Palabras clave
Células Madre Adultas
Pulpa Dental
Separación Celular
Citometría de Flujo
Células Madre Mesenquimatosas
Pulpa Dental
Separación Celular
Citometría de Flujo
Células Madre Mesenquimatosas
Cómo citar
Establecimiento e implementación de un protocolo simplificado de expansión y cultivo de Células Madre de Pulpa Dental Humana (DPSCh). (2021). Odontoestomatología, 23(38). https://doi.org/10.22592/ode2021n37e207
Resumen
Objetivos: Establecer e implementar un protocolo simplificado de extracción, aislamiento primario y cultivo de células madre derivadas de la pulpa dental humana (DPSCh). Analizar cuantitativamente y cualitativamente las células aisladas.
Metodología: 10 terceros molares sanos donados por pacientes que concurrieron a la Facultad de Odontología, UdelaR y otorgaron su consentimiento escrito fueron procesados antes de las 48 hs. Se realizó la fractura de la pieza para la obtención del tejido pulpar y se procesó por el método explante. Se analizó viabilidad celular y expresión de marcadores por citometría de flujo en pasajes 4 y 12 y se corroboró mediante inmunocitoquímica.
Resultados: Las células obtenidas presentaron una vitalidad mayor al 90% en todos los pasajes, observándose una morfología característica y expresión de marcadores de células madre mesenquimales CD90, C105, CD73, CD29 y 166 mediante citometría de flujo en ambos pasajes.
Conclusiones: Se logró establecer un protocolo de aislamiento y expansión celular, con alta tasa de éxito de una población de DPSCh.
Referencias
1. Kim T-W, Che J-H, Yun J-W. Use of stem cells as alternative methods to animal experimentation in predictive toxicology. Regul Toxicol Pharmacol. 2019 Jul;105(January):15–29.
2.Daley GQ. Stem cells and the evolving notion of cellular identity. Philos Trans B. 2015 Oct 19;370(1680):20140376.
3.Shyh-Chang N, Ng H-H. The metabolic programming of stem cells. Genes Dev. 2017 Feb 15;31(4):336–46.
4.Kolios G, Moodley Y. Introduction to Stem Cells and Regenerative Medicine. Respiration. 2013;85(1):3–10.
5.Li N, Hua J. Interactions between mesenchymal stem cells and the immune system. Cell Mol Life Sci. 2017 Jul 18;74(13):2345–60.
6.Mortada I, Mortada R, Al Bazzal M. Dental Pulp Stem Cells and Neurogenesis. In: Advs Exp Medicine, Biology-Neuroscience and respiration. 2017. p. 63–75.
7.Keating A. Mesenchymal Stromal Cells: New Directions. Cell Stem Cell. 2012 Jun;10(6):709–16.
8.Caplan AI. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017 Jun;6(6):1445–51.
9.Peng L, Ye L, Zhou X. Mesenchymal Stem Cells and Tooth Engineering. Int J Oral Sci. 2009 Mar;1(1):6–12.
10.Ji ST, Kim H, Yun J, Chung JS, Kwon S-M. Promising Therapeutic Strategies for Mesenchymal Stem Cell-Based Cardiovascular Regeneration: From Cell Priming to Tissue Engineering. Stem Cells Int. 2017;2017:1–13.
11.Honda MJ, Watanabe E, Mikami Y, Saito Y, Toriumi T, Shirakawa T, et al. Mesenchymal Dental Stem Cells for Tissue Regeneration. Int J Oral Maxillofac Implants. 2013 Jan;28(6):e451–60.
12.Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci. 2000 Dec 5;97(25):13625–30.
13.Ulmer FL, Winkel A, Kohorst P, Stiesch M. Stem cells--prospects in dentistry. Rev Mens suisse d’odonto-stomatologie 2010;120(10):860–83.
14.Sloan AJ, Waddington RJ. Dental pulp stem cells: what, where, how? Int J Paediatr Dent. 2009 Jan;19(1):61–70.
15.Honda MJ, Imaizumi M, Tsuchiya S, Morsczeck C. Dental follicle stem cells and tissue engineering. J Oral Sci. 2010 Dec;52(4):541–52.
16.Orimoto A, Kyakumoto S, Eitsuka T, Nakagawa K, Kiyono T, Fukuda T. Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition. Papaccio G, editor. PLoS One. 2020 Mar 2;15(3):e0229996.
17.Luzuriaga J, Pastor-Alonso O, Encinas JM, Unda F, Ibarretxe G, Pineda JR. Human Dental Pulp Stem Cells Grown in Neurogenic Media Differentiate Into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain. Front Physiol. 2019 Mar 28;10(March):1–18.
18.Pai V, Vishwanath V, Prasanna J, Nadig R, Nadig R, Karthik J. Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: An in vitro study. J Conserv Dent. 2013;16(5):423.
19.Lan X, Sun Z, Chu C, Boltze J, Li S. Dental Pulp Stem Cells: An Attractive Alternative for Cell Therapy in Ischemic Stroke. Front Neurol. 2019 Aug 2;10(JUL):1–10.
20.Mead B, Logan A, Berry M, Leadbeater W, Scheven BA. Concise Review: Dental Pulp Stem Cells: A Novel Cell Therapy for Retinal and Central Nervous System Repair. Stem Cells. 2017 Jan;35(1):61–7.
21.Huang GT-J, Gronthos S, Shi S. Mesenchymal Stem Cells Derived from Dental Tissues vs . Those from Other Sources: Their Biology and Role in Regenerative Medicine. J Dent Res. 2009 Sep 18;88(9):792–806.
22.Longoni A, Utomo L, van Hooijdonk I, Bittermann G, Vetter V, Kruijt Spanjer E, et al. The chondrogenic differentiation potential of dental pulp stem cells. Eur Cells Mater. 2020 Feb 21;39:121–35.
23.Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem Cell Properties of Human Dental Pulp Stem Cells. J Dent Res. 2002 Aug 13;81(8):531–5.
24.La Noce M, Paino F, Spina A, Naddeo P, Montella R, Desiderio V, et al. Dental pulp stem cells: State of the art and suggestions for a true translation of research into therapy. J Dent. 2014 Jul;42(7):761–8.
25.Inostroza C, Vega-Letter AM, Brizuela C, Castrillón L, Saint Jean N, Duran CM, et al. Mesenchymal Stem Cells Derived from Human Inflamed Dental Pulp Exhibit Impaired Immunomodulatory Capacity In Vitro. J Endod. 2020 Aug 1;46(8):1091-1098.e2.
26.Chen Y, Li X, Wu J, Lu W, Xu W, Wu B. Dental pulp stem cells from human teeth with deep caries displayed an enhanced angiogenesis potential in vitro. J Dent Sci. 2021 Jan 1;16(1):318–26.
27.Ferrúa CP, Centeno EGZ, Rosa LC da, Amaral CC do, Severo RF, Sarkis-Onofre R, et al. How has dental pulp stem cells isolation been conducted? A scoping review. Braz Oral Res. 2017 Dec 18;31:1–9.
28.Glickman GN. AAE Consensus Conference on Diagnostic Terminology: Background and Perspectives. J Endod. 2009 Dec;35(12):1619–20.
29.Yang X, Li L, Xiao L, Zhang D. Recycle the dental fairy’s package: overview of dental pulp stem cells. Stem Cell Res Ther. 2018 Dec 13;9(1):347.
30.Scheller EL, Krebsbach PH, Konh DH. Tissue engineering: state of the art in oral rehabilitation. J Oral Rehabil. 2009 May;36(5):368–89.
31.Yalvac ME, Ramazanoglu M, Rizvanov AA, Sahin F, Bayrak OF, Salli U, et al. Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis. Pharmacogenomics J. 2010 Apr;10(2):105–13.
32.Atari M, Barajas M, Hernández-Alfaro F, Gil C, Fabregat M, Ferrés Padró E, et al. Isolation of pluripotent stem cells from human third molar dental pulp. Histol Histopathol. 2011;26(8):1057–70.
33.Pereira LO, Rubini MR, Silva JR, Oliveira DM, Silva ICR, Poças-Fonseca MJ, et al. Comparison of stem cell properties of cells isolated from normal and inflamed dental pulps. Int Endod J. 2012 Dec;45(12):1080–90.
34.Alongi DJ, Yamaza T, Song Y, Fouad AF, Romberg EE, Shi S, et al. Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential. Regen Med. 2010 Jul;5(4):617–31.
35.Karamzadeh R, Eslaminejad MB, Aflatoonian R. Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods. J Vis Exp. 2012 Nov 24;69(69).
36.Catón J, Bostanci N, Remboutsika E, De Bari C, Mitsiadis TA. Future dentistry: cell therapy meets tooth and periodontal repair and regeneration. J Cell Mol Med. 2011 May;15(5):1054–65.
37.Hilkens P, Gervois P, Fanton Y, Vanormelingen J, Martens W, Struys T, et al. Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells. Cell Tissue Res. 2013 Jul 29;353(1):65–78.
38.Raoof M, Yaghoobi MM, Derakhshani A, Kamal-Abadi AM, Ebrahimi B, Abbasnejad M, et al. A modified efficient method for dental pulp stem cell isolation. Dent Res J (Isfahan). 2014 Mar;11(2):244–50.
39.Suchanek J, Soukup T, Visek B, Ivancakova R, Kucerova L, Mokry J. Dental pulp stem cells and their characterization. Biomed Pap. 2009 Mar 1;153(1):31–5.
40.Perry BC, Zhou D, Wu X, Yang F-C, Byers MA, Chu T-MG, et al. Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use. Tissue Eng Part C Methods. 2008 Jun;14(2):149–56.
41.Martin-Piedra MA, Garzon I, Oliveira AC, Alfonso-Rodriguez CA, Carriel V, Scionti G, et al. Cell viability and proliferation capability of long-term human dental pulp stem cell cultures. Cytotherapy. 2014 Feb;16(2):266–77.
42.Conde MCM, Chisini LA, Grazioli G, Francia A, Carvalho RV de, Alcázar JCB, et al. Does Cryopreservation Affect the Biological Properties of Stem Cells from Dental Tissues? A Systematic Review. Braz Dent J. 2016 Dec;27(6):633–40.
43.Wall ME, Bernacki SH, Loboa EG. Effects of Serial Passaging on the Adipogenic and Osteogenic Differentiation Potential of Adipose-Derived Human Mesenchymal Stem Cells. Tissue Eng. 2007 Jun;13(6):1291–8.
44.Ruparel NB, de Almeida JFA, Henry MA, Diogenes A. Characterization of a Stem Cell of Apical Papilla Cell Line: Effect of Passage on Cellular Phenotype. J Endod. 2013 Mar;39(3):357–63.
45.Vemuri MC, Chase LG, Rao MS. Mesenchymal Stem Cell Assays and Applications. In: Methods. Totowa, NJ: Humana Press; 2011. p. 3–8. (Methods in Molecular Biology; vol. 698).
46.Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F., Krause DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006 Aug;8(4):315–7.
2.Daley GQ. Stem cells and the evolving notion of cellular identity. Philos Trans B. 2015 Oct 19;370(1680):20140376.
3.Shyh-Chang N, Ng H-H. The metabolic programming of stem cells. Genes Dev. 2017 Feb 15;31(4):336–46.
4.Kolios G, Moodley Y. Introduction to Stem Cells and Regenerative Medicine. Respiration. 2013;85(1):3–10.
5.Li N, Hua J. Interactions between mesenchymal stem cells and the immune system. Cell Mol Life Sci. 2017 Jul 18;74(13):2345–60.
6.Mortada I, Mortada R, Al Bazzal M. Dental Pulp Stem Cells and Neurogenesis. In: Advs Exp Medicine, Biology-Neuroscience and respiration. 2017. p. 63–75.
7.Keating A. Mesenchymal Stromal Cells: New Directions. Cell Stem Cell. 2012 Jun;10(6):709–16.
8.Caplan AI. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017 Jun;6(6):1445–51.
9.Peng L, Ye L, Zhou X. Mesenchymal Stem Cells and Tooth Engineering. Int J Oral Sci. 2009 Mar;1(1):6–12.
10.Ji ST, Kim H, Yun J, Chung JS, Kwon S-M. Promising Therapeutic Strategies for Mesenchymal Stem Cell-Based Cardiovascular Regeneration: From Cell Priming to Tissue Engineering. Stem Cells Int. 2017;2017:1–13.
11.Honda MJ, Watanabe E, Mikami Y, Saito Y, Toriumi T, Shirakawa T, et al. Mesenchymal Dental Stem Cells for Tissue Regeneration. Int J Oral Maxillofac Implants. 2013 Jan;28(6):e451–60.
12.Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci. 2000 Dec 5;97(25):13625–30.
13.Ulmer FL, Winkel A, Kohorst P, Stiesch M. Stem cells--prospects in dentistry. Rev Mens suisse d’odonto-stomatologie 2010;120(10):860–83.
14.Sloan AJ, Waddington RJ. Dental pulp stem cells: what, where, how? Int J Paediatr Dent. 2009 Jan;19(1):61–70.
15.Honda MJ, Imaizumi M, Tsuchiya S, Morsczeck C. Dental follicle stem cells and tissue engineering. J Oral Sci. 2010 Dec;52(4):541–52.
16.Orimoto A, Kyakumoto S, Eitsuka T, Nakagawa K, Kiyono T, Fukuda T. Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition. Papaccio G, editor. PLoS One. 2020 Mar 2;15(3):e0229996.
17.Luzuriaga J, Pastor-Alonso O, Encinas JM, Unda F, Ibarretxe G, Pineda JR. Human Dental Pulp Stem Cells Grown in Neurogenic Media Differentiate Into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain. Front Physiol. 2019 Mar 28;10(March):1–18.
18.Pai V, Vishwanath V, Prasanna J, Nadig R, Nadig R, Karthik J. Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: An in vitro study. J Conserv Dent. 2013;16(5):423.
19.Lan X, Sun Z, Chu C, Boltze J, Li S. Dental Pulp Stem Cells: An Attractive Alternative for Cell Therapy in Ischemic Stroke. Front Neurol. 2019 Aug 2;10(JUL):1–10.
20.Mead B, Logan A, Berry M, Leadbeater W, Scheven BA. Concise Review: Dental Pulp Stem Cells: A Novel Cell Therapy for Retinal and Central Nervous System Repair. Stem Cells. 2017 Jan;35(1):61–7.
21.Huang GT-J, Gronthos S, Shi S. Mesenchymal Stem Cells Derived from Dental Tissues vs . Those from Other Sources: Their Biology and Role in Regenerative Medicine. J Dent Res. 2009 Sep 18;88(9):792–806.
22.Longoni A, Utomo L, van Hooijdonk I, Bittermann G, Vetter V, Kruijt Spanjer E, et al. The chondrogenic differentiation potential of dental pulp stem cells. Eur Cells Mater. 2020 Feb 21;39:121–35.
23.Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem Cell Properties of Human Dental Pulp Stem Cells. J Dent Res. 2002 Aug 13;81(8):531–5.
24.La Noce M, Paino F, Spina A, Naddeo P, Montella R, Desiderio V, et al. Dental pulp stem cells: State of the art and suggestions for a true translation of research into therapy. J Dent. 2014 Jul;42(7):761–8.
25.Inostroza C, Vega-Letter AM, Brizuela C, Castrillón L, Saint Jean N, Duran CM, et al. Mesenchymal Stem Cells Derived from Human Inflamed Dental Pulp Exhibit Impaired Immunomodulatory Capacity In Vitro. J Endod. 2020 Aug 1;46(8):1091-1098.e2.
26.Chen Y, Li X, Wu J, Lu W, Xu W, Wu B. Dental pulp stem cells from human teeth with deep caries displayed an enhanced angiogenesis potential in vitro. J Dent Sci. 2021 Jan 1;16(1):318–26.
27.Ferrúa CP, Centeno EGZ, Rosa LC da, Amaral CC do, Severo RF, Sarkis-Onofre R, et al. How has dental pulp stem cells isolation been conducted? A scoping review. Braz Oral Res. 2017 Dec 18;31:1–9.
28.Glickman GN. AAE Consensus Conference on Diagnostic Terminology: Background and Perspectives. J Endod. 2009 Dec;35(12):1619–20.
29.Yang X, Li L, Xiao L, Zhang D. Recycle the dental fairy’s package: overview of dental pulp stem cells. Stem Cell Res Ther. 2018 Dec 13;9(1):347.
30.Scheller EL, Krebsbach PH, Konh DH. Tissue engineering: state of the art in oral rehabilitation. J Oral Rehabil. 2009 May;36(5):368–89.
31.Yalvac ME, Ramazanoglu M, Rizvanov AA, Sahin F, Bayrak OF, Salli U, et al. Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis. Pharmacogenomics J. 2010 Apr;10(2):105–13.
32.Atari M, Barajas M, Hernández-Alfaro F, Gil C, Fabregat M, Ferrés Padró E, et al. Isolation of pluripotent stem cells from human third molar dental pulp. Histol Histopathol. 2011;26(8):1057–70.
33.Pereira LO, Rubini MR, Silva JR, Oliveira DM, Silva ICR, Poças-Fonseca MJ, et al. Comparison of stem cell properties of cells isolated from normal and inflamed dental pulps. Int Endod J. 2012 Dec;45(12):1080–90.
34.Alongi DJ, Yamaza T, Song Y, Fouad AF, Romberg EE, Shi S, et al. Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential. Regen Med. 2010 Jul;5(4):617–31.
35.Karamzadeh R, Eslaminejad MB, Aflatoonian R. Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods. J Vis Exp. 2012 Nov 24;69(69).
36.Catón J, Bostanci N, Remboutsika E, De Bari C, Mitsiadis TA. Future dentistry: cell therapy meets tooth and periodontal repair and regeneration. J Cell Mol Med. 2011 May;15(5):1054–65.
37.Hilkens P, Gervois P, Fanton Y, Vanormelingen J, Martens W, Struys T, et al. Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells. Cell Tissue Res. 2013 Jul 29;353(1):65–78.
38.Raoof M, Yaghoobi MM, Derakhshani A, Kamal-Abadi AM, Ebrahimi B, Abbasnejad M, et al. A modified efficient method for dental pulp stem cell isolation. Dent Res J (Isfahan). 2014 Mar;11(2):244–50.
39.Suchanek J, Soukup T, Visek B, Ivancakova R, Kucerova L, Mokry J. Dental pulp stem cells and their characterization. Biomed Pap. 2009 Mar 1;153(1):31–5.
40.Perry BC, Zhou D, Wu X, Yang F-C, Byers MA, Chu T-MG, et al. Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use. Tissue Eng Part C Methods. 2008 Jun;14(2):149–56.
41.Martin-Piedra MA, Garzon I, Oliveira AC, Alfonso-Rodriguez CA, Carriel V, Scionti G, et al. Cell viability and proliferation capability of long-term human dental pulp stem cell cultures. Cytotherapy. 2014 Feb;16(2):266–77.
42.Conde MCM, Chisini LA, Grazioli G, Francia A, Carvalho RV de, Alcázar JCB, et al. Does Cryopreservation Affect the Biological Properties of Stem Cells from Dental Tissues? A Systematic Review. Braz Dent J. 2016 Dec;27(6):633–40.
43.Wall ME, Bernacki SH, Loboa EG. Effects of Serial Passaging on the Adipogenic and Osteogenic Differentiation Potential of Adipose-Derived Human Mesenchymal Stem Cells. Tissue Eng. 2007 Jun;13(6):1291–8.
44.Ruparel NB, de Almeida JFA, Henry MA, Diogenes A. Characterization of a Stem Cell of Apical Papilla Cell Line: Effect of Passage on Cellular Phenotype. J Endod. 2013 Mar;39(3):357–63.
45.Vemuri MC, Chase LG, Rao MS. Mesenchymal Stem Cell Assays and Applications. In: Methods. Totowa, NJ: Humana Press; 2011. p. 3–8. (Methods in Molecular Biology; vol. 698).
46.Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F., Krause DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006 Aug;8(4):315–7.