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Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer

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Abstract

The periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10–20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 μg/ml ascorbic acid and 2 mM β-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL.

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References

  • Abdallah BM, Haack-Sorensen M, Burns JS, Elsnab B, Jakob F, Hokland P, Kassem M (2005) Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite of extensive proliferation. Biochem Biophys Res Commun 326:527–538

    Article  PubMed  CAS  Google Scholar 

  • Arora PD, McCulloch CA (1994) Dependence of collagen remodelling on alpha-smooth muscle actin expression by fibroblasts. J Cell Physiol 159:161–175

    Article  PubMed  CAS  Google Scholar 

  • Beertsen W, McCulloch CA, Sodek J (1997) The periodontal ligament: a unique, multifunctional connective tissue. Periodontol 2000 13:20–40

    Article  PubMed  CAS  Google Scholar 

  • Berkovitz BKB, Shore RC (1995) Cells of the periodontal ligament. In: Berkovitz BKB, Moxham BJ, Newman HJ (ed) The periodontal ligament in health and disease. Mosby-Wolfe, London, pp 9–33

    Google Scholar 

  • Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279:349–352

    Article  PubMed  CAS  Google Scholar 

  • Carnes DL, Maeder CL, Graves DT (1997) Cells with osteoblastic phenotypes can be explanted from human gingiva and periodontal ligament. J Periodontol 68:701–707

    PubMed  CAS  Google Scholar 

  • Colgin LM, Reddel RR (1999) Telomere maintenance mechanisms and cellular immortalization. Curr Opin Genet Dev 9:97–103

    Article  PubMed  CAS  Google Scholar 

  • Crosby AH, Edwards SJ, Murray JC, Dixon MJ (1995) Genomic organization of the human osteopontin gene: exclusive of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II. Genomics 27:155–160

    Article  PubMed  CAS  Google Scholar 

  • Gollahon LS, Kraus E, Wu TA, Yim SO, Strong LC, Shay JW, Tainsky MA (1998) Telomerase activity during spontaneous immortalization of Li-Fraumeni syndrome skin fibroblasts. Oncogene 17:709–717

    Article  PubMed  CAS  Google Scholar 

  • Gould TR, Melcher AH, Brunette DM (1977) Location of progenitor cells in periodontal ligament of mouse molar stimulated by wounding. Anat Rec 188:133–141

    Article  PubMed  CAS  Google Scholar 

  • Gould TR, Melcher AH, Brunette DM (1980) Migration and division of progenitor cell populations in periodontal ligament after wounding. J Periodontal Res 15:20–42

    Article  PubMed  CAS  Google Scholar 

  • Gould TR, Brunette DM, Dorey J (1982) Cell turnover in the periodontal ligament determined by continuous infusion of H3-thymidine using osmotic minipumps. J Periodontal Res 17:662–668

    Article  PubMed  CAS  Google Scholar 

  • Gronthos S, Chen S, Wang CY, Robey PG, Shi S (2003) Telomerase accelerates osteogenesis of bone marrow stromal stem cells by upregulation of CBFA1, osterix, and osteocalcin. J Bone Miner Res 18:716–722

    Article  PubMed  CAS  Google Scholar 

  • Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA (1999) Creation of human tumor cells with defined genetic elements. Nature 400:464–468

    Article  PubMed  CAS  Google Scholar 

  • Itahana K, Dimri GP, Hara E, Itahana Y, Zou Y, Desprez PY, Campisiet J (2002) A role for p53 in maintaining and establishing the quiescence growth arrest in human cells. J Biol Chem 277:18206–18214

    Article  PubMed  CAS  Google Scholar 

  • Kamata N, Fujimoto R, Tomonari M, Taki M, Nagayama M, Yasumoto S (2004) Immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase. J Oral Pathol Med 33:417–423

    Article  PubMed  CAS  Google Scholar 

  • Kanaya T, Kyo S, Hamada K, Takakura M, Kitagawa Y, Harada H, Inoue M (2000) Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription. Clin Cancer Res 6:1239–1247

    PubMed  CAS  Google Scholar 

  • Karimbux NY, Rosenblum ND, Nishimura I (1992) Site-specific expression of collagen I and XII mRNAs in the rat periodontal ligament at two developmental stages. J Dent Res 71:1355–1362

    PubMed  CAS  Google Scholar 

  • Kato C, Kojima T, Komaki M, Mimori K, Duarte WR, Takenaga K, Ishikawa I (2005) S100A4 inhibition by RNAi up-regulates osteoblast related genes in periodontal ligament cells. Biochem Biophys Res Commun 326:147–153

    Article  PubMed  CAS  Google Scholar 

  • Kawano Y, Kobune M, Yamaguchi M, Nakamura K, Ito Y, Sasaki K, Takahashi S, Nakamura T, Chiba H, Sato T, Matsunaga T, Azuma H, Ikebuchi K, Ikeda H, Kato J, Niitsu Y, Hamada H (2003) Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)-transfected human stromal cells. Blood 101:532–540

    Article  PubMed  CAS  Google Scholar 

  • Kiefer MC, Saphire AC, Bauer DM, Barr PJ (1990) The cDNA and derived amino acid sequences of human bovine bone Gla protein. Nucleic Acids Res 18:1909

    Article  PubMed  CAS  Google Scholar 

  • Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011–2015

    Article  PubMed  CAS  Google Scholar 

  • Kudo Y, Hiraoka M, Kitagawa S, Miyauchi M, Kakuo S, Zhao M, Ide T, Tanaka T (2002) Establishment of human cementifying fibroma cell lines by transfection with temperature-sensitive simian virus-40 T-antigen gene and hTERT gene. Bone 30:712–717

    Article  PubMed  CAS  Google Scholar 

  • Kusumoto M, Ogawa T, Mizumoto K, Ueno H, Niiyama H, Sato N, Nakamura M, Tanaka M (1999) Adenovirus-mediated p53 gene transduction inhibits telomerase activity independent of its effects on cell cycle arrest and apoptosis in human pancreatic cancer cells. Clin Cancer Res 5:2140–2147

    PubMed  CAS  Google Scholar 

  • Lekic P, McCulloch CA (1996) Periodontal ligament cell populations: the central role of fibroblasts in creating a unique tissue. Anat Rec 245:327–341

    Article  PubMed  CAS  Google Scholar 

  • Lekic P, Rojas J, Birek C, Tenenbaum H, McCulloch CA (2001) Phenotypic comparison of periodontal ligament cells in vivo and in vitro. J Periodontal Res 36:71–79

    Article  PubMed  CAS  Google Scholar 

  • Levine AJ (1990) The p53 protein and its interactions with the oncogene products of the small DNA tumor viruses. Virology 177:419–426

    Article  PubMed  CAS  Google Scholar 

  • Li H, Bartold PM, Young WG, Xiao Y, Waters MJ (2001) Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium. J Bone Miner Res 16:1068–1076

    Article  PubMed  CAS  Google Scholar 

  • MacNeil RL, Berry JE, Strayhorn CL, Shigeyama Y, Somerman MJ (1998) Expression of type I and XII collagen during development of the periodontal ligament in the mouse. Arch Oral Biol 43:779–787

    Article  PubMed  CAS  Google Scholar 

  • Maeda H, Wada N, Nakamuta H, Akamine A (2004) Human periapical granulation tissue contains osteogenic cells. Cell Tissue Res 315:203–208

    Article  PubMed  Google Scholar 

  • Maeda H, Wada N, Fujii S, Akamine A (2005) Fibroblastic cells from human periapical granulation tissue preferentially form calcified matrices in decalcified and boiled rat bone. Cell Tissue Res 320:135–140

    Article  PubMed  Google Scholar 

  • Matsuda N, Kumar NM, Ramakrishnan PR, Lin WL, Genco RJ, Cho MI (1993) Evidence for up-regulation of epidermal growth-factor receptors on rat periodontal ligament fibroblastic cells associated with stabilization of phenotype in vitro. Arch Oral Biol 38:559–569

    Article  PubMed  CAS  Google Scholar 

  • McCulloch CA (1985) Progenitor cell populations in the periodontal ligament of mice. Anat Rec 211:258–262

    Article  PubMed  CAS  Google Scholar 

  • McCulloch CA, Bordin S (1991) Role of fibroblast subpopulations in periodontal physiology and pathology. J Periodontal Res 26:144–154

    Article  PubMed  CAS  Google Scholar 

  • McCulloch CA, Melcher AH (1982) Continuous labelling of the periodontal ligament of mice. J Periodontal Res 18:231–241

    Article  Google Scholar 

  • McCulloch CA, Melcher AH (1983) Cell density and cell generation in the periodontal ligament of mice. Am J Anat 167:43–58

    Article  PubMed  CAS  Google Scholar 

  • McCulloch CA, Nemeth E, Lowenberg B, Melcher AH (1987) Paravascular cells in endosteal spaces of alveolar bone contribute to periodontal ligament cell populations. Anat Rec 219:233–242

    Article  PubMed  CAS  Google Scholar 

  • Melcher AH (1976) On the repair potential of periodontal tissues. J Periodontol 47:256–260

    PubMed  CAS  Google Scholar 

  • Melcher AH (1984) Cellular activity in adaptation of the periodontium In: McNamara JA Jr, Ribbens KA (ed) Malocclusion and the periodontium. University of Michigan Press, Michigan, pp 1–15

    Google Scholar 

  • Nakayama J, Tahara H, Tahara E, Saito M, Ito K, Nakamura H, Nakanishi T, Tahara E, Ide T, Ishikawa F (1998) Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 18:65–68

    Article  PubMed  CAS  Google Scholar 

  • Nemoto E, Shimonishi M, Nitta Y, Shimauchi H (2004) The involvement of platelet-derived growth factor receptors and insulin-like growth factor-1 receptors signaling during mineralized nodule formation by human periodontal ligament cells. J Periodontal Res 39:388–397

    Article  PubMed  CAS  Google Scholar 

  • Ogawa E, Maruyama M, Kagoshima H, Inuzuka M, Lu J, Satake M, Shigesada K, Ito Y (1993) PEBP2/PEA2 represents a family of transcription factors homologous to products of the Drosophila runt gene and human AML1 gene. Proc Natl Acad Sci USA 90:6859–6863

    Article  PubMed  CAS  Google Scholar 

  • Reichenberger E, Baur S, Sukotjo C, Olsen BR, Karimbux NY, Nishimura I (2000) Collagen XII mutation disrupts matrix structure of periodontal ligament and skin. J Dent Res 79:1962–1968

    Article  PubMed  CAS  Google Scholar 

  • Roberts WE, Mozsary PG, Klingler E (1982) Nuclear size as a cell-kinetic maker for osteoblast differentiation. Am J Anat 165:373–384

    Article  PubMed  CAS  Google Scholar 

  • Ryu MS (1995) Telomeres, telomerase, and immortality. J Natl Cancer Inst 87:884–894

    Article  Google Scholar 

  • Saito M, Nakagawa K, Hamada K, Hirose S, Harada H, Kohno S, Nagato S, Ohnishi T (2004) Introduction of p16INK4a inhibits telomerase activity through transcriptional suppression of human telomerase reverse transcriptase expression in human gliomas. Int J Oncol 24:1213–1220

    PubMed  CAS  Google Scholar 

  • Shats I, Milyavsky M, Tang X, Stambolsky P, Erez N, Brosh R, Kogan I, Braunstein I, Tzukerman M, Ginsberg D, Rotter V (2004) p53-dependent down-regulation of telomerase is mediated by p21waf1. J Biol Chem 279:50976–50985

    Article  PubMed  CAS  Google Scholar 

  • Shay JW, Wright WE, Werbin H (1991) Defining the molecular mechanisms of human cell immortalization. Biochim Biophys Acta 1072:1–7

    PubMed  CAS  Google Scholar 

  • Shay JW, Zou Y, Hiyama E, Wright WE (2001) Telomerase and cancer. Hum Mol Genet 10:677–685

    Article  PubMed  CAS  Google Scholar 

  • Stampfer MR, Garbe J, Nijjar T, Wigington D, Swisshelm K, Yaswen P (2003) Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines. Oncogene 22:5238–5251

    Article  PubMed  CAS  Google Scholar 

  • Wada N, Maeda H, Tanabe K, Tsuda E, Yano K, Nakamuta H, Akamine A (2001) Periodontal ligament cells secrete the factor that inhibits osteoclastic differentiation and function: the factor is osteoprotegerin/osteoclastogenesis inhibitory factor. J Periodontal Res 36:56–63

    Article  PubMed  CAS  Google Scholar 

  • Wada N, Maeda H, Yoshimine Y, Akamine A (2004) Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF-kappa B ligand in periodontal ligament fibroblasts through the induction interleukin-1 beta and tumor necrosis factor-alpha. Bone 35:629–635

    Article  CAS  Google Scholar 

  • Xu D, Wang Q, Gruber A, Bjorkholm M, Chen Z, Zaid A, Selivanova G, Peterson C, Wiman KG, Pisa P (2000) Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells. Oncogene 19:5123–5133

    Article  PubMed  CAS  Google Scholar 

  • Yamada S, Murakami S, Matoba R, Ozawa Y, Yokokoji T, Nakahira Y, Ikezawa K, Takayama S, Matsubara K, Okada H (2001) Expression profile of active genes in human periodontal ligament and isolation of PLAP-1, a novel SLRP family gene. Gene 275:279–286

    Article  PubMed  CAS  Google Scholar 

  • Yamashita Y, Sato M, Noguchi T (1987) Alkaline phosphatase in the periodontal ligament of the rabbit and macaque monkey. Arch Oral Biol 32:677–678

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812–8582, Japan

    Shinsuke Fujii, Hidefumi Maeda, Naohisa Wada & Akifumi Akamine

  2. Department of Occupational Therapy, School of Health Science, Kibi International University, Takahashi, Okayama, 716-8508, Japan

    Yoshio Kano

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  1. Shinsuke Fujii
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  2. Hidefumi Maeda
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  3. Naohisa Wada
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  4. Yoshio Kano
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  5. Akifumi Akamine
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Correspondence to Hidefumi Maeda.

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This work was supported by grants-in-aid (project 15689024, 16209056, 17659599, and 17791359) for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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Fujii, S., Maeda, H., Wada, N. et al. Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer. Cell Tissue Res 324, 117–125 (2006). https://doi.org/10.1007/s00441-005-0101-4

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