Effects of lysophosphatidic acid on human periodontal ligament stem cells from teeth extracted from dental patients
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摘要
Despite their potential applications in future regenerative medicine, periodontal ligament stem cells(PDLSCs) are difficult to obtain in large amounts from patients. Therefore, maintaining sternness while expanding the cell numbers for medical use is the key to transitioning PDLSCs from the bench to the clinic. Lysophosphatidic acid(LPA), which is present in the human body and saliva, is a signaling molecule derived from phospholipids. In this study, we examined the effects of LPA on sternness maintenance in human PDLSCs. Several spindle-shaped and fibroblast-like periodontal ligament stem-like cell lines were established from PDLSC isolation. Among these cell lines, the most morphologically appropriate cell line was characterized. The expression levels of OCT4, NANOG(a stem cell marker), and CD90(a mesenchymal stem cell marker) were high. However, CD73(a negative marker of mesenchymal stem cells) expression was not observed. Notably, immunofluorescence analysis identified the expression of STRO-1, CD146(a mesenchymal stem cell marker), and sex determining region Y-box 2 at the protein level. In addition, lipid droplets were stained by Oil red O after the induction of adipogenesis for 21 days, and mineralized nodules were stained by Alizarin Red S after the induction of osteogenesis for 14 days. Alkaline phosphate staining also demonstrated the occurrence of osteogenesis. In summary, we established a human PDLSC line, which could be applied as a cell source for tissue regeneration in dental patients. However, further studies are needed to determine the detailed effects of LPA on PDLSCs.
Despite their potential applications in future regenerative medicine, periodontal ligament stem cells(PDLSCs) are difficult to obtain in large amounts from patients. Therefore, maintaining sternness while expanding the cell numbers for medical use is the key to transitioning PDLSCs from the bench to the clinic. Lysophosphatidic acid(LPA), which is present in the human body and saliva, is a signaling molecule derived from phospholipids. In this study, we examined the effects of LPA on sternness maintenance in human PDLSCs. Several spindle-shaped and fibroblast-like periodontal ligament stem-like cell lines were established from PDLSC isolation. Among these cell lines, the most morphologically appropriate cell line was characterized. The expression levels of OCT4, NANOG(a stem cell marker), and CD90(a mesenchymal stem cell marker) were high. However, CD73(a negative marker of mesenchymal stem cells) expression was not observed. Notably, immunofluorescence analysis identified the expression of STRO-1, CD146(a mesenchymal stem cell marker), and sex determining region Y-box 2 at the protein level. In addition, lipid droplets were stained by Oil red O after the induction of adipogenesis for 21 days, and mineralized nodules were stained by Alizarin Red S after the induction of osteogenesis for 14 days. Alkaline phosphate staining also demonstrated the occurrence of osteogenesis. In summary, we established a human PDLSC line, which could be applied as a cell source for tissue regeneration in dental patients. However, further studies are needed to determine the detailed effects of LPA on PDLSCs.
Despite their potential applications in future regenerative medicine, periodontal ligament stem cells(PDLSCs) are difficult to obtain in large amounts from patients. Therefore, maintaining sternness while expanding the cell numbers for medical use is the key to transitioning PDLSCs from the bench to the clinic. Lysophosphatidic acid(LPA), which is present in the human body and saliva, is a signaling molecule derived from phospholipids. In this study, we examined the effects of LPA on sternness maintenance in human PDLSCs. Several spindle-shaped and fibroblast-like periodontal ligament stem-like cell lines were established from PDLSC isolation. Among these cell lines, the most morphologically appropriate cell line was characterized. The expression levels of OCT4, NANOG(a stem cell marker), and CD90(a mesenchymal stem cell marker) were high. However, CD73(a negative marker of mesenchymal stem cells) expression was not observed. Notably, immunofluorescence analysis identified the expression of STRO-1, CD146(a mesenchymal stem cell marker), and sex determining region Y-box 2 at the protein level. In addition, lipid droplets were stained by Oil red O after the induction of adipogenesis for 21 days, and mineralized nodules were stained by Alizarin Red S after the induction of osteogenesis for 14 days. Alkaline phosphate staining also demonstrated the occurrence of osteogenesis. In summary, we established a human PDLSC line, which could be applied as a cell source for tissue regeneration in dental patients. However, further studies are needed to determine the detailed effects of LPA on PDLSCs.
引文
[1] Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet, 2004, 364(9429):149-155.
[2] Shi S, Bartold PM, Miura M, et al. The efficacy of mesenchymal stem cells to regenerate and repair dental structures[J]. Orthod Craniofac Res, 2005, 8(3):191-199.
[3] Gay IC, Chen S, MacDougall M. Isolation and characterization of multipotent human periodontal ligament stem cells[J].Orthod Craniofac Res, 2007, 10(3):149-160.
[4] Huang GT,Gronthos S,Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources:their biology and role in regenerative medicine[J]. J Dent Res, 2009, 88(9):792-806.
[5] Thesleff I, Sharpe P. Signalling networks regulating dental development[J]. Mech Dev, 1997, 67(2):111-123.
[6] Huang CY, Pelaez D, Dominguez-Bendala J, et al. Plasticity of stem cells derived from adult periodontal ligament[J]. Regen Med, 2009, 4(6):809-821.
[7] Liu Y, Zheng Y, Ding G, et al. Periodontal ligament stem cellmediated treatment for periodontitis in miniature swine[J]. Stem Cells, 2008, 26(4):1065-1073.
[8] Park JY, Jeon SH, Choung PH. Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis[J]. Cell Transplant, 2011, 20(2):271-285.
[9] Iwata T, Yamato M, Zhang Z, et al. Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use[J]. J Clin Periodontol, 2010, 37(12):1088-1099.
[10] Feng F, Akiyama K, Liu Y, et al. Utility of PDL progenitors for in vivo tissue regeneration:a report of 3 cases[J]. Oral Dis,2010, 16(1):20-28.
[11] Zhu W, Liang M. Periodontal ligament stem cells:current status, concerns, and future prospects[J]. Stem Cells Int, 2015,2015:972313.
[12] Aikawa S, Hashimoto T, Kano K, et al. Lysophosphatidic acid as a lipid mediator with multiple biological actions[J]. J Biochem, 2015, 157(2):81-89.
[13] van Leeuwen FN, Giepmans BN, van Meeteren LA, et al.Lysophosphatidic acid:mitogen and motility factor[J].Biochem Soc Trans, 2003, 31(Pt 6):1209-1212.
[14] Ishii I, Fukushima N, Ye X, et al. Lysophospholipid receptors:signaling and biology[J]. Annu Rev Biochem, 2004, 73:321-354.
[15] Sheng X, Yung YC, Chen A, et al. Lysophosphatidic acid signalling in development[J]. Development, 2015, 142(8):1390-1395.
[16] Zhao Y, Natarajan V. Lysophosphatidic acid(LPA)and its receptors:role in airway inflammation and remodeling[J].Biochim Biophys Acta, 2013, 1831(1):86-92.
[17] Kostic I, Fidalgo-Carvalho I, Aday S, et al. Lysophosphatidic acid enhances survival of human CD34(+)cells in ischemic conditions[J]. Sci Rep, 2015, 5:16406.
[18] Sugiura T, Nakane S, Kishimoto S, et al. Lysophosphatidic acid, a growth factor-like lipid, in the saliva[J]. J Lipid Res,2002, 43(12):2049-2055.
[19] Bathena SP, Huang J, Nunn ME, et al. Quantitative determination of lysophosphatidic acids(LPAs)in human saliva and gingival crevicular fluid(GCF)by LC-MS/MS[J]. J Pharm Biomed Anal,2011, 56(2):402-407.
[20] Cheng R, Shao MY, Yang H, et al. The effect of lysophosphatidic acid and Rho-associated kinase patterning on adhesion of dental pulp cells[J]. Int Endod J, 2011, 44(1):2-8.
[21] Cheng R, Cheng L, Shao MY, et al. Roles of lysophosphatidic acid and the Rho-associated kinase pathway in the migration of dental pulp cells[J]. Exp Cell Res, 2010, 316(6):1019-1027.
[22] Pan H, Cheng L, Yang H, et al. Lysophosphatidic acid rescueshuman dental pulp cells from ischemia-induced apoptosis[J], J Endod, 2014, 40(2):217-222.
[23] George J, Headen KV, Ogunleye AO, et al. Lysophosphatidic Acid signals through specific lysophosphatidic Acid receptor subtypes to control key regenerative responses of human gingival and periodontal ligament fibroblasts[J]. JPeriodontol,2009, 80(8):1338-1347.
[24] Mo JS, Park HW, Guan KL. The Hippo signaling pathway in stem cell biology and cancer[J]. EMBO Rep, 2014,15(6):642-656.
[25] Ramalho-Santos M,Yoon S,Matsuzaki Y,et al.“Sternness”:transcriptional profiling of embryonic and adult stem cells[J].Science, 2002, 298(5593):597-600.
[26] Qin H, Blaschke K, Wei G, et al. Transcriptional analysis of pluripotency reveals the Hippo pathway as a barrier to reprogramming[J]. Hum Mol Genet, 2012, 21(9):2054-2067.
[27] Qin H, Diaz A, Blouin L, et al. Systematic identification of barriers to human iPSC generation[J]. Cell, 2014, 158(2):449-461.
[28] Qin H, Hejna M, Liu Y, et al. YAP induces human naive pluripotency[J]. Cell Rep, 2016, 14(10):2301-2312.
[29] Lindholm PF, Hwang YS. LPA increases tumor growth and bone destruction through enhancement of osteoclastogenic cytokines[J]. Anticancer Res, 2016, 36(1):61-70.
[30] Yi KJ, So KH, Hata Y, et al. The regulation of oxytocin receptor gene expression during adipogenesis[J]. J Neuroendocrinol,2015, 27(5):335-342.
[31]Trubiani O, Piattelli A, Gatta V, et al. Assessment of an efficient xeno-free culture system of human periodontal ligament stem cells[J]. Tissue Eng Part C Methods, 2015, 21(1):52-64.
[32] Pitson SM, Pebay A. Regulation of stem cell pluripotency and neural differentiation by lysophospholipids[J]. Neurosignals,2009, 17(4):242-254.
[33] Saunders JA, Rogers LC, Klomsiri C, et al. Reactive oxygen species mediate lysophosphatidic acid induced signaling in ovarian cancer cells[J]. Free Radic Biol Med, 2010, 49(12):2058-2067.
[34] Chen Q, Olashaw N, Wu J. Participation of reactive oxygen species in the lysophosphatidic acid-stimulated mitogenactivated protein kinase kinase activation pathway[J]. J Biol Chem, 1995, 270(48):28499-28502.
[35] Idzko M, Laut M, Panther E, et al. Lysophosphatidic acid induces chemotaxis, oxygen radical production, CDllb upregulation, Ca~(2+)mobilization, and actin reorganization in human eosinophils via pertussis toxin-sensitive G proteins[J]. J Immunol, 2004, 172(7):4480-4485.
[36] Lin CC, Lin CE, Lin YC, et al. Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells[J]. Biochem Biophys Res Commun, 2013, 440(4):564-569.
[37] Kang S, Han J, Song SY, et al. Lysophosphatidic acid increases the proliferation and migration of adiposederived stem cells via the generation of reactive oxygen species[J]. Mol Med Rep,2015, 12(4):5203-5210.
[38] Cerutis DR, Weston MD, Alnouti Y, et al. A major human oral lysophosphatidic acid species, LPA 18:1, regulates novel genes in human gingival fibroblasts[J]. J Periodontol, 2015, 86(5):713-725.
[39] Hsueh YJ, Chen HC, Wu SE, et al. Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways[J]. Mol Ther Methods Clin Dev, 2015, 2:15014.
[40] Cuizhu T, Yong W, Weiting G, et al. Effects of YAP-small interfering RNA on the proliferation and apoptosis of human periodontal ligament stem cells[J]. Hua Xi Kou Qiang Yi Xue Za Zhi(in Chinese), 2015, 33(6):622-626.
[2] Shi S, Bartold PM, Miura M, et al. The efficacy of mesenchymal stem cells to regenerate and repair dental structures[J]. Orthod Craniofac Res, 2005, 8(3):191-199.
[3] Gay IC, Chen S, MacDougall M. Isolation and characterization of multipotent human periodontal ligament stem cells[J].Orthod Craniofac Res, 2007, 10(3):149-160.
[4] Huang GT,Gronthos S,Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources:their biology and role in regenerative medicine[J]. J Dent Res, 2009, 88(9):792-806.
[5] Thesleff I, Sharpe P. Signalling networks regulating dental development[J]. Mech Dev, 1997, 67(2):111-123.
[6] Huang CY, Pelaez D, Dominguez-Bendala J, et al. Plasticity of stem cells derived from adult periodontal ligament[J]. Regen Med, 2009, 4(6):809-821.
[7] Liu Y, Zheng Y, Ding G, et al. Periodontal ligament stem cellmediated treatment for periodontitis in miniature swine[J]. Stem Cells, 2008, 26(4):1065-1073.
[8] Park JY, Jeon SH, Choung PH. Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis[J]. Cell Transplant, 2011, 20(2):271-285.
[9] Iwata T, Yamato M, Zhang Z, et al. Validation of human periodontal ligament-derived cells as a reliable source for cytotherapeutic use[J]. J Clin Periodontol, 2010, 37(12):1088-1099.
[10] Feng F, Akiyama K, Liu Y, et al. Utility of PDL progenitors for in vivo tissue regeneration:a report of 3 cases[J]. Oral Dis,2010, 16(1):20-28.
[11] Zhu W, Liang M. Periodontal ligament stem cells:current status, concerns, and future prospects[J]. Stem Cells Int, 2015,2015:972313.
[12] Aikawa S, Hashimoto T, Kano K, et al. Lysophosphatidic acid as a lipid mediator with multiple biological actions[J]. J Biochem, 2015, 157(2):81-89.
[13] van Leeuwen FN, Giepmans BN, van Meeteren LA, et al.Lysophosphatidic acid:mitogen and motility factor[J].Biochem Soc Trans, 2003, 31(Pt 6):1209-1212.
[14] Ishii I, Fukushima N, Ye X, et al. Lysophospholipid receptors:signaling and biology[J]. Annu Rev Biochem, 2004, 73:321-354.
[15] Sheng X, Yung YC, Chen A, et al. Lysophosphatidic acid signalling in development[J]. Development, 2015, 142(8):1390-1395.
[16] Zhao Y, Natarajan V. Lysophosphatidic acid(LPA)and its receptors:role in airway inflammation and remodeling[J].Biochim Biophys Acta, 2013, 1831(1):86-92.
[17] Kostic I, Fidalgo-Carvalho I, Aday S, et al. Lysophosphatidic acid enhances survival of human CD34(+)cells in ischemic conditions[J]. Sci Rep, 2015, 5:16406.
[18] Sugiura T, Nakane S, Kishimoto S, et al. Lysophosphatidic acid, a growth factor-like lipid, in the saliva[J]. J Lipid Res,2002, 43(12):2049-2055.
[19] Bathena SP, Huang J, Nunn ME, et al. Quantitative determination of lysophosphatidic acids(LPAs)in human saliva and gingival crevicular fluid(GCF)by LC-MS/MS[J]. J Pharm Biomed Anal,2011, 56(2):402-407.
[20] Cheng R, Shao MY, Yang H, et al. The effect of lysophosphatidic acid and Rho-associated kinase patterning on adhesion of dental pulp cells[J]. Int Endod J, 2011, 44(1):2-8.
[21] Cheng R, Cheng L, Shao MY, et al. Roles of lysophosphatidic acid and the Rho-associated kinase pathway in the migration of dental pulp cells[J]. Exp Cell Res, 2010, 316(6):1019-1027.
[22] Pan H, Cheng L, Yang H, et al. Lysophosphatidic acid rescueshuman dental pulp cells from ischemia-induced apoptosis[J], J Endod, 2014, 40(2):217-222.
[23] George J, Headen KV, Ogunleye AO, et al. Lysophosphatidic Acid signals through specific lysophosphatidic Acid receptor subtypes to control key regenerative responses of human gingival and periodontal ligament fibroblasts[J]. JPeriodontol,2009, 80(8):1338-1347.
[24] Mo JS, Park HW, Guan KL. The Hippo signaling pathway in stem cell biology and cancer[J]. EMBO Rep, 2014,15(6):642-656.
[25] Ramalho-Santos M,Yoon S,Matsuzaki Y,et al.“Sternness”:transcriptional profiling of embryonic and adult stem cells[J].Science, 2002, 298(5593):597-600.
[26] Qin H, Blaschke K, Wei G, et al. Transcriptional analysis of pluripotency reveals the Hippo pathway as a barrier to reprogramming[J]. Hum Mol Genet, 2012, 21(9):2054-2067.
[27] Qin H, Diaz A, Blouin L, et al. Systematic identification of barriers to human iPSC generation[J]. Cell, 2014, 158(2):449-461.
[28] Qin H, Hejna M, Liu Y, et al. YAP induces human naive pluripotency[J]. Cell Rep, 2016, 14(10):2301-2312.
[29] Lindholm PF, Hwang YS. LPA increases tumor growth and bone destruction through enhancement of osteoclastogenic cytokines[J]. Anticancer Res, 2016, 36(1):61-70.
[30] Yi KJ, So KH, Hata Y, et al. The regulation of oxytocin receptor gene expression during adipogenesis[J]. J Neuroendocrinol,2015, 27(5):335-342.
[31]Trubiani O, Piattelli A, Gatta V, et al. Assessment of an efficient xeno-free culture system of human periodontal ligament stem cells[J]. Tissue Eng Part C Methods, 2015, 21(1):52-64.
[32] Pitson SM, Pebay A. Regulation of stem cell pluripotency and neural differentiation by lysophospholipids[J]. Neurosignals,2009, 17(4):242-254.
[33] Saunders JA, Rogers LC, Klomsiri C, et al. Reactive oxygen species mediate lysophosphatidic acid induced signaling in ovarian cancer cells[J]. Free Radic Biol Med, 2010, 49(12):2058-2067.
[34] Chen Q, Olashaw N, Wu J. Participation of reactive oxygen species in the lysophosphatidic acid-stimulated mitogenactivated protein kinase kinase activation pathway[J]. J Biol Chem, 1995, 270(48):28499-28502.
[35] Idzko M, Laut M, Panther E, et al. Lysophosphatidic acid induces chemotaxis, oxygen radical production, CDllb upregulation, Ca~(2+)mobilization, and actin reorganization in human eosinophils via pertussis toxin-sensitive G proteins[J]. J Immunol, 2004, 172(7):4480-4485.
[36] Lin CC, Lin CE, Lin YC, et al. Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells[J]. Biochem Biophys Res Commun, 2013, 440(4):564-569.
[37] Kang S, Han J, Song SY, et al. Lysophosphatidic acid increases the proliferation and migration of adiposederived stem cells via the generation of reactive oxygen species[J]. Mol Med Rep,2015, 12(4):5203-5210.
[38] Cerutis DR, Weston MD, Alnouti Y, et al. A major human oral lysophosphatidic acid species, LPA 18:1, regulates novel genes in human gingival fibroblasts[J]. J Periodontol, 2015, 86(5):713-725.
[39] Hsueh YJ, Chen HC, Wu SE, et al. Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways[J]. Mol Ther Methods Clin Dev, 2015, 2:15014.
[40] Cuizhu T, Yong W, Weiting G, et al. Effects of YAP-small interfering RNA on the proliferation and apoptosis of human periodontal ligament stem cells[J]. Hua Xi Kou Qiang Yi Xue Za Zhi(in Chinese), 2015, 33(6):622-626.