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Comparison between osteoblasts derived from human dental pulp stem cells and osteosarcoma cell lines
Cell Biology International 32 (2008) 733e738 www.elsevier.com/locate/cellbi
Comparison between osteoblasts derived from human dental pulp stem cells and osteosarcoma cell lines Annalisa Palmieri a, Furio Pezzetti a, Antonio Graziano b, D’Aquino Riccardo b, Ilaria Zollino c, Giorgio Brunelli c, Marcella Martinelli a, Marzia Arlotti a, Francesco Carinci c,* a
Centre of Molecular Genetics, CARISBO Foundation, Institute of Histology and General Embryology, School of Medicine, University of Bologna, Bologna, Italy b Dental Clinic, Second University of Naples, Naples, Italy c Chair of Maxillofacial Surgery, School of Medicine, University of Ferrara, Arcispedale S. Anna, Corso Giovecca 203, 44100 Ferrara, Italy Received 18 October 2007; revised 14 December 2007; accepted 25 February 2008
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 STRO1 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 19.2 K 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. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Stem cells; Dental pulp; Autologous bone; Microarray; Tumour
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 * Corresponding author. Tel./fax: þ39 0532 455582. E-mail address: [email protected] (F. Carinci). URL: http://www.carinci.org
stem cells c-kit, CD34 and CD45 were detected. Mesenchymal stem/progenitor cell populations that are c-kit- and CD34positive 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
1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.02.003
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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 18e37 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 a-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-kitþ/STRO-1þ/ CD34þ/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 19.2 K 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 postprocess 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 (Tstatistic 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. 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 GMPdependent 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 cellecell recognition, signalling and for cell movement). Interesting down-regulated genes in cell development include: NTRK3 e a member of the neurotrophic tyrosine receptor kinase-NTRK family e 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 e 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
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Table 1 Down-regulate genes in OHDP vs. OCs Name
Symbol
Cytoband
Score (d )
Structure specific recognition protein 1 Procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1 N-Deacetylase/N-sulfotransferase 3 Tropomodulin 2 (neuronal) Nuclear factor I/A CD36 molecule (thrombospondin receptor) LIM domain only 2 (rhombotin-like 1) ATP-binding cassette, sub-family B member 7 Hypothetical protein LOC647197 Zinc finger and BTB domain containing 1 Peter pan homolog (Drosophila) Sex comb on midleg homolog 1 (Drosophila) PHD finger protein 10 Carboxypeptidase D M-phase phosphoprotein 6 G protein-coupled receptor associated sorting protein 2 Trinucleotide repeat containing 4 Protein kinase, cGMP-dependent, type I Mercaptopyruvate sulfurtransferase Amidohydrolase domain containing 1 Poly(A) binding protein, cytoplasmic 1 PR domain containing 2, with ZNF domain Neurotrophic tyrosine kinase, receptor, type 3 Hypothetical protein FLJ12681 Tetratricopeptide repeat domain 3 Reticulon 4 receptor-like 1 COBL-like 1 Serine/arginine repetitive matrix 2 Chromosome 20 open reading frame 20 Syndecan 3 (N-syndecan) Zinc and ring finger 3 Discs, large homolog 2, chapsyn-110 RAB22A, member RAS oncogene family Pappalysin 2 Myeloid cell nuclear differentiation antigen CD48 molecule Neuronal PAS domain protein 2 Protein tyrosine phosphatase, receptor type, N polypeptide 2 Syndecan binding protein (syntenin) MON2 homolog (S. cerevisiae) Family with sequence similarity 13, member C1 Runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) Sidekick homolog 1 (chicken) Sin3A-associated protein, 30kDa Triple functional domain (PTPRF interacting) Acetyl-Coenzyme A carboxylase alpha X-ray repair complementing defective repair in Chinese hamster cells 5 GRIP and coiled-coil domain containing 2 Jagged 1 (Alagille syndrome) Serum response factor binding protein 1 Caspase 8, apoptosis-related cysteine peptidase Mitogen-activated protein kinase kinase kinase 4 Moesin Dihydrolipoamide dehydrogenase Sepiapterin reductase Sushi domain containing 1 RAB11 family interacting protein 5 (class I) Chromosome 10 open reading frame 76 KIAA0350 protein Astrotactin Chitobiase, di-N-acetyl-
SSRP1 PLOD1 NDST3 TMOD2 NFIA CD36 LMO2 ABCB7 LOC647197 ZBTB1 PPAN SCMH1 PHF10 CPD MPHOSPH6 GPRASP2 TNRC4 PRKG1 MPST AMDHD1 PABPC1 PRDM2 NTRK3 LA16c-360B4.1 TTC3 RTN4RL1 COBLL1 SRRM2 C20orf20 SDC3 ZNRF3 DLG2 RAB22A PAPPA2 MNDA CD48 NPAS2 PTPRN2 SDCBP MON2 FAM13C1 RUNX1
11q12 1p36.3ep36.2 4q26 15q21.1eq21.2 1p31.3ep31.2 7q11.2 11p13 Xq12eq13 14q32.2 14q23.3 19p13 1p34 6q27 17p11.1eq11.2 16q23.3 Xq22.1 1q21 10q11.2 22q13.1 12q23.1 8q22.2eq23 1p36.21 15q25 16p13.3 21q22.2 17p13.3 2q24.3 16p13.3 20q13.33 1pter-p22.3 22q12.1 11q14.1 20q13.32 1q23eq25 1q22 1q21.3eq22 2q11.2 7q36 8q12 12q14.1 10q21.1 21q22.3
5.83 5.62 5.20 5.16 5.02 4.88 4.75 4.56 4.55 4.35 4.29 4.27 4.23 4.19 4.18 4.08 4.07 4.02 3.78 3.73 3.69 3.67 3.65 3.54 3.48 3.46 3.44 3.43 3.39 3.37 3.27 3.22 3.22 3.19 3.18 3.09 3.09 3.04 3.02 3.01 3.00 2.98
SDK1 SAP30 TRIO ACACA XRCC5
7p22.2 4q34.1 5p15.1ep14 17q21 2q35
2.98 2.94 2.93 2.93 2.88
GCC2 JAG1 SRFBP1 CASP8 MAP3K4 MSN DLD SPR SUSD1 RAB11FIP5 C10orf76 KIAA0350 ASTN CTBS
2q12.3 20p12.1ep11.23 5q23.1 2q33eq34 6q26 Xq11.2eq12 7q31eq32 2p14ep12 9q31.3eq33.1 2p13ep12 10q24.32 16p13.13 1q25.2 1p22
2.86 2.76 2.75 2.73 2.73 2.71 2.71 2.67 2.54 2.53 2.52 2.51 2.51 2.51 (continued on next page)
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Table 1 (continued ) Name
Symbol
Cytoband
Score (d )
Ribosomal protein L6 Formyl peptide receptor-like 1 Hypothetical protein MGC10646 Aryl hydrocarbon receptor interacting protein Adenosine deaminase, RNA-specific, B1 (RED1 homolog rat) X (inactive)-specific transcript Phosphoribosyl pyrophosphate synthetase 2 RAB22A, member RAS oncogene family SMC6 structural maintenance of chromosomes 6-like 1 (yeast) Spectrin, beta, erythrocytic (includes spherocytosis, clinical type I) Hypothetical protein FLJ30596 Periphilin 1 Caldesmon 1 SAM domain, SH3 domain and nuclear localisation signals, 1 Sodium channel, voltage-gated, type IV, beta DKFZP686A01247 hypothetical protein F-box and WD-40 domain protein 11
RPL6 FPRL1 MGC 10646 AIP ADARB1 XIST PRPS2 RAB22A SMC6L1 SPTB FLJ30596 PPHLN1 CALD1 SAMSN1 SCN4B DKFZP686A01247 FBXW11
12q24.1 19q13.3eq13.4 4q21.21 11q13.3 21q22.3 Xq13.2 Xp22.3ep22.2 20q13.32 2p24.2 14q23eq24.2 5p13.2 12q12 7q33 21q11 11q23.3 4p13 5q35.1
2.49 2.46 2.45 2.45 2.40 2.33 2.32 2.28 2.27 2.26 2.25 2.24 2.23 2.23 2.22 2.20 2.19
Table 2 Up-regulate genes in OHDP vs. OCs Name
Symbol
Cytoband
Score (d )
Hypothetical protein MGC4692 Decorin HLA-B associated transcript 1 Beta-site APP-cleaving enzyme 1 Prenylcysteine oxidase 1 MORN repeat containing 2 Collagen, type VI, alpha 3 Glypican 3 Nudix (nucleoside diphosphate linked moiety X)-type motif 13 Methyl-CpG binding domain protein 2 WD repeat domain 20 ADAM metallopeptidase domain 12 (meltrin alpha) Zinc finger protein 38 F-box and WD-40 domain protein 7 (archipelago homolog, Drosophila) Vimentin HERPUD family member 2 50 -30 exoribonuclease 1 Transcribed locus Chromosome 11 open reading frame 69 GTP binding protein 5 (putative) TRAF-type zinc finger domain containing 1 Protein kinase, cAMP-dependent, regulatory, type II, alpha Chromosome 20 open reading frame 11 Adenylate cyclase 1 (brain) Myosin VI Tyrosine kinase, non-receptor, 2 Amine oxidase, copper containing 2 (retina-specific) Stathmin 1/oncoprotein 18 Aldehyde dehydrogenase 8 family, member A1 Centrosomal protein 78kDa Hypothetical protein DKFZp761I2123 Syntaxin 17 Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 Zinc finger protein 16 Solute carrier family 39, member 14 Castor homolog 1, zinc finger (Drosophila) Forkhead box P1 Zinc finger protein 93 Fibrinogen-like 1
MGC4692 DCN BAT1 BACE1 PCYOX1 MORN2 COL6A3 GPC3 NUDT13 MBD2 WDR20 ADAM12 ZNF38 FBXW7
12q21.33 6p21.3 11q23.2eq23.3 2p13.3 2p22.1 2q37 Xq26.1 10q22.1 18q21 14q32.31 10q26.3 7q22.1 4q31.3
4.67 4.53 4.31 4.31 4.25 4.06 4.02 4.00 3.95 3.91 3.73 3.72 3.59 3.52
VIM HERPUD2 XRN1
10p13 7p14.2 3q23
C11orf69 GTPBP5 TRAFD1 PRKAR2A C20orf11 ADCY1 MYO6 TNK2 AOC2 STMN1 ALDH8A1 CEP78 DKFZp761I2123 STX17 SLC11A2
11p13 20q13.33 12q 3p21.3ep21.2 20q13.33 7p13ep12 6q13 3q29 17q21 1p36.1ep35 6q23.2 9q21.2 7p13 9q31.1 12q13
3.43 3.38 3.33 3.29 3.25 3.18 3.11 3.08 3.08 3.04 3.03 2.94 2.84 2.77 2.75 2.74 2.74 2.71 2.61
ZNF16 SLC39A14 CASZ1 FOXP1 ZNF93 FGL1
8q24 8p21.3 1p36.22 3p14.1 19p12 8p22ep21.3
2.61 2.49 2.37 2.29 2.29 2.28
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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.
involving cellecell and cellematrix 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 15 mm 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 (AlHajj 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 downregulated 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
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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). References Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100:3983e8. Barclay AN, Jackson DI, Willis AC, Williams AF. The leukocyte-common antigen (L-CA) family. Adv Exp Med Biol 1988;237:3e7. Carinci F, Volinia S, Pezzetti F, Francioso F, Tosi L, Piattelli A. Titaniumecell interaction: analysis of gene expression profiling. J Biomed Mater Res Appl Biomater 2003;66:341e6. Carinci F, Pezzetti F, Volinia S, Francioso F, Arcelli D, Farina E, et al. Zirconium oxide: analysis of MG63 osteoblast-like cell response by means of a microarray technology. Biomaterials 2004a;25:215e28. Carinci F, Pezzetti F, Volinia S, Francioso F, Arcelli D, Marchesini J, et al. Analysis of MG63 osteoblastic-cell response to a new nanoporous implant surface by means of a microarray technology. Clin Oral Implants Res 2004b;15:180e6. Carinci F, Pezzetti F, Volinia S, Laino G, Arcelli D, Caramelli E, et al. P-15 cell-binding domain derived from collagen: analysis of MG63 osteoblastic-cell response by means of a microarray technology. J Periodontol 2004c;75:66e83. Carinci F, Piattelli A, Stabellini G, Palmieri A, Scapoli L, Laino G, et al. Calcium sulfate: analysis of MG63 osteoblast-like cell response by means of a microarray technology. J Biomed Mater Res 2004d;71:260e7. D’Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A, et al. Human postnatal dental pulp cells co-differentiate into osteoblasts
and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ 2007;14:1162e71. Graziano A, d’Aquino R, Angelis MG, De Francesco F, Giordano A, Laino G, et al. Scaffold’s surface geometry significantly affects human stem cell bone tissue engineering. J Cell Physiol 2007a;l:12. Graziano A, d’Aquino R, Cusella-De Angelis MG, Laino G, Piattelli A, Pacifici M, et al. Concave pit-containing scaffold surfaces improve stem cell-derived osteoblast performance and lead to significant bone tissue formation. PLoS ONE 2007b;2:e496. Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1þ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994;84: 4164e73. Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, et al. Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A 2003;100:15178e83. Laino G, d’Aquino R, Graziano A, Lanza V, Carinci F, Naro F, et al. A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue (LAB). J Bone Miner Res 2005;20:1394e402. Laino G, Graziano A, d’Aquino R, Pirozzi G, Lanza V, Valiante S, et al. An approachable human adult stem cell source for hard-tissue engineering. J Cell Physiol 2006a;206:693e701. Laino G, Carinci F, Graziano A, d’Aquino R, Lanza V, De Rosa A, et al. In vitro bone production using stem cells derived from human dental pulp. J Craniofac Surg 2006b;17:511e5. Papaccio G, Graziano A, d’Aquino R, Graziano MF, Pirozzi G, Menditti D, et al. Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol 2006;208:319e25. Simmons PJ, Torok-Storb B. CD34 expression by stromal precursors in normal human adult bone marrow. Blood 1991;78:2848e53. Singh SK, Clarke ID, Hide T, Dirks PB. Cancer stem cells in nervous system tumors. Oncogene 2004;23:7267e73. Smith N, Dong Y, Lian JB, Pratap J, Kingsley PD, van Wijnen AJ, et al. Overlapping expression of Runx1(Cbfa2) and Runx2(Cbfa1) transcription factors supports cooperative induction of skeletal development. J Cell Physiol 2005;203:133e43. Tsukahara S, Miyazawa N, Akagawa H, Forejtova S, Pavelka K, Tanaka T, et al. COL6A1, the candidate gene for ossification of the posterior longitudinal ligament, is associated with diffuse idiopathic skeletal hyperostosis in Japanese. Spine 2005;30:2321e4. Wiener JR, Windham TC, Estrella VC, Parikh NU, Thall PF, Deavers MT, et al. Activated SRC protein tyrosine kinase is overexpressed in late-stage human ovarian cancers. Gynecol Oncol 2003;88:73e9. Yamashiro T, Wang XP, Li Z, Oya S, Aberg T, Fukunaga T, et al. Possible roles of Runx1 and Sox9 in incipient intramembranous ossification. J Bone Miner Res 2004;19:1671e7. Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425:836e40.
Comparison between osteoblasts derived from human dental pulp stem cells and osteosarcoma cell lines Annalisa Palmieri a, Furio Pezzetti a, Antonio Graziano b, D’Aquino Riccardo b, Ilaria Zollino c, Giorgio Brunelli c, Marcella Martinelli a, Marzia Arlotti a, Francesco Carinci c,* a
Centre of Molecular Genetics, CARISBO Foundation, Institute of Histology and General Embryology, School of Medicine, University of Bologna, Bologna, Italy b Dental Clinic, Second University of Naples, Naples, Italy c Chair of Maxillofacial Surgery, School of Medicine, University of Ferrara, Arcispedale S. Anna, Corso Giovecca 203, 44100 Ferrara, Italy Received 18 October 2007; revised 14 December 2007; accepted 25 February 2008
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 STRO1 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 19.2 K 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. Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Stem cells; Dental pulp; Autologous bone; Microarray; Tumour
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 * Corresponding author. Tel./fax: þ39 0532 455582. E-mail address: [email protected] (F. Carinci). URL: http://www.carinci.org
stem cells c-kit, CD34 and CD45 were detected. Mesenchymal stem/progenitor cell populations that are c-kit- and CD34positive 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
1065-6995/$ - see front matter Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.02.003
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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 18e37 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 a-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-kitþ/STRO-1þ/ CD34þ/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 19.2 K 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 postprocess 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 (Tstatistic 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. 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 GMPdependent 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 cellecell recognition, signalling and for cell movement). Interesting down-regulated genes in cell development include: NTRK3 e a member of the neurotrophic tyrosine receptor kinase-NTRK family e 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 e 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
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Table 1 Down-regulate genes in OHDP vs. OCs Name
Symbol
Cytoband
Score (d )
Structure specific recognition protein 1 Procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1 N-Deacetylase/N-sulfotransferase 3 Tropomodulin 2 (neuronal) Nuclear factor I/A CD36 molecule (thrombospondin receptor) LIM domain only 2 (rhombotin-like 1) ATP-binding cassette, sub-family B member 7 Hypothetical protein LOC647197 Zinc finger and BTB domain containing 1 Peter pan homolog (Drosophila) Sex comb on midleg homolog 1 (Drosophila) PHD finger protein 10 Carboxypeptidase D M-phase phosphoprotein 6 G protein-coupled receptor associated sorting protein 2 Trinucleotide repeat containing 4 Protein kinase, cGMP-dependent, type I Mercaptopyruvate sulfurtransferase Amidohydrolase domain containing 1 Poly(A) binding protein, cytoplasmic 1 PR domain containing 2, with ZNF domain Neurotrophic tyrosine kinase, receptor, type 3 Hypothetical protein FLJ12681 Tetratricopeptide repeat domain 3 Reticulon 4 receptor-like 1 COBL-like 1 Serine/arginine repetitive matrix 2 Chromosome 20 open reading frame 20 Syndecan 3 (N-syndecan) Zinc and ring finger 3 Discs, large homolog 2, chapsyn-110 RAB22A, member RAS oncogene family Pappalysin 2 Myeloid cell nuclear differentiation antigen CD48 molecule Neuronal PAS domain protein 2 Protein tyrosine phosphatase, receptor type, N polypeptide 2 Syndecan binding protein (syntenin) MON2 homolog (S. cerevisiae) Family with sequence similarity 13, member C1 Runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) Sidekick homolog 1 (chicken) Sin3A-associated protein, 30kDa Triple functional domain (PTPRF interacting) Acetyl-Coenzyme A carboxylase alpha X-ray repair complementing defective repair in Chinese hamster cells 5 GRIP and coiled-coil domain containing 2 Jagged 1 (Alagille syndrome) Serum response factor binding protein 1 Caspase 8, apoptosis-related cysteine peptidase Mitogen-activated protein kinase kinase kinase 4 Moesin Dihydrolipoamide dehydrogenase Sepiapterin reductase Sushi domain containing 1 RAB11 family interacting protein 5 (class I) Chromosome 10 open reading frame 76 KIAA0350 protein Astrotactin Chitobiase, di-N-acetyl-
SSRP1 PLOD1 NDST3 TMOD2 NFIA CD36 LMO2 ABCB7 LOC647197 ZBTB1 PPAN SCMH1 PHF10 CPD MPHOSPH6 GPRASP2 TNRC4 PRKG1 MPST AMDHD1 PABPC1 PRDM2 NTRK3 LA16c-360B4.1 TTC3 RTN4RL1 COBLL1 SRRM2 C20orf20 SDC3 ZNRF3 DLG2 RAB22A PAPPA2 MNDA CD48 NPAS2 PTPRN2 SDCBP MON2 FAM13C1 RUNX1
11q12 1p36.3ep36.2 4q26 15q21.1eq21.2 1p31.3ep31.2 7q11.2 11p13 Xq12eq13 14q32.2 14q23.3 19p13 1p34 6q27 17p11.1eq11.2 16q23.3 Xq22.1 1q21 10q11.2 22q13.1 12q23.1 8q22.2eq23 1p36.21 15q25 16p13.3 21q22.2 17p13.3 2q24.3 16p13.3 20q13.33 1pter-p22.3 22q12.1 11q14.1 20q13.32 1q23eq25 1q22 1q21.3eq22 2q11.2 7q36 8q12 12q14.1 10q21.1 21q22.3
5.83 5.62 5.20 5.16 5.02 4.88 4.75 4.56 4.55 4.35 4.29 4.27 4.23 4.19 4.18 4.08 4.07 4.02 3.78 3.73 3.69 3.67 3.65 3.54 3.48 3.46 3.44 3.43 3.39 3.37 3.27 3.22 3.22 3.19 3.18 3.09 3.09 3.04 3.02 3.01 3.00 2.98
SDK1 SAP30 TRIO ACACA XRCC5
7p22.2 4q34.1 5p15.1ep14 17q21 2q35
2.98 2.94 2.93 2.93 2.88
GCC2 JAG1 SRFBP1 CASP8 MAP3K4 MSN DLD SPR SUSD1 RAB11FIP5 C10orf76 KIAA0350 ASTN CTBS
2q12.3 20p12.1ep11.23 5q23.1 2q33eq34 6q26 Xq11.2eq12 7q31eq32 2p14ep12 9q31.3eq33.1 2p13ep12 10q24.32 16p13.13 1q25.2 1p22
2.86 2.76 2.75 2.73 2.73 2.71 2.71 2.67 2.54 2.53 2.52 2.51 2.51 2.51 (continued on next page)
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Table 1 (continued ) Name
Symbol
Cytoband
Score (d )
Ribosomal protein L6 Formyl peptide receptor-like 1 Hypothetical protein MGC10646 Aryl hydrocarbon receptor interacting protein Adenosine deaminase, RNA-specific, B1 (RED1 homolog rat) X (inactive)-specific transcript Phosphoribosyl pyrophosphate synthetase 2 RAB22A, member RAS oncogene family SMC6 structural maintenance of chromosomes 6-like 1 (yeast) Spectrin, beta, erythrocytic (includes spherocytosis, clinical type I) Hypothetical protein FLJ30596 Periphilin 1 Caldesmon 1 SAM domain, SH3 domain and nuclear localisation signals, 1 Sodium channel, voltage-gated, type IV, beta DKFZP686A01247 hypothetical protein F-box and WD-40 domain protein 11
RPL6 FPRL1 MGC 10646 AIP ADARB1 XIST PRPS2 RAB22A SMC6L1 SPTB FLJ30596 PPHLN1 CALD1 SAMSN1 SCN4B DKFZP686A01247 FBXW11
12q24.1 19q13.3eq13.4 4q21.21 11q13.3 21q22.3 Xq13.2 Xp22.3ep22.2 20q13.32 2p24.2 14q23eq24.2 5p13.2 12q12 7q33 21q11 11q23.3 4p13 5q35.1
2.49 2.46 2.45 2.45 2.40 2.33 2.32 2.28 2.27 2.26 2.25 2.24 2.23 2.23 2.22 2.20 2.19
Table 2 Up-regulate genes in OHDP vs. OCs Name
Symbol
Cytoband
Score (d )
Hypothetical protein MGC4692 Decorin HLA-B associated transcript 1 Beta-site APP-cleaving enzyme 1 Prenylcysteine oxidase 1 MORN repeat containing 2 Collagen, type VI, alpha 3 Glypican 3 Nudix (nucleoside diphosphate linked moiety X)-type motif 13 Methyl-CpG binding domain protein 2 WD repeat domain 20 ADAM metallopeptidase domain 12 (meltrin alpha) Zinc finger protein 38 F-box and WD-40 domain protein 7 (archipelago homolog, Drosophila) Vimentin HERPUD family member 2 50 -30 exoribonuclease 1 Transcribed locus Chromosome 11 open reading frame 69 GTP binding protein 5 (putative) TRAF-type zinc finger domain containing 1 Protein kinase, cAMP-dependent, regulatory, type II, alpha Chromosome 20 open reading frame 11 Adenylate cyclase 1 (brain) Myosin VI Tyrosine kinase, non-receptor, 2 Amine oxidase, copper containing 2 (retina-specific) Stathmin 1/oncoprotein 18 Aldehyde dehydrogenase 8 family, member A1 Centrosomal protein 78kDa Hypothetical protein DKFZp761I2123 Syntaxin 17 Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 Zinc finger protein 16 Solute carrier family 39, member 14 Castor homolog 1, zinc finger (Drosophila) Forkhead box P1 Zinc finger protein 93 Fibrinogen-like 1
MGC4692 DCN BAT1 BACE1 PCYOX1 MORN2 COL6A3 GPC3 NUDT13 MBD2 WDR20 ADAM12 ZNF38 FBXW7
12q21.33 6p21.3 11q23.2eq23.3 2p13.3 2p22.1 2q37 Xq26.1 10q22.1 18q21 14q32.31 10q26.3 7q22.1 4q31.3
4.67 4.53 4.31 4.31 4.25 4.06 4.02 4.00 3.95 3.91 3.73 3.72 3.59 3.52
VIM HERPUD2 XRN1
10p13 7p14.2 3q23
C11orf69 GTPBP5 TRAFD1 PRKAR2A C20orf11 ADCY1 MYO6 TNK2 AOC2 STMN1 ALDH8A1 CEP78 DKFZp761I2123 STX17 SLC11A2
11p13 20q13.33 12q 3p21.3ep21.2 20q13.33 7p13ep12 6q13 3q29 17q21 1p36.1ep35 6q23.2 9q21.2 7p13 9q31.1 12q13
3.43 3.38 3.33 3.29 3.25 3.18 3.11 3.08 3.08 3.04 3.03 2.94 2.84 2.77 2.75 2.74 2.74 2.71 2.61
ZNF16 SLC39A14 CASZ1 FOXP1 ZNF93 FGL1
8q24 8p21.3 1p36.22 3p14.1 19p12 8p22ep21.3
2.61 2.49 2.37 2.29 2.29 2.28
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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.
involving cellecell and cellematrix 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 15 mm 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 (AlHajj 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 downregulated 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
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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). References Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100:3983e8. Barclay AN, Jackson DI, Willis AC, Williams AF. The leukocyte-common antigen (L-CA) family. Adv Exp Med Biol 1988;237:3e7. Carinci F, Volinia S, Pezzetti F, Francioso F, Tosi L, Piattelli A. Titaniumecell interaction: analysis of gene expression profiling. J Biomed Mater Res Appl Biomater 2003;66:341e6. Carinci F, Pezzetti F, Volinia S, Francioso F, Arcelli D, Farina E, et al. Zirconium oxide: analysis of MG63 osteoblast-like cell response by means of a microarray technology. Biomaterials 2004a;25:215e28. Carinci F, Pezzetti F, Volinia S, Francioso F, Arcelli D, Marchesini J, et al. Analysis of MG63 osteoblastic-cell response to a new nanoporous implant surface by means of a microarray technology. Clin Oral Implants Res 2004b;15:180e6. Carinci F, Pezzetti F, Volinia S, Laino G, Arcelli D, Caramelli E, et al. P-15 cell-binding domain derived from collagen: analysis of MG63 osteoblastic-cell response by means of a microarray technology. J Periodontol 2004c;75:66e83. Carinci F, Piattelli A, Stabellini G, Palmieri A, Scapoli L, Laino G, et al. Calcium sulfate: analysis of MG63 osteoblast-like cell response by means of a microarray technology. J Biomed Mater Res 2004d;71:260e7. D’Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A, et al. Human postnatal dental pulp cells co-differentiate into osteoblasts
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