Odontology DOI 10.1007/s10266-013-0145-y
ORIGINAL ARTICLE
Cementogenic potential of multipotential mesenchymal stem cells purified from the human periodontal ligament Daisuke Torii • Kiyoshi Konishi • Nobuyuki Watanabe Shinichi Goto • Takeki Tsutsui
•
Received: 26 July 2013 / Accepted: 15 December 2013 Ó The Society of The Nippon Dental University 2014
Abstract The periodontal ligament (PDL) consists of a group of specialized connective tissue fibers embedded in the alveolar bone and cementum that are believed to contain progenitors for mineralized tissue-forming cell lineages. These progenitors may contribute to regenerative cell therapy or tissue engineering methods aimed at recovery of tissue formation and functions lost in periodontal degenerative changes. Some reports using immortal clonal cell lines of cementoblasts, which are cells containing mineralized tissue-forming cell lineages, have shown that their phenotypic alteration and gene expression are associated with mineralization. Immortal, multipotential PDL-derived cell lines may be useful biological tools for evaluating differentiation-inducing agents. In this study, we confirmed the gene expression and mineralization potential of primary and immortal human PDL cells and characterized their immunophenotype. Following incubation with mineralization induction medium containing b-glycerophosphate, ascorbic acid, and dexamethasone, normal human PDL (Pel) cells and an immortal
D. Torii T. Tsutsui Department of Pharmacology, The Nippon Dental University School of Life Dentistry at Tokyo, Tokyo, Japan K. Konishi (&) Department of Microbiology, The Nippon Dental University School of Life Dentistry at Tokyo, Tokyo, Japan e-mail: [email protected] N. Watanabe Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan S. Goto Department of Dental Materials Science, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan
derivative line (Pelt) cells showed higher levels of mineralization compared with cells grown in normal growth medium. Both cell types were positive for putative surface antigens of mesenchymal cells (CD44, CD73, CD90, and CD105). They were also positive for stage-specific embryonic antigen-3, a marker of multipotential stem cells. Furthermore, PDL cells expressed cementum attachment protein and cementum protein 1 when cultured with recombinant human bone morphogenetic protein-2 or -7. The results suggest that normal and immortal human PDL cells contain multipotential mesenchymal stem cells with cementogenic potential. Keywords
Periodontal ligament Cementum Stem cells
Introduction Preventing tooth loss related to periodontal disease and prosthodontic treatment after tooth loss are important for improving the quality of life in patients with periodontal disease. Conventional surgical periodontal procedures such as guided tissue regeneration techniques and bone grafting have been conducted according to the degree of tissue deficit. However, these procedures involve problems such as increased surgical stress to healthy tissues with autologous bone grafting and adaptation of a graft substitute containing tempered material or animal-derived components. Furthermore, these procedures do not lead to complete reconstruction of periodontal tissue containing alveolar bone, connective tissue, periodontal ligament (PDL), and cementum, which are all lost during degenerative changes. On the other hand, for regenerative medicine in other organs, allogeneic stem cell transplantation therapy using a side population of cells that commonly reside in the interstitial spaces of the
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kidney leads to recovery of kidney function in mice [1]. Therefore, regulating the appropriate differentiation of stem cells that exist in periodontal tissues will lead to repair of cementum, bone, or the PDL, which are lost in periodontal diseases. Mesenchymal stem cells are a type of adult stem cell thought to contribute to regeneration of tissues such as bone, cartilage, and adipose tissue [2]. PDL cells are also mesenchymal cells that originate from the dental follicle that forms during the cap stage of tooth germ development from an ectomesenchymal progenitor cell population that is derived from the cranial neural crest [3]. Dental follicle cells are thought to contribute to the formation of periodontal tissues [4] and contain undifferentiated, lineagecommitted cells in their population [5]. However, their immunophenotypical characteristics and stem cell hierarchy have not yet been clarified. A previous report showed that human dental follicle cells strongly express two putative cementoblast markers, cementum attachment protein (CAP) and cementum protein-23 (CP-23). They are also reported to express cementum protein 1 (CEMP1) when stimulated with bone morphogenetic protein (BMP)-2 or -7 [6]. BMP-2 can recruit progenitors contained in PDL to the cementoblastic lineage and regulate differentiation of the cementoblastic lineage by inducing expression of CAP [7]. CAP is also expressed by cementoblasts as a 65-kDa protein and may play a role in cementogenesis [8]. The other marker, CP-23, is strongly expressed on cementoblasts, cell subpopulations of the PDL, and cells located around blood vessels in the PDL [9]. Another study demonstrated that CP-23 not only is a marker protein for cementoblast-related cells but also regulates cementoblast commitment of PDL cells [10]. In this study, we analyzed normal human PDL cells (Pel cells) [11] and a line of immortal derivatives Pelt cells those isogenic to Pel cells except for hTERT gene [12] for expression of surface markers, and evaluated their mineralization potential. When stimulated with BMP-2 or -7, these cultured human PDL cells showed high-level expression of the above cementoblast markers. They also showed formation of mineralized nodules following incubation with mineralization induction medium. Furthermore, we confirmed a small population of cells among the human PDL cells that was positive for putative surface antigens of mesenchymal stem cells. Our results suggest that these immortal human PDL cells retained the properties of the original stem cell population and the potential for mineralization. They could be useful tools for innovative drug development or for studying regenerative medicine for patients with periodontal diseases.
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Materials and methods Cell culture Pel cells derived from normal adult human PDL, and Pelt cells, which are immortal derivatives, were established as described previously [11, 12]. Culture medium for normal growth was a-minimum essential medium (a-MEM) (GIBCO/Life Technologies, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan), 1.0 mM pyruvic acid (Sigma-Aldrich, Tokyo, Japan), 0.2 mM serine, 0.1 mM aspartic acid, 0.22 % NaHCO3 (Wako Pure Chemical Industries, Osaka, Japan), 100 units/ml penicillin, and 100 lg/ml streptomycin (GIBCO/Life Technologies). Cells were maintained in a humidified atmosphere of 5 % CO2/95 % air at 37 °C. The medium was changed every 3–4 days. When the cells reached confluence, they were subcultured at a split ratio of 1:2–1:4 by gentle trypsinization with 0.05 % trypsin– EDTA solution (GIBCO/Life Technologies) for 3 min at room temperature. The number of population doublings of Pel cells used in these experiments was between 11 and 16. Flow cytometry and cell sorting PDL cells were incubated with 100 ll fluorescence-conjugated antibody for 1 h at 4 °C in Ca2?- and Mg2?-free phosphate-buffered saline [PBS(-)] containing 0.5 % bovine serum albumin (BSA) and 2 mM EDTA. The FACSAriaTM III Flow Cytometry System (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the expression of cell surface antigens. Alexa FluorÒ 647-conjugated anti-CD90 (1:10; cat. 9905-53, AbD Serotec, Oxford, UK), peridinin–chlorophyll–protein complex with cyanin-5.5 (PerCP-Cy5.5)-conjugated anti-CD44 (1:80; cat. 45-0441-80), phycoerythrin (PE)-conjugated anti-CD73 (1:20; cat. 12-0739-41), PECy7-conjugated anti-CD105 (1:20; cat. 25-1057-41, eBioscience, San Diego, CA, USA), and PE-conjugated anti-stage-specific embryonic antigen 3 (SSEA-3, 1:20; cat. 330311, BioLegend, San Diego, CA, USA) antibodies were used for staining. Induction of cell differentiation PDL cells were plated at a density of 1000 cells/well in 8-well glass chamber slide system (SPL Life Sciences, Gyeonggi-do, Korea) and cultured in normal growth medium until they reached confluence. The medium was replaced with a-MEM with 2 % FBS containing 100 ng/ml
Odontology
recombinant human BMP (rhBMP)-2 or -7 (R&D Systems, Minneapolis, MN, USA). Then, the cells were cultured in 2 weeks under the BMP stimulus. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Total RNA in PDL cells was isolated with NucleoSpinÒ RNA II according to the manufacturer’s instructions (Macherey– Nagel, Du¨ren, Germany). Total RNA (2 lg) was subjected to RT using the high capacity RNA-to-cDNA Kit (Applied Biosystems/Life Technologies). For qRT-PCR, TaqManÒ gene expression assays and the StepOnePlusTM Real-Time PCR System (Applied Biosystems/Life Technologies) were used on the human target genes, CAP (assay ID: Hs00171965_m1, gene bank number: NM_01421.3, amplicon length: 74) and CEMP1 (assay ID: Hs04185363_s1, gene bank number: NM_001048212.3, amplicon length: 72; Applied Biosystems/ Life Technologies). mRNA expression of b-actin (4326315E0710012; Applied Biosystems/Life Technologies) served as an internal control for normalization. Relative standard curves were analyzed with StepOne Software v2.1 (Applied Biosystems/Life Technologies). The experiments were performed in quadruplicate. Mineralization assay PDL cells were plated at a density of 2.5 9 104 cells/cm2 in 12-well plates in normal growth medium and incubated until they reached confluence. They were cultured in mineralization induction medium containing 10 % FBS, 10 mM sodium bglycerophosphate n-hydrate (Wako Pure Chemical Industries), 10 nM dexamethasone (Wako Pure Chemical Industries), and 100 lM L-ascorbic acid phosphate magnesium salt n-hydrate (Wako Pure Chemical Industries) and stained with 2 % alizarin red S (pH 4.2) as described previously [13]. Electron probe microanalysis (EPMA) After 3 weeks of growth in mineralization induction medium, PDL cells were fixed with 4 % paraformaldehyde (PFA) for 15 min. Calcium and phosphorus in cultured cells were detected with JXA-8900 EPMA (JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV.
with 1 % BSA in PBS for 30 min, and then incubated with primary antibodies for 1 h. Primary antibodies were diluted 1:200 for anti-CAP (sc-53947, Santa Cruz Biotechnology, Dallas, TX, USA) [8] and 1:100 for anti-CEMP1 (sc-164032) [9]. Cells were incubated with Alexa FluorÒ 647-conjugated donkey anti-goat IgG and Alexa FluorÒ 568-conjugated goat anti-mouse IgG (Molecular Probes/ Life Technologies) for 1 h. Cell nuclei were stained with diamidino-2-phenylindole (DAPI). For negative controls, primary antibodies were replaced with an appropriate isotype-matched negative IgG or IgM. Fluorescence staining was visualized with confocal microscopy (LSM 700, Carl Zeiss, Oberkochen, Germany). ImageJ software was used to count the stained PDL cells. Statistical analysis Data were compared using the Student’s t test or Fisher’s exact test, assuming double-sided independent variance and with p \ 0.05 considered to be significant.
Results Characterization of Pel and Pelt Pel and Pelt showed spindle-shaped morphology (data not shown), and more than 99 % were positive for CD44, 73, 90, and 105 with flow cytometry analysis (Fig. 1). In addition, 0.8 and 0.4 % of Pel and Pelt, respectively, were positive for SSEA-3 (SSEA-3?) (Fig. 1). Formation of mineralized nodules After 3 weeks of culture in mineralization induction medium containing b-glycerophosphate, dexamethasone, and L-ascorbic acid, Pel and Pelt were positive for alizarin red S staining and formed mineralized nodules (Fig. 2b, c). Conversely, Pel and Pelt cultured in normal growth medium were negative for alizarin red S staining (Fig. 2a, d). To evaluate depositions of calcium and phosphorus, both cell types with or without mineralized nodules were assayed with EPMA. Calcium and phosphorus were detected only in Pel and Pelt with nodules (Fig. 3).
Immunocytochemistry Gene expression related to cementogenesis For fluorescence microscopy analysis, trypsinized PDL cells were seeded on glass slide chambers (2.5 9 103 cells/cm2). After 2 weeks of differentiation induction in medium containing 2 % FBS and 100 ng/ml rhBMP-7, cells were fixed with 4 % PFA for 15 min, permeabilized with 0.1 % Triton X-100 for 5 min, blocked
The expressions of CAP were 1.55 and 2.0 times higher in Pel and Pelt stimulated with BMP-2, respectively, than in control cells (Fig. 4a, c). Pel and Pelt stimulated with BMP-7 showed 2.95- and 2.65-fold higher CAP expression, respectively, than control cells (Fig. 4a, c). Similarly, the
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Fig. 1 Flow cytometric analysis of Pel and Pelt revealed the expression of cell surface markers associated with mesenchymal stromal cells, CD73, CD90 and CD105. A small percentage of both cells were positive for stage-specific embryonic antigen (SSEA)-3
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Fig. 1 continued
Fig. 2 Mineralized nodule formation of Pel (a–c) and Pelt (d–f). Alizarin red staining shows the mineralization in both cells. Scale bars 100 lm
Control
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Control.
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B
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Odontology Fig. 3 Calcium and phosphorus deposition in mineralized nodule-like structure formed by Pel (a, b) or Pelt (c, d). SEM images show that no nodule formation was observed in both cells cultured in control medium (a, c). The nodule-like structures were formed in cells cultured in induction medium (b, d) contained calcium and phosphorus (data not shown). Scale bars 100 lm
Relative Expression
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Relative Expression
Fig. 4 Cementogenesis-related gene expression in Pel and Pelt. Quantitative RT-PCR analysis presented the up-regulated expression of cementum attachment protein (CAP) (a, c) and cementum protein 1 (CEMP1) (b, d) in both Pel (a, b) and Pelt (c, d) cultured with rhBMP-2 or rhBMP-7. b-actin was used as an internal control. Statistical significance was determined using the Student’s t test (*p \ 0.05 vs. control)
0
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Pelt (SSEA-3+/CD44+/CD105+)
Pel (SSEA-3+/CD44+/CD105+) A
B
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Fig. 5 Expression of cementoblastic phenotypes in SSEA-3/CD44/ CD105 triple-positive cells sorted from Pel or Pelt. Both sorted cells were fibroblastic (a, b) and positive for cementum attachment protein
(CAP) and cementum protein 1 (CEMP1) when cultured with rhBMP7. Scale bars 100 lm
expressions of CEMP1 were 1.45 and 1.85 times higher in Pel and Pelt stimulated with BMP-2, respectively, than in control cells (Fig. 4b, d). Pel and Pelt stimulated with BMP-7 showed 1.65- and 2.85-fold higher CEMP1 expression, respectively, than control cells (Fig. 4b, d).
Cementogenic potential of SSEA-3-positive cells 90.9 % of the isolated SSEA-3? Pel and 99.6 % of the isolated SSEA-3? Pelt expressed CAP and CEMP1 when stimulated with rhBMP-7. Respectively, 4.9 and 1.4 % of
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Pelt (SSEA–3+/CD105+)
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Fig. 5 continued
those cultured without rhBMP-7 expressed CAP and CEMP1 (Fig. 5).
Discussion To characterize the cultured human PDL cells, we analyzed expression of cell surface antigens on Pel and Pelt cells. As shown using flow cytometry (Fig. 1), more than 99 % of the cells were positive for CD44, 73, 90, and 105. Although these cell surface markers have been reported to be mesenchymal stem cell markers in the dental follicle and the PDL [14, 15], we cannot assume that almost all Pel and Pelt cells possess stemness because they were heterogeneous cell populations derived from human PDL. In recent studies, a subset of adult stem cells whose properties are similar in some respects to those of induced pluripotent stem cells (iPS cells) exists among adult human fibroblasts [16] and contributes to the generation of iPS
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cells [17]. Human iPS cells can also be established from PDL fibroblasts with low reprogramming efficiency [18], and reports suggest that those PDL cells also contain a somatic stem cell population possessing multilineage differentiation potential. Another report shows that SSEA-4positive clonal cells derived from human PDL have adipogenic, osteogenic, and chondrogenic potential [19]. Hence, SSEAs may be specific markers for identification of stem cells in the PDL. In the present study, we found BMP-induced cementogenesis in SSEA-3/CD44/CD105 triple-positive cells isolated from normal and immortal human PDL cells (Fig. 5). As reported previously, CAP production was up-regulated in human periodontal fibroblasts by BMP-2 [7]. On the other hand, cementogenesis may also be regulated by BMP-7 expression [20, 21]. Furthermore, rhNoggin, a BMP antagonist, abolishes alkaline phosphatase activity, mineralization, and expression of both CAP and CP-23 in cultured human dental follicle cells. However, during
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stimulation with rhBMP-2, rhNoggin blocks Smad-1 phosphorylation, but not MAPK phosphorylation. Thus, cementogenesis may be affected by both exogenous and endogenous BMP pathways [22]. However, analysis of cementogenesis in SSEA-3/CD44/CD105 triple-positive cells derived from the human PDL has not been reported before. Therefore, this finding provides new insight into regenerative medicine using dental stem/progenitor cells. Our results demonstrate that normal and immortal human PDL cells have mineralization and cementogenic potential and contain a small cell population expressing the markers of multipotential mesenchymal stem cells. Both cell types may be useful for biological applications using PDL-derived mesenchymal stem cells. Acknowledgments This work was supported in part by a Grant-inAid for Young Scientists (No. 25862060 to D. T.) from the Japan Society for the Promotion of Science (JSPS) and a Research Grant (2012–2013 to K. K., T. T., D. T.) from the Nippon Dental University. Conflict of interest of interest.
The authors declare that they have no conflict
Ethical Standard This study was approved by the Committee of Ethics, the Nippon Dental University School of Life Dentistry at Tokyo.
References 1. Hishikawa K, Marumo T, Miura S, Nakanishi A, Matsuzaki Y, Shibata K, Ichiyanagi T, Kohike H, Komori T, Takahashi I, Takase O, Imai N, Yoshikawa M, Inowa T, Hayashi M, Nakaki T, Nakauchi H, Okano H, Fujita T. Musculin/MyoR is expressed in kidney side population cells and can regulate their function. J Cell Biol. 2005;169:921–8. 2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7. 3. Chai Y, Jiang X, Ito Y, Bringas P Jr, Han J, Rowitch DH, Soriano P, McMahon AP, Sucov HM. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 2000;127:1671–9. 4. Bosshardt DD, Schroeder HE. Cementogenesis reviewed: a comparison between human premolars and rodent molars. Anat Rec. 1996;245:267–92. 5. Morsczeck C, Gotz W, Schierholz J, Zeilhofer F, Kuhn U, Mohl C, Sippel C, Hoffmann KH. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol. 2005;24:155–65. 6. Ke´moun P, Laurencin-Dalicieux S, Rue J, Farges JC, Gennero I, Conte-Auriol F, Briand-Mesange F, Gadelorge M, Arzate H, Narayanan AS, Brunel G, Salles JP. Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro. Cell Tissue Res. 2007;329:283–94. 7. Pitaru S, Pritzki A, Bar-Kana I, Grosskopf A, Savion N, Narayanan AS. Bone morphogenetic protein 2 induces the expression of cementum attachment protein in human periodontal ligament clones. Connect Tissue Res. 2002;43:257–64.
8. Saito M, Iwase M, Maslan S, Nozaki N, Yamauchi M, Handa K, Takahashi O, Sato S, Kawase T, Teranaka T, Narayanan AS. Expression of cementum-derived attachment protein in bovine tooth germ during cementogenesis. Bone. 2001;29:242–8. 9. Alvarez-Pe´rez MA, Narayanan S, Zeichner-David M, Rodrı´guez Carmona B, Arzate H. Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23). Bone. 2006;38:409–19. 10. Komaki M, Iwasaki K, Arzate H, Narayanan AS, Izumi Y, Morita I. Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells. J Cell Physiol. 2012;227:649–57. 11. Omori N, Kobayashi H, Tsutsui T. Quantitative comparison of cytocidal effects of tetracyclines and fluoroquinolones on human periodontal ligament fibroblasts. J Periodontal Res. 1999; 34:290–5. 12. Tsutsui T, Kumakura S, Yamamoto A, Kanai H, Tamura Y, Kato T, Anpo M, Tahara H, Barrett JC. Association of p16INK4a and pRb inactivation with immortalization of human cells. Carcinogenesis. 2002;23:2111–7. 13. Inaba T, Kobayashi T, Tsutsui TW, Ogawa M, Uchida M, Tsutsui T. Expression status of mRNA for sex hormone receptors in human dental pulp cells and the response to sex hormones in the cells. Arch Oral Biol. 2013;58:943–50. 14. Yalvac ME, Ramazanoglu M, Gumru OZ, Sahin F, Palota´s A, Rizvanov AA. Comparison and optimisation of transfection of human dental follicle cells, a novel source of stem cells, with different chemical methods and electro-poration. Neurochem Res. 2009;34:1272–7. 15. Tamaki Y, Nakahara T, Ishikawa H, Sato S. In vitro analysis of mesenchymal stem cells derived from human teeth and bone marrow. Odontology. 2012;. doi:10.1007/s10266-012-0075-0. 16. Kuroda Y, Kitada M, Wakao S, Nishikawa K, Tanimura Y, Makinoshima H, Goda M, Akashi H, Inutsuka A, Niwa A, Shigemoto T, Nabeshima Y, Nakahata T, Nabeshima Y, Fujiyoshi Y, Dezawa M. Unique multipotent cells in adult human mesenchymal cell populations. Proc Natl Acad Sci USA. 2010;107: 8639–43. 17. Wakao S, Kitada M, Kuroda Y, Shigemoto T, Matsuse D, Akashi H, Tanimura Y, Tsuchiyama K, Kikuchi T, Goda M, Nakahata T, Fujiyoshi Y, Dezawa M. Multilineage-differentiating stressenduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proc Natl Acad Sci USA. 2011;108:9875–80. 18. Nomura Y, Ishikawa M, Yashiro Y, Sanggarnjanavanich S, Yamaguchi T, Arai C, Noda K, Takano Y, Nakamura Y, Hanada N. Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells. Histochem Cell Biol. 2012;137:719–32. 19. Fukushima H, Kawanabe N, Murata S, Ishihara Y, Yanagita T, Balam TA, Yamashiro T. SSEA-4 is a marker of human deciduous periodontal ligament stem cells. J Dent Res. 2012;91: 955–60. 20. Hakki SS, Foster BL, Nagatomo KJ, Bozkurt SB, Hakki EE, Somerman MJ, Nohutcu RM. Bone morphogenetic protein-7 enhances cementoblast function in vitro. J Periodontol. 2010;81: 1663–74. 21. Kitagawa M, Ao M, Miyauchi M, Abiko Y, Takata T. F-spondin regulates the differentiation of human cementoblast-like (HCEM) cells via BMP7 expression. Biochem Biophys Res Commun. 2012;418:229–33. 22. Silve´rio KG, Davidson KC, James RG, Adams AM, Foster BL, Nociti FH Jr, Somerman MJ, Moon RT. Wnt/b-catenin pathway regulates bone morphogenetic protein (BMP2)-mediated differentiation of dental follicle cells. J Periodontal Res. 2012;47:309–19.
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ORIGINAL ARTICLE
Cementogenic potential of multipotential mesenchymal stem cells purified from the human periodontal ligament Daisuke Torii • Kiyoshi Konishi • Nobuyuki Watanabe Shinichi Goto • Takeki Tsutsui
•
Received: 26 July 2013 / Accepted: 15 December 2013 Ó The Society of The Nippon Dental University 2014
Abstract The periodontal ligament (PDL) consists of a group of specialized connective tissue fibers embedded in the alveolar bone and cementum that are believed to contain progenitors for mineralized tissue-forming cell lineages. These progenitors may contribute to regenerative cell therapy or tissue engineering methods aimed at recovery of tissue formation and functions lost in periodontal degenerative changes. Some reports using immortal clonal cell lines of cementoblasts, which are cells containing mineralized tissue-forming cell lineages, have shown that their phenotypic alteration and gene expression are associated with mineralization. Immortal, multipotential PDL-derived cell lines may be useful biological tools for evaluating differentiation-inducing agents. In this study, we confirmed the gene expression and mineralization potential of primary and immortal human PDL cells and characterized their immunophenotype. Following incubation with mineralization induction medium containing b-glycerophosphate, ascorbic acid, and dexamethasone, normal human PDL (Pel) cells and an immortal
D. Torii T. Tsutsui Department of Pharmacology, The Nippon Dental University School of Life Dentistry at Tokyo, Tokyo, Japan K. Konishi (&) Department of Microbiology, The Nippon Dental University School of Life Dentistry at Tokyo, Tokyo, Japan e-mail: [email protected] N. Watanabe Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan S. Goto Department of Dental Materials Science, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan
derivative line (Pelt) cells showed higher levels of mineralization compared with cells grown in normal growth medium. Both cell types were positive for putative surface antigens of mesenchymal cells (CD44, CD73, CD90, and CD105). They were also positive for stage-specific embryonic antigen-3, a marker of multipotential stem cells. Furthermore, PDL cells expressed cementum attachment protein and cementum protein 1 when cultured with recombinant human bone morphogenetic protein-2 or -7. The results suggest that normal and immortal human PDL cells contain multipotential mesenchymal stem cells with cementogenic potential. Keywords
Periodontal ligament Cementum Stem cells
Introduction Preventing tooth loss related to periodontal disease and prosthodontic treatment after tooth loss are important for improving the quality of life in patients with periodontal disease. Conventional surgical periodontal procedures such as guided tissue regeneration techniques and bone grafting have been conducted according to the degree of tissue deficit. However, these procedures involve problems such as increased surgical stress to healthy tissues with autologous bone grafting and adaptation of a graft substitute containing tempered material or animal-derived components. Furthermore, these procedures do not lead to complete reconstruction of periodontal tissue containing alveolar bone, connective tissue, periodontal ligament (PDL), and cementum, which are all lost during degenerative changes. On the other hand, for regenerative medicine in other organs, allogeneic stem cell transplantation therapy using a side population of cells that commonly reside in the interstitial spaces of the
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kidney leads to recovery of kidney function in mice [1]. Therefore, regulating the appropriate differentiation of stem cells that exist in periodontal tissues will lead to repair of cementum, bone, or the PDL, which are lost in periodontal diseases. Mesenchymal stem cells are a type of adult stem cell thought to contribute to regeneration of tissues such as bone, cartilage, and adipose tissue [2]. PDL cells are also mesenchymal cells that originate from the dental follicle that forms during the cap stage of tooth germ development from an ectomesenchymal progenitor cell population that is derived from the cranial neural crest [3]. Dental follicle cells are thought to contribute to the formation of periodontal tissues [4] and contain undifferentiated, lineagecommitted cells in their population [5]. However, their immunophenotypical characteristics and stem cell hierarchy have not yet been clarified. A previous report showed that human dental follicle cells strongly express two putative cementoblast markers, cementum attachment protein (CAP) and cementum protein-23 (CP-23). They are also reported to express cementum protein 1 (CEMP1) when stimulated with bone morphogenetic protein (BMP)-2 or -7 [6]. BMP-2 can recruit progenitors contained in PDL to the cementoblastic lineage and regulate differentiation of the cementoblastic lineage by inducing expression of CAP [7]. CAP is also expressed by cementoblasts as a 65-kDa protein and may play a role in cementogenesis [8]. The other marker, CP-23, is strongly expressed on cementoblasts, cell subpopulations of the PDL, and cells located around blood vessels in the PDL [9]. Another study demonstrated that CP-23 not only is a marker protein for cementoblast-related cells but also regulates cementoblast commitment of PDL cells [10]. In this study, we analyzed normal human PDL cells (Pel cells) [11] and a line of immortal derivatives Pelt cells those isogenic to Pel cells except for hTERT gene [12] for expression of surface markers, and evaluated their mineralization potential. When stimulated with BMP-2 or -7, these cultured human PDL cells showed high-level expression of the above cementoblast markers. They also showed formation of mineralized nodules following incubation with mineralization induction medium. Furthermore, we confirmed a small population of cells among the human PDL cells that was positive for putative surface antigens of mesenchymal stem cells. Our results suggest that these immortal human PDL cells retained the properties of the original stem cell population and the potential for mineralization. They could be useful tools for innovative drug development or for studying regenerative medicine for patients with periodontal diseases.
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Materials and methods Cell culture Pel cells derived from normal adult human PDL, and Pelt cells, which are immortal derivatives, were established as described previously [11, 12]. Culture medium for normal growth was a-minimum essential medium (a-MEM) (GIBCO/Life Technologies, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan), 1.0 mM pyruvic acid (Sigma-Aldrich, Tokyo, Japan), 0.2 mM serine, 0.1 mM aspartic acid, 0.22 % NaHCO3 (Wako Pure Chemical Industries, Osaka, Japan), 100 units/ml penicillin, and 100 lg/ml streptomycin (GIBCO/Life Technologies). Cells were maintained in a humidified atmosphere of 5 % CO2/95 % air at 37 °C. The medium was changed every 3–4 days. When the cells reached confluence, they were subcultured at a split ratio of 1:2–1:4 by gentle trypsinization with 0.05 % trypsin– EDTA solution (GIBCO/Life Technologies) for 3 min at room temperature. The number of population doublings of Pel cells used in these experiments was between 11 and 16. Flow cytometry and cell sorting PDL cells were incubated with 100 ll fluorescence-conjugated antibody for 1 h at 4 °C in Ca2?- and Mg2?-free phosphate-buffered saline [PBS(-)] containing 0.5 % bovine serum albumin (BSA) and 2 mM EDTA. The FACSAriaTM III Flow Cytometry System (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the expression of cell surface antigens. Alexa FluorÒ 647-conjugated anti-CD90 (1:10; cat. 9905-53, AbD Serotec, Oxford, UK), peridinin–chlorophyll–protein complex with cyanin-5.5 (PerCP-Cy5.5)-conjugated anti-CD44 (1:80; cat. 45-0441-80), phycoerythrin (PE)-conjugated anti-CD73 (1:20; cat. 12-0739-41), PECy7-conjugated anti-CD105 (1:20; cat. 25-1057-41, eBioscience, San Diego, CA, USA), and PE-conjugated anti-stage-specific embryonic antigen 3 (SSEA-3, 1:20; cat. 330311, BioLegend, San Diego, CA, USA) antibodies were used for staining. Induction of cell differentiation PDL cells were plated at a density of 1000 cells/well in 8-well glass chamber slide system (SPL Life Sciences, Gyeonggi-do, Korea) and cultured in normal growth medium until they reached confluence. The medium was replaced with a-MEM with 2 % FBS containing 100 ng/ml
Odontology
recombinant human BMP (rhBMP)-2 or -7 (R&D Systems, Minneapolis, MN, USA). Then, the cells were cultured in 2 weeks under the BMP stimulus. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Total RNA in PDL cells was isolated with NucleoSpinÒ RNA II according to the manufacturer’s instructions (Macherey– Nagel, Du¨ren, Germany). Total RNA (2 lg) was subjected to RT using the high capacity RNA-to-cDNA Kit (Applied Biosystems/Life Technologies). For qRT-PCR, TaqManÒ gene expression assays and the StepOnePlusTM Real-Time PCR System (Applied Biosystems/Life Technologies) were used on the human target genes, CAP (assay ID: Hs00171965_m1, gene bank number: NM_01421.3, amplicon length: 74) and CEMP1 (assay ID: Hs04185363_s1, gene bank number: NM_001048212.3, amplicon length: 72; Applied Biosystems/ Life Technologies). mRNA expression of b-actin (4326315E0710012; Applied Biosystems/Life Technologies) served as an internal control for normalization. Relative standard curves were analyzed with StepOne Software v2.1 (Applied Biosystems/Life Technologies). The experiments were performed in quadruplicate. Mineralization assay PDL cells were plated at a density of 2.5 9 104 cells/cm2 in 12-well plates in normal growth medium and incubated until they reached confluence. They were cultured in mineralization induction medium containing 10 % FBS, 10 mM sodium bglycerophosphate n-hydrate (Wako Pure Chemical Industries), 10 nM dexamethasone (Wako Pure Chemical Industries), and 100 lM L-ascorbic acid phosphate magnesium salt n-hydrate (Wako Pure Chemical Industries) and stained with 2 % alizarin red S (pH 4.2) as described previously [13]. Electron probe microanalysis (EPMA) After 3 weeks of growth in mineralization induction medium, PDL cells were fixed with 4 % paraformaldehyde (PFA) for 15 min. Calcium and phosphorus in cultured cells were detected with JXA-8900 EPMA (JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV.
with 1 % BSA in PBS for 30 min, and then incubated with primary antibodies for 1 h. Primary antibodies were diluted 1:200 for anti-CAP (sc-53947, Santa Cruz Biotechnology, Dallas, TX, USA) [8] and 1:100 for anti-CEMP1 (sc-164032) [9]. Cells were incubated with Alexa FluorÒ 647-conjugated donkey anti-goat IgG and Alexa FluorÒ 568-conjugated goat anti-mouse IgG (Molecular Probes/ Life Technologies) for 1 h. Cell nuclei were stained with diamidino-2-phenylindole (DAPI). For negative controls, primary antibodies were replaced with an appropriate isotype-matched negative IgG or IgM. Fluorescence staining was visualized with confocal microscopy (LSM 700, Carl Zeiss, Oberkochen, Germany). ImageJ software was used to count the stained PDL cells. Statistical analysis Data were compared using the Student’s t test or Fisher’s exact test, assuming double-sided independent variance and with p \ 0.05 considered to be significant.
Results Characterization of Pel and Pelt Pel and Pelt showed spindle-shaped morphology (data not shown), and more than 99 % were positive for CD44, 73, 90, and 105 with flow cytometry analysis (Fig. 1). In addition, 0.8 and 0.4 % of Pel and Pelt, respectively, were positive for SSEA-3 (SSEA-3?) (Fig. 1). Formation of mineralized nodules After 3 weeks of culture in mineralization induction medium containing b-glycerophosphate, dexamethasone, and L-ascorbic acid, Pel and Pelt were positive for alizarin red S staining and formed mineralized nodules (Fig. 2b, c). Conversely, Pel and Pelt cultured in normal growth medium were negative for alizarin red S staining (Fig. 2a, d). To evaluate depositions of calcium and phosphorus, both cell types with or without mineralized nodules were assayed with EPMA. Calcium and phosphorus were detected only in Pel and Pelt with nodules (Fig. 3).
Immunocytochemistry Gene expression related to cementogenesis For fluorescence microscopy analysis, trypsinized PDL cells were seeded on glass slide chambers (2.5 9 103 cells/cm2). After 2 weeks of differentiation induction in medium containing 2 % FBS and 100 ng/ml rhBMP-7, cells were fixed with 4 % PFA for 15 min, permeabilized with 0.1 % Triton X-100 for 5 min, blocked
The expressions of CAP were 1.55 and 2.0 times higher in Pel and Pelt stimulated with BMP-2, respectively, than in control cells (Fig. 4a, c). Pel and Pelt stimulated with BMP-7 showed 2.95- and 2.65-fold higher CAP expression, respectively, than control cells (Fig. 4a, c). Similarly, the
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Fig. 1 Flow cytometric analysis of Pel and Pelt revealed the expression of cell surface markers associated with mesenchymal stromal cells, CD73, CD90 and CD105. A small percentage of both cells were positive for stage-specific embryonic antigen (SSEA)-3
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Fig. 1 continued
Fig. 2 Mineralized nodule formation of Pel (a–c) and Pelt (d–f). Alizarin red staining shows the mineralization in both cells. Scale bars 100 lm
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Fig. 4 Cementogenesis-related gene expression in Pel and Pelt. Quantitative RT-PCR analysis presented the up-regulated expression of cementum attachment protein (CAP) (a, c) and cementum protein 1 (CEMP1) (b, d) in both Pel (a, b) and Pelt (c, d) cultured with rhBMP-2 or rhBMP-7. b-actin was used as an internal control. Statistical significance was determined using the Student’s t test (*p \ 0.05 vs. control)
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Pelt (SSEA-3+/CD44+/CD105+)
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Fig. 5 Expression of cementoblastic phenotypes in SSEA-3/CD44/ CD105 triple-positive cells sorted from Pel or Pelt. Both sorted cells were fibroblastic (a, b) and positive for cementum attachment protein
(CAP) and cementum protein 1 (CEMP1) when cultured with rhBMP7. Scale bars 100 lm
expressions of CEMP1 were 1.45 and 1.85 times higher in Pel and Pelt stimulated with BMP-2, respectively, than in control cells (Fig. 4b, d). Pel and Pelt stimulated with BMP-7 showed 1.65- and 2.85-fold higher CEMP1 expression, respectively, than control cells (Fig. 4b, d).
Cementogenic potential of SSEA-3-positive cells 90.9 % of the isolated SSEA-3? Pel and 99.6 % of the isolated SSEA-3? Pelt expressed CAP and CEMP1 when stimulated with rhBMP-7. Respectively, 4.9 and 1.4 % of
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Pelt (SSEA–3+/CD105+)
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those cultured without rhBMP-7 expressed CAP and CEMP1 (Fig. 5).
Discussion To characterize the cultured human PDL cells, we analyzed expression of cell surface antigens on Pel and Pelt cells. As shown using flow cytometry (Fig. 1), more than 99 % of the cells were positive for CD44, 73, 90, and 105. Although these cell surface markers have been reported to be mesenchymal stem cell markers in the dental follicle and the PDL [14, 15], we cannot assume that almost all Pel and Pelt cells possess stemness because they were heterogeneous cell populations derived from human PDL. In recent studies, a subset of adult stem cells whose properties are similar in some respects to those of induced pluripotent stem cells (iPS cells) exists among adult human fibroblasts [16] and contributes to the generation of iPS
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cells [17]. Human iPS cells can also be established from PDL fibroblasts with low reprogramming efficiency [18], and reports suggest that those PDL cells also contain a somatic stem cell population possessing multilineage differentiation potential. Another report shows that SSEA-4positive clonal cells derived from human PDL have adipogenic, osteogenic, and chondrogenic potential [19]. Hence, SSEAs may be specific markers for identification of stem cells in the PDL. In the present study, we found BMP-induced cementogenesis in SSEA-3/CD44/CD105 triple-positive cells isolated from normal and immortal human PDL cells (Fig. 5). As reported previously, CAP production was up-regulated in human periodontal fibroblasts by BMP-2 [7]. On the other hand, cementogenesis may also be regulated by BMP-7 expression [20, 21]. Furthermore, rhNoggin, a BMP antagonist, abolishes alkaline phosphatase activity, mineralization, and expression of both CAP and CP-23 in cultured human dental follicle cells. However, during
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stimulation with rhBMP-2, rhNoggin blocks Smad-1 phosphorylation, but not MAPK phosphorylation. Thus, cementogenesis may be affected by both exogenous and endogenous BMP pathways [22]. However, analysis of cementogenesis in SSEA-3/CD44/CD105 triple-positive cells derived from the human PDL has not been reported before. Therefore, this finding provides new insight into regenerative medicine using dental stem/progenitor cells. Our results demonstrate that normal and immortal human PDL cells have mineralization and cementogenic potential and contain a small cell population expressing the markers of multipotential mesenchymal stem cells. Both cell types may be useful for biological applications using PDL-derived mesenchymal stem cells. Acknowledgments This work was supported in part by a Grant-inAid for Young Scientists (No. 25862060 to D. T.) from the Japan Society for the Promotion of Science (JSPS) and a Research Grant (2012–2013 to K. K., T. T., D. T.) from the Nippon Dental University. Conflict of interest of interest.
The authors declare that they have no conflict
Ethical Standard This study was approved by the Committee of Ethics, the Nippon Dental University School of Life Dentistry at Tokyo.
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