Restricted accessResearch articleFirst published online 2014-06
Bone Morphogenetic Protein-9 Effectively Induces Osteo/Odontoblastic Differentiation of the Reversibly Immortalized Stem Cells of Dental Apical Papilla
Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPARγ2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration.
Get full access to this article
View all access options for this article.
References
1.
HuangGT, GronthosS and ShiS. (2009). Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res, 88:792–806.
2.
BluteauG, LuderHU, De BariC and MitsiadisTA. (2008). Stem cells for tooth engineering. Eur Cell Mater, 16:1–9.
3.
RaiS, KaurM and KaurS. (2013). Applications of stem cells in interdisciplinary dentistry and beyond: an overview. Ann Med Health Sci Res, 3:245–254.
4.
HuangGT. (2011). Dental pulp and dentin tissue engineering and regeneration: advancement and challenge. Front Biosci (Elite Ed), 3:788–800.
5.
GoldbergM. (2011). Pulp healing and regeneration: more questions than answers. Adv Dent Res, 23:270–274.
6.
SmithAJ, SmithJG, SheltonRM and CooperPR. (2012). Harnessing the natural regenerative potential of the dental pulp. Dent Clin North Am, 56:589–601.
7.
TziafasD and KodonasK. (2010). Differentiation potential of dental papilla, dental pulp, and apical papilla progenitor cells. J Endod, 36:781–789.
8.
ProckopDJ. (1997). Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 276:71–74.
9.
RastegarF, ShenaqD, HuangJ, ZhangW, ZhangBQ, HeBC, ChenL, ZuoGW, LuoQ, et al. (2010). Mesenchymal stem cells: molecular characteristics and clinical applications. World J Stem Cells, 2:67–80.
10.
ShenaqDS, RastegarF, PetkovicD, ZhangBQ, HeBC, ChenL, ZuoGW, LuoQ, ShiQ, et al. (2010). Mesenchymal progenitor cells and their orthopedic applications: forging a path towards clinical trials. Stem Cells Int, 2010:519028.
11.
SonoyamaW, LiuY, YamazaT, TuanRS, WangS, ShiS and HuangGT. (2008). Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod, 34:166–171.
12.
ChengH, JiangW, PhillipsFM, HaydonRC, PengY, ZhouL, LuuHH, AnN, BreyerB, et al. (2003). Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am, 85-A:1544–1552.
13.
KangQ, SunMH, ChengH, PengY, MontagAG, DeyrupAT, JiangW, LuuHH, LuoJ, et al. (2004). Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther, 11:1312–1320.
14.
LuuHH, SongWX, LuoX, ManningD, LuoJ, DengZL, SharffKA, MontagAG, HaydonRC and HeTC. (2007). Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res, 25:665–677.
15.
KangQ, SongWX, LuoQ, TangN, LuoJ, LuoX, ChenJ, BiY, HeBC, et al. (2009). A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. Stem Cells Dev, 18:545–559.
16.
LutherG, WagnerER, ZhuG, KangQ, LuoQ, LamplotJ, BiY, LuoX, LuoJ, et al. (2011). BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potentia. Curr Gene Ther, 11:229–240.
17.
WestermanKA and LeboulchP. (1996). Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A, 93:8971–8976.
18.
BiY, HuangJ, HeY, ZhuGH, SuY, HeBC, LuoJ, WangY, KangQ, et al. (2009). Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells. J Cell Biochem, 108:295–303.
19.
HuangJ, BiY, ZhuGH, HeY, SuY, HeBC, WangY, KangQ, ChenL, et al. (2009). Retinoic acid signalling induces the differentiation of mouse fetal liver-derived hepatic progenitor cells. Liver Int, 29:1569–1581.
YangK, ChenJ, JiangW, HuangE, CuiJ, KimSH, HuN, LiuH, ZhangW, et al. (2012). Conditional immortalization establishes a repertoire of mouse melanocyte progenitors with distinct melanogenic differentiation potential. J Invest Dermatol, 132:2479–2483.
22.
LiM, ChenY, BiY, JiangW, LuoQ, HeY, SuY, LiuX, CuiJ, et al. (2013). Establishment and characterization of the reversibly immortalized mouse fetal heart progenitors. Int J Med Sci, 10:1035–1046.
23.
WangX, CuiJ, ZhangBQ, ZhangH, BiY, KangQ, WangN, BieP, YangZ, et al. (2013). Decellularized liver scaffolds effectively support the proliferation and differentiation of mouse fetal hepatic progenitors. J Biomed Mater Res A [Epub ahead of print]; DOI: 10.1002/jbm.a.34764.
24.
LuoX, ChenJ, SongWX, TangN, LuoJ, DengZL, SharffKA, HeG, BiY, et al. (2008). Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects. Lab Invest, 88:1264–1277.
25.
HaydonRC, ZhouL, FengT, BreyerB, ChengH, JiangW, IshikawaA, PeabodyT, MontagA, SimonMA and HeTC. (2002). Nuclear receptor agonists as potential differentiation therapy agents for human osteosarcoma. Clin Cancer Res, 8:1288–1294.
LuoJ, DengZL, LuoX, TangN, SongWX, ChenJ, SharffKA, LuuHH, HaydonRC, et al. (2007). A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc, 2:1236–1247.
28.
LuoQ, KangQ, SiW, JiangW, ParkJK, PengY, LiX, LuuHH, LuoJ, et al. (2004). Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J Biol Chem, 279:55958–55968.
RastegarF, GaoJL, ShenaqD, LuoQ, ShiQ, KimSH, JiangW, WagnerER, HuangE, et al. (2010). Lysophosphatidic acid acyltransferase beta (LPAATbeta) promotes the tumor growth of human osteosarcoma. PLoS One, 5:e14182.
31.
SuY, WagnerER, LuoQ, HuangJ, ChenL, HeBC, ZuoGW, ShiQ, ZhangBQ, et al. (2011). Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma. Oncogene, 30:3907–3917.
32.
HuangE, ZhuG, JiangW, YangK, GaoY, LuoQ, GaoJL, KimSH, LiuX, et al. (2012). Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells. J Bone Miner Res, 27:1566–1575.
33.
HeBC, ChenL, ZuoGW, ZhangW, BiY, HuangJ, WangY, JiangW, LuoQ, et al. (2010). Synergistic antitumor effect of the activated PPARgamma and retinoid receptors on human osteosarcoma. Clin Cancer Res, 16:2235–2245.
34.
HeBC, GaoJL, ZhangBQ, LuoQ, ShiQ, KimSH, HuangE, GaoY, YangK, et al. (2011). Tetrandrine inhibits Wnt/beta-catenin signaling and suppresses tumor growth of human colorectal cancer. Mol Pharmacol, 79:211–219.
35.
HuN, JiangD, HuangE, LiuX, LiR, LiangX, KimSH, ChenX, GaoJL, et al. (2013). BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells. J Cell Sci, 126:532–541.
36.
LuuHH, KangQ, ParkJK, SiW, LuoQ, JiangW, YinH, MontagAG, SimonMA, et al. (2005). An orthotopic model of human osteosarcoma growth and spontaneous pulmonary metastasis. Clin Exp Metastasis, 22:319–329.
37.
SuY, LuoX, HeBC, WangY, ChenL, ZuoGW, LiuB, BiY, HuangJ, et al. (2009). Establishment and characterization of a new highly metastatic human osteosarcoma cell line. Clin Exp Metastasis, 26:599–610.
38.
ZhuGH, HuangJ, BiY, SuY, TangY, HeBC, HeY, LuoJ, WangY, et al. (2009). Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells. Differentiation 78. 195–204.
39.
SharffKA, SongWX, LuoX, TangN, LuoJ, ChenJ, BiY, HeBC, HuangJ, et al. (2009). Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. J Biol Chem, 284:649–659.
40.
LuoJ, TangM, HuangJ, HeBC, GaoJL, ChenL, ZuoGW, ZhangW, LuoQ, et al. (2010). TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells. J Biol Chem, 285:29588–29598.
41.
ChenL, JiangW, HuangJ, HeBC, ZuoGW, ZhangW, LuoQ, ShiQ, ZhangBQ, et al. (2010). Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation. J Bone Miner Res, 25:2447–2459.
42.
TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK and YamanakaS. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.
43.
DominiciM, Le BlancK, MuellerI, Slaper-CortenbachI, MariniF, KrauseD, DeansR, KeatingA, ProckopD and HorwitzE. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8:315–317.
44.
BakopoulouA, LeyhausenG, VolkJ, TsiftsoglouA, GarefisP, KoidisP and GeurtsenW. (2011). Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP). Arch Oral Biol, 56:709–721.
45.
SaitoM, HandaK, KiyonoT, HattoriS, YokoiT, TsubakimotoT, HaradaH, NoguchiT, ToyodaM, SatoS and TeranakaT. (2005). Immortalization of cementoblast progenitor cells with Bmi-1 and TERT. J Bone Miner Res, 20:50–57.
46.
YokoiT, SaitoM, KiyonoT, IsekiS, KosakaK, NishidaE, TsubakimotoT, HaradaH, EtoK, NoguchiT and TeranakaT. (2007). Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo. Cell Tissue Res, 327:301–311.
47.
vom BrockeJ, SchmeiserHH, ReinboldM and HollsteinM. (2006). MEF immortalization to investigate the ins and outs of mutagenesis. Carcinogenesis, 27:2141–2147.
48.
GeserickC, TejeraA, Gonzalez-SuarezE, KlattP and BlascoMA. (2006). Expression of mTert in primary murine cells links the growth-promoting effects of telomerase to transforming growth factor-beta signaling. Oncogene, 25:4310–4319.
49.
PrivesC. (1990). The replication functions of SV40 T antigen are regulated by phosphorylation. Cell, 61:735–738.
50.
GeeCJ and HarrisH. (1979). Tumorigenicity of cells transformed by Simian virus 40 and of hybrids between such cells and normal diploid cells. J Cell Sci, 36:223–240.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
