and structural integrity of the ECM architecture (Cen et al., 2008). Ascorbic acid can stimulate distinguishable expression of Col 1 in DPSCs because these cells had a structure that was enriched with ECM (Park et al., 2010). The expression of COL1A1 might be due to the addition of L-ascorbic acid 2-phosphate into the culture medium during the culturing process. L-ascorbic acid 2-phosphate can stimulate collagen accumulation (Hata and Senoo, 1989). The use of L-ascorbic acid 2-phosphate in the culture medium also increases the production of type I and type III collagen peptide in cultured rabbit keratinocytes (Saika et al., 1992). LGALS1 was also overexpressed in DPSCs compared to SHED. High levels of Gal-1, a b-galactoside binding secreted protein, correlate with aggressiveness of tumours and acquisition of a metastatic phenotype of tumours, resulting in a poor prognosis (Jung et al., 2007). Gal-1 also promotes proliferation and migration of glioblastoma cell lines in vitro (Camby et al., 2002), acts as pro-angiogenic factor in tumour angiogenesis (Le Mercier et al., 2008), promotes proliferation and migration of endothelial cells (Thijssen et al., 2006) and vascular smooth muscle cells (Moiseeva et al., 2000), leading to influences on the formation of new blood vessels. Crosstalk exists between tumour-derived suppressive factors; TGF-b triggers Gal-1 expression via (mothers against decapentaplegic homolog) SMAD3/SMAD4 dependent pathway (Daroqui et al., 2007) and in turn, Gal-1 induces IL-10 production in monocytes and T-cells (van der Leij et al., 2007). Moreover, 587
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Gal-1 is a key regulator of murine CD4þCD25þFoxp3 regulatory T-cells (Garin et al., 2007) which is essential in suppression of anti-cancer immunity (Orentas et al., 2006). Gal-1 also inhibits the immune response at the level of leukocyte infiltration; it decreases the adhesion and extravasation of lymphocytes and neutrophils through activate dendothelium (Cooper et al., 2008). In addition, Gal-1 expression in tumour cells or the surrounding stroma intimately regulates the function and vitality of infiltrating-Tcells (Gandhi et al., 2007). Gal-1 has many prospects for therapeutic applications. Gal-1 has critical importance in neuronal cell differentiation and survival in the central and the peripheral nervous systems, plus the establishment and maintenance of T-cell tolerance and homeostasis in vivo. Gal-1 expression or overexpression in tumours or the surrounding tissue can also be considered a sign of their malignant progression. The targeted overexpression (or delivery) of Gal-1 should be selected as a novel approach for the treatment of inflammation-related diseases, such as graft versus host disease (GVHD) (Baum et al., 2003), arthritis (Rabinovich et al., 1999), colitis (Santucci et al., 2003), and nephritis (Tsuchiyama et al., 2000). This gene could be also seen as a possible therapeutic aim in some neurodegenerative diseases (Kadoya and Horie, 2005) and muscular dystrophies (Goldring et al., 2002). Limiting the migration of cancer cells by depress-expressing the Gal-1 in the cancer cells restores a level of sensitivity to cell death, and also cytotoxic drugs (Lefranc et al., 2005). Hence, anti-Gal-1 compounds are needed to fight migrating cancer cells (NangiaMakker et al., 2002; Sorme et al., 2003; Ingrassia et al., 2006). We have shown the differences between SHED and DPSCs with regard to their characteristics, immunocytology, proliferation and gene expression. These results can be used to determine and measure their functional activities. The specific gene expression patterns of SHED and DPSCs, and the underlying differences that exist between them, might be useful in determining the functional roles of these relevant genes pertaining to its specific protein expression. This knowledge may have application in stem cell therapies, using stem cells obtained from human dental pulp as multipotent cell sources for genetic and tissue engineering technology. Conflict of interest statement The authors declare that they have no competing interests. Acknowledgements and funding We would like to express our gratitude to the clinicians, staff, patients and patients’ families for their cooperation in this study. This study was supported by the Universiti Sains Malaysia Short term grant (304/PPSG/6139051). 588
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