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Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2008) 32, 733–738 (Printed in Great Britain)
Comparison between osteoblasts derived from human dental pulp stem cells and osteosarcoma cell lines
Annalisa Palmieria, Furio Pezzettia, Antonio Grazianob, D'Aquino Riccardob, Ilaria Zollinoc, Giorgio Brunellic, Marcella Martinellia, Marzia Arlottia and Francesco Carincic*
aCentre of Molecular Genetics, CARISBO Foundation, Institute of Histology and General Embryology, School of Medicine, University of Bologna, Bologna, Italy
bDental Clinic, Second University of Naples, Naples, Italy
cChair of Maxillofacial Surgery, School of Medicine, University of Ferrara, Arcispedale S. Anna, Corso Giovecca 203, 44100 Ferrara, Italy


Abstract

Stem cells derived from human dental pulp are able to differentiate into osteoblasts and are a potential source of autologous bone. The aim of this study was to compare genes differentially expressed in osteoblastoids from human dental pulp (OHDP) to osteosarcoma cells (OCs).

Human dental pulp was extracted and immersed in a digestive solution. Cells were cultured and selected using c-kit, CD34, CD45 and STRO-1 antibodies. In parallel, two OCs (i.e., SAOS2 and TE85) were cultured. RNA was extracted from different populations of cells and cDNA was used for the hybridisation of human 19.2K DNA microarrays.

We identified several differences in gene expression between OHDP and OCs. Some down-regulated OHDP genes, such as RUNX1, MAP4K4 and PRDM2, are involved in bone development, cell motility and transcript regulation. Gene expression in OHDP is significantly different from that in OCs, suggesting differences in cell function and activity between these cells.


Keywords: Stem cells, Dental pulp, Autologous bone, Microarray, Tumour.

*Corresponding author. Tel./fax: +39 0532 455582.


1 Introduction

Stem cells derived from human dental pulp are able to differentiate into osteoblastoid cells and are a potential source of autologous bone produced in vitro (Laino et al., 2005, 2006a,b).

A new and highly enriched population of stem cells derived from dental pulp of both deciduous and permanent teeth was isolated, cultured and successively selected using FACS (Laino et al., 2005, 2006a,b). Cells obtained from dental pulp were cultured and successively selected using a fluorescence activated cell sorter (FACS). Immunoreactivity profiles of the cultured cells were performed and specific antigens for the stromal stem cells c-kit, CD34 and CD45 were detected. Mesenchymal stem/progenitor cell populations that are c-kit- and CD34-positive and CD45 negative were isolated. These cells proliferate extensively under standard culture conditions, have a long life span and maintain their multipotential capabilities for generations (Laino et al., 2006a,b; Papaccio et al., 2006).

Osteoblasts derived from human pulp stem cells (ODHPS) express osteocalcin and flk-1 (VEGF-R2) (D'Aquino et al., 2007; Graziano et al., 2007a,b). Interestingly, endotheliocytes that form vessel walls and stem cells synergically differentiate into osteoblasts and endotheliocytes (D'Aquino et al., 2007) When ODHPS obtained in vitro were transplanted into immunocompromised rats, they generated a tissue structure with an integral blood supply similar to that of human adult bone (D'Aquino et al., 2007).

Stem cells are of great interest for tissue regeneration, tissue-based clinical therapies and transplantation, but due to their characteristics of self-renewal and unlimited replication they are also appealing candidates for the definition of ‘cells of origin’ for cancer. The discovery that subpopulations of cells having stem cell characteristics were found in tumour biopsies from brain and breast cancers provides support for the cancer stem cell hypothesis (Al-Hajj et al., 2003; Hemmati et al., 2003; Singh et al., 2004).

The analysis of the differences between osteoblastoids from human dental pulp (OHDP) and osteosarcoma cells (OCs) will help to detect the distinctive genes between OHDP and sarcomas. This information might be useful to test in vitro-produced bone tissue before autografting in order to avoid potential cancer cells transplantation. By using microarray slides containing 19,200 different oligonucleotides we compared the gene profiles of OHDP and OCs.

2 Materials and methods

Cell selection, culture, proliferation and osteoblast differentiation were performed as previously described (Laino et al., 2006b). Briefly, human dental pulp was extracted using a dentinal excavator or a Gracey curette from permanent teeth (34 molars) in healthy subjects (aged 18–37 years, 13 females and 21 males) following informed consent. The removed pulp was immersed in a digestive solution. Once digested, the solution was filtered. After filtration, cells were immersed in α-MEM culture medium to which FBS, l-glutamine and antibiotics were added. The cell suspension was then centrifuged and the pellet was re-suspended in the same culture medium and placed in flasks for growth.

The cytometric analysis was performed between days 15 and 22 of culture, depending on the cell proliferation rate, using the following mouse anti-human antibodies: c-kit (Barclay et al., 1988), CD34 (Simmons and Torok-Storb, 1991), CD45 (Zhang et al., 2003), and STRO-1 (Gronthos et al., 1994).

Thirty days after isolation, cells (c-kit+/STRO-1+/CD34+/CD45j) started to differentiate into osteoblasts and produce an extracellular matrix. After 3 weeks, in order to characterize differentiated osteoblasts, they were detached for differentiating markers, including CD44 and RUNX2. RNA was extracted when stem cells were completely differentiated in osteoblasts, which was after 2 months. In parallel we cultured OCs (i.e., SAOS2 and TE85). RNA was extracted when the cells were sub-confluent.

RNA extraction and cDNA synthesis were performed as described in previous reports (Carinci et al., 2003, 2004a,b,c,d). Mono-reactive Cy3 and Cy5 esters were used for indirect cDNA labelling. Human 19.2K DNA microarrays were used. A GenePix 4000a DNA micro-array scanner was used to scan the slides, and data were extracted with GenePix Pro. After removing the local background, normalisation was performed. GP3 perl script was used in order to post-process raw data files from the scanning procedure. Z-Score normalisation, a trimmed mean of 75% and a threshold value of three were used to filter the GenePix raw data.

The SAM (Significance Analysis of Microarray) program was then performed and an SAM score was obtained (T-statistic value) (Carinci et al., 2003, 2004a,b,c,d).

3 Results

In comparing OHDP to OCs, it was found that 56 genes were down-regulated whereas 98 genes were up-regulated. The genes differentially expressed are reported in Tables 1 and 2; the SAM plot is reported in Fig. 1. We briefly analyzed some of those with better-known functions.


Table 1.

Down-regulate genes in OHDP vs. OCs


Table 2.

Up-regulate genes in OHDP vs. OCs


Fig. 1

SAM (statistical analysis of microarray) plot of OHDP vs. OCs. Expected differentially expressed genes are reported in the x axis whereas observed differentially expressed genes are in the y axis. Down-regulated genes (green dots) are located in the lower left side of the diagram; up-regulated genes (red dots) are in the upper right side; genes with different expression but statistically not significant are black dots. Parallel lines drawn from the lower-left to upper-right squares are the cut-off limits. The solid line indicates the equal value of observed and expected differentially expressed genes.


NameSymbolCytobandScore (d)
Hypothetical protein MGC4692MGC4692−4.67
DecorinDCN12q21.33−4.53
HLA-B associated transcript 1BAT16p21.3−4.31
Beta-site APP-cleaving enzyme 1BACE111q23.2–q23.3−4.31
Prenylcysteine oxidase 1PCYOX12p13.3−4.25
MORN repeat containing 2MORN22p22.1−4.06
Collagen, type VI, alpha 3COL6A32q37−4.02
Glypican 3GPC3Xq26.1−4.00
Nudix (nucleoside diphosphate linked moiety X)-type motif 13NUDT1310q22.1−3.95
Methyl-CpG binding domain protein 2MBD218q21−3.91
WD repeat domain 20WDR2014q32.31−3.73
ADAM metallopeptidase domain 12 (meltrin alpha)ADAM1210q26.3−3.72
Zinc finger protein 38ZNF387q22.1−3.59
F-box and WD-40 domain protein 7 (archipelago homolog, Drosophila)FBXW74q31.3−3.52
VimentinVIM10p13−3.43
HERPUD family member 2HERPUD27p14.2−3.38
5′-3′ exoribonuclease 1XRN13q23−3.33
Transcribed locus−3.29
Chromosome 11 open reading frame 69C11orf6911p13−3.25
GTP binding protein 5 (putative)GTPBP520q13.33−3.18
TRAF-type zinc finger domain containing 1TRAFD112q−3.11
Protein kinase, cAMP-dependent, regulatory, type II, alphaPRKAR2A3p21.3–p21.2−3.08
Chromosome 20 open reading frame 11C20orf1120q13.33−3.08
Adenylate cyclase 1 (brain)ADCY17p13–p12−3.04
Myosin VIMYO66q13−3.03
Tyrosine kinase, non-receptor, 2TNK23q29−2.94
Amine oxidase, copper containing 2 (retina-specific)AOC217q21−2.84
Stathmin 1/oncoprotein 18STMN11p36.1–p35−2.77
Aldehyde dehydrogenase 8 family, member A1ALDH8A16q23.2−2.75
Centrosomal protein 78kDaCEP789q21.2−2.74
Hypothetical protein DKFZp761I2123DKFZp761I21237p13−2.74
Syntaxin 17STX179q31.1−2.71
Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2SLC11A212q13−2.61
Zinc finger protein 16ZNF168q24−2.61
Solute carrier family 39, member 14SLC39A148p21.3−2.49
Castor homolog 1, zinc finger (Drosophila)CASZ11p36.22−2.37
Forkhead box P1FOXP13p14.1−2.29
Zinc finger protein 93ZNF9319p12−2.29
Fibrinogen-like 1FGL18p22–p21.3−2.28


NameSymbolCytobandScore (d)
Structure specific recognition protein 1SSRP111q125.83
Procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1PLOD11p36.3–p36.25.62
N-Deacetylase/N-sulfotransferase 3NDST34q265.20
Tropomodulin 2 (neuronal)TMOD215q21.1–q21.25.16
Nuclear factor I/ANFIA1p31.3–p31.25.02
CD36 molecule (thrombospondin receptor)CD367q11.24.88
LIM domain only 2 (rhombotin-like 1)LMO211p134.75
ATP-binding cassette, sub-family B member 7ABCB7Xq12–q134.56
Hypothetical protein LOC647197LOC64719714q32.24.55
Zinc finger and BTB domain containing 1ZBTB114q23.34.35
Peter pan homolog (Drosophila)PPAN19p134.29
Sex comb on midleg homolog 1 (Drosophila)SCMH11p344.27
PHD finger protein 10PHF106q274.23
Carboxypeptidase DCPD17p11.1–q11.24.19
M-phase phosphoprotein 6MPHOSPH616q23.34.18
G protein-coupled receptor associated sorting protein 2GPRASP2Xq22.14.08
Trinucleotide repeat containing 4TNRC41q214.07
Protein kinase, cGMP-dependent, type IPRKG110q11.24.02
Mercaptopyruvate sulfurtransferaseMPST22q13.13.78
Amidohydrolase domain containing 1AMDHD112q23.13.73
Poly(A) binding protein, cytoplasmic 1PABPC18q22.2–q233.69
PR domain containing 2, with ZNF domainPRDM21p36.213.67
Neurotrophic tyrosine kinase, receptor, type 3NTRK315q253.65
Hypothetical protein FLJ12681LA16c-360B4.116p13.33.54
Tetratricopeptide repeat domain 3TTC321q22.23.48
Reticulon 4 receptor-like 1RTN4RL117p13.33.46
COBL-like 1COBLL12q24.33.44
Serine/arginine repetitive matrix 2SRRM216p13.33.43
Chromosome 20 open reading frame 20C20orf2020q13.333.39
Syndecan 3 (N-syndecan)SDC31pter-p22.33.37
Zinc and ring finger 3ZNRF322q12.13.27
Discs, large homolog 2, chapsyn-110DLG211q14.13.22
RAB22A, member RAS oncogene familyRAB22A20q13.323.22
Pappalysin 2PAPPA21q23–q253.19
Myeloid cell nuclear differentiation antigenMNDA1q223.18
CD48 moleculeCD481q21.3–q223.09
Neuronal PAS domain protein 2NPAS22q11.23.09
Protein tyrosine phosphatase, receptor type, N polypeptide 2PTPRN27q363.04
Syndecan binding protein (syntenin)SDCBP8q123.02
MON2 homolog (S. cerevisiae)MON212q14.13.01
Family with sequence similarity 13, member C1FAM13C110q21.13.00
Runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene)RUNX121q22.32.98
Sidekick homolog 1 (chicken)SDK17p22.22.98
Sin3A-associated protein, 30kDaSAP304q34.12.94
Triple functional domain (PTPRF interacting)TRIO5p15.1–p142.93
Acetyl-Coenzyme A carboxylase alphaACACA17q212.93
X-ray repair complementing defective repair in Chinese hamster cells 5XRCC52q352.88
GRIP and coiled-coil domain containing 2GCC22q12.32.86
Jagged 1 (Alagille syndrome)JAG120p12.1–p11.232.76
Serum response factor binding protein 1SRFBP15q23.12.75
Caspase 8, apoptosis-related cysteine peptidaseCASP82q33–q342.73
Mitogen-activated protein kinase kinase kinase 4MAP3K46q262.73
MoesinMSNXq11.2–q122.71
Dihydrolipoamide dehydrogenaseDLD7q31–q322.71
Sepiapterin reductaseSPR2p14–p122.67
Sushi domain containing 1SUSD19q31.3–q33.12.54
RAB11 family interacting protein 5 (class I)RAB11FIP52p13–p122.53
Chromosome 10 open reading frame 76C10orf7610q24.322.52
KIAA0350 proteinKIAA035016p13.132.51
AstrotactinASTN1q25.22.51
Chitobiase, di-N-acetyl-CTBS1p222.51
Ribosomal protein L6RPL612q24.12.49
Formyl peptide receptor-like 1FPRL119q13.3–q13.42.46
Hypothetical protein MGC10646MGC 106464q21.212.45
Aryl hydrocarbon receptor interacting proteinAIP11q13.32.45
Adenosine deaminase, RNA-specific, B1 (RED1 homolog rat)ADARB121q22.32.40
X (inactive)-specific transcriptXISTXq13.22.33
Phosphoribosyl pyrophosphate synthetase 2PRPS2Xp22.3–p22.22.32
RAB22A, member RAS oncogene familyRAB22A20q13.322.28
SMC6 structural maintenance of chromosomes 6-like 1 (yeast)SMC6L12p24.22.27
Spectrin, beta, erythrocytic (includes spherocytosis, clinical type I)SPTB14q23–q24.22.26
Hypothetical protein FLJ30596FLJ305965p13.22.25
Periphilin 1PPHLN112q122.24
Caldesmon 1CALD17q332.23
SAM domain, SH3 domain and nuclear localisation signals, 1SAMSN121q112.23
Sodium channel, voltage-gated, type IV, betaSCN4B11q23.32.22
DKFZP686A01247 hypothetical proteinDKFZP686A012474p132.20
F-box and WD-40 domain protein 11FBXW115q35.12.19


3.1 Down-regulated genes in OHDP (Table 1)

Many down-regulated genes participate in cell differentiation: JAG1, a calcium ion binding protein involved in haematopoiesis; PPHLN1, which is important for epithelial differentiation and epidermal integrity; PRKG1, a GMP-dependent protein kinase that may play roles in physiological processes such as relaxation of vascular smooth muscle and inhibition of platelet aggregation; and CASP8, a protein involved in the programmed cell death induced by FAS and various apoptotic stimuli.

Other down-regulated genes are cell adhesion molecules (such as SDK1, CD36 and FPRL1) or cell motility proteins such as SDCBP (whose function is cytoskeletal-membrane organization) and MSN (a member of the ERM family, important for cell–cell recognition, signalling and for cell movement).

Interesting down-regulated genes in cell development include: NTRK3 – a member of the neurotrophic tyrosine receptor kinase-NTRK family – mutations in this gene have been associated with medulloblastomas, secretory breast carcinomas and other cancers; RUNX1, a heterodimeric transcription factor that binds to the core element of many enhancers and promoters – chromosomal translocations involving this gene are well-documented and have been associated with several types of leukemia; and MAP4K4, a kinase that mediates the TNF-alpha signalling pathway.

PRDM2 is a zinc finger protein that can bind to retinoblastoma protein and estrogens receptor.

3.2 Up-regulated genes in OHDP (Table 2)

Many up-regulated genes mediate signal transduction. FGL1 is a member of the fibrinogen family; GPC3 is a cell surface heparan sulfate proteoglycan that may play a role in the control of cell division and growth regulation; and PRKAR2A is a signalling molecule that has been shown to regulate protein transport from endosomes to the Golgi apparatus and further to the endoplasmic reticulum.

Other up-regulated genes that encode for cell differentiation proteins are MYO6 and ADAM12. Myosin VI is an actin-based molecular motor involved in intracellular vesicle and organelle transport. ADAM12 is a disintegrin and metalloprotease implicated in a variety of biological processes involving cell–cell and cell–matrix interactions, including fertilization, muscle development and neurogenesis.

Additional up-regulated genes are related to cell cycle regulations, like XRN1 (which may play a role in mRNA metabolism and cytoplasmic functions like meiosis, telomere maintenance and microtubule assembly) and STMN1 (involved in the regulation of the microtubule filament system). These proteins may also play a role in the control of cell division and growth regulation.

4 Discussion

OHDP were obtained and characterized from deciduous and adult teeth (Laino et al., 2005, 2006a,b) and were selected by using different markers specific to stromal stem cells. In culture, they proliferated and differentiated into osteoblastoids still capable of self-renewing and then, under appropriate conditions, into osteoblasts forming living bone (Laino et al., 2005, 2006a,b; Papaccio et al., 2006). In vitro mineralized tissue up to 15mm thick was obtained (Laino et al., 2006a). This hard tissue can be useful for autologous transplants, a cure needed for several pathologies requiring bone repair (D'Aquino et al., 2007; Graziano et al., 2007a,b). However, this procedure may hold a relevant risk insight. Indeed, cell selection by UV laser beam and subsequent in vitro expansion beyond immune system control can theoretically induce cell transformation and the positive selection of cancer cells (Al-Hajj et al., 2003; Hemmati et al., 2003; Singh et al., 2004).

The aim of this study was to perform almost genome-wide screening for genes differentially expressed in OHDP vs. OCs using a cDNA microarray technique that is able to provide a comparative analysis of the RNA expression of thousands of genes simultaneously. Interesting, RUNX1 is down-regulated in OHDP. RUNX1 is essential for haematopoiesis, but also contains RUNX binding sites in its promoter region, suggesting a possible cross-regulation with RUNX2 and potential regulatory roles in bone development.

Smith et al. (2005) demonstrated that RUNX1 and RUNX2 are expressed in different stages of skeletal development, with a possible role for RUNX1 in mediating early events of endochondral and intramembranous bone formation, while RUNX2 is a potent inducer in the late stages of chondrocyte and osteoblast differentiation.

Another study conducted by Yamashiro et al. (2004) found that RUNX1 expression is down-regulated on the terminal differentiation of osteoblasts, suggesting that RUNX1 may play a role in early osteogenesis. These results support the thesis that the down regulation of RUNX1 in ODPH is due to the terminal differentiation of these cells in osteoblasts.

Other down-regulated genes in OHDP are MAP4K4 and PRDM2. MAP4K4 mediates the TNF-alpha signalling pathway. Activation of members of the MAPK family is the major mechanism for the transduction of promigratory stimuli. The protein is involved in developmental cell migration (Wiener et al., 2003) and is reported to augment cellular motility and invasion of rat intestinal epithelial cells in the presence of hepatocyte growth factor. PRDM2 is a down-regulated tumour suppressor gene. PRDM2 encodes a zinc finger protein that binds to retinoblastoma protein. It plays a role in transcriptional regulation during neuronal differentiation and the pathogenesis of retinoblastoma (Tsukahara et al., 2005).

In conclusion, OHDP and OCs have different genetic portraits with a higher expression of genes involved in cell mobility and kinetics in osteosarcomas. We believe that a comprehensive characterization of OHDP could lead to significant findings. The neoplastic proliferation of cancer stem cells is likely to be driven by mutations that inappropriately activate pathways which promote the self-renewal of normal stem cells. The analysis of the differences between OHDP and OCs could help to elucidate the pathways and genes involved in tumour development and maintenance. Moreover, the reported data can be useful for comparing in vitro-produced bone tissue before grafting tissue. A characterization of the genetic profiling of OHDP and OCs is required to avoid the risk of transplanting cancer cells together with bone tissue.

Acknowledgments

This study was partially supported, by grants from FAR (F.C.) and PRIN 2005 (F.C. prot. 2005067555_002).

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Received 18 October 2007/14 December 2007; accepted 25 February 2008

doi:10.1016/j.cellbi.2008.02.003


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)