과잉치 치수 세포의 계대별 석회화 비교
Comparison of Mineralization in Each Passage of Dental Pulp Stem Cells from Supernumerary Tooth
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초록
본 연구는 상악 정중과잉치에서 얻은 치수유래 줄기세포(human dental pulp stem cells from supernumerary tooth, sDPSCs)를 대상으로 분화능의 변화 특성을 알아보고자 하였다. 전신 병력이 없는 6세 남자아이 상악 중절치와 측절치 사이에 매복 된 과잉치를 발치하여 sDPSCs를 얻었다. 세포들을 16계대까지배양하였고, 1 - 8계대는 Young군으로 9 - 16계대는 Old군으로 나누었다. 각 계대 배양에 소요된 시간은 Young군에서는 2.25 ± 0.46 일, Old군에서는 3.25 ± 0.46일을 보였으며, 이는 통계적으로 유의한 차이를 보였다(p < 0.05). 각 계대 별로 세포 형태를 관찰하고 분화를 유도한 후 Alizarin-red solution 염색을 통해 골모세포(odontoblast)로 분화되는 정도를 관찰하였다. 1, 8, 9계대에서 분화제를 처리하지 않은 세포의 형태에서는 방추형의 섬유모세포와 유사한 길쭉한 형태로 과립(nodule)이 적었지만, 16계대에서는 세포의 크기가커지고 넓적한 형태로 변하고 과립도 많아졌다. 1계대는 분화 7일부터 과립이 관찰되며, 8계대에서는 14일 동안 분화제를 처리한 후과립이 명확히 관찰되었다. 그러나 9계대 이후에서는 과립의 빈도가 상당히 감소되었다. ARS 염색에서는 1, 8계대는 진한 붉은색으로염색되었으나, 9, 16계대는 염색이 옅게 되었다. 이를 통해 sDPSCs는 8계대 이전의 세포를 줄기세포의 원천으로 우선 고려하는 것이 좋다고 사료된다.
Abstract
The purpose of this study was to evaluate the difference of differentiation potential in each passage of dental pulp stem cells from supernumerary tooth (sDPSCs). The sDPSCs were obtained from a healthy 6-year-old male patient under the guidelines and got the informed consent. Cells were cultured until passage number 16 and divided into two groups; 1 - 8 passages as a young group and 9 - 16 passages as an old group. It was taken 2.25 ± 0.46 days in a young group and 3.25 ± 0.46 days in an old group to propagate cells of each passage until confluence and there were statistically significant differences between two groups (p < 0.05). In every passage, cell morphology was observed with microscope and evaluated the capacity to form high levels of minerals by alizarin red solution staining after treating differentiation medium. Fibroblast-like, spindle shaped, elongated cells and a few nodules were found in uninduced cultures of passage number 1, 8 and 9. But at 16 passage culture, cell size became larger and broader and observed with more nodules. After inducing differentiation, mineralized nodules were detected at the first passage of 7th day culture whereas at the 8 passage culture, nodules were seen clearly at 14th day culture. In addition, the amount of mineralized nodules were remarkably decreased after passage 9. From the data presented in this study, it is recommended to use sDPSCs of passage number within 8 for utilizing as stem cells.
참고문헌 (36)
- Smith A, "A glossary for stem-cell biology", Nature, 441, pp.1060~1060 (2006)
- Evans MJ, "Establishment in culture of pluripotential cells from mouse embryos", Nature, 292, pp.154~156 (1981)
- Almeida-Porada G, "Adult stem cell plasticity and methods of detection", Rev Clin Exp Hematol, 5, pp.26~41 (2001)
- Temple S, "Stem cell plasticity-building the brain of our dreams", Nature Reviews Neuroscience, 2, pp.513~520 (2001)
- Matsubara T, "A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix", Biochem Biophys Res Commun, 313, pp.503~508 (2004)
- Baxter MA, "Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion", Stem Cells, 22, pp.675~682 (2004)
- Bianco P, "Bone marrow stromal stem cells: nature, biology, and potential applications", Stem Cells, 19, pp.180~192 (2001)
- Bruder SP, "Growth kinetics, selfrenewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation", J Cell Biochem, 64, pp.278~294 (1997)
- Gronthos S, "Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo", Proc Natl Acad Sci USA, 97, pp.13625~13630 (2000)
- Seo BM, "Investigation of multipotent postnatal stem cells from human periodontal ligament", Lancet, 364, pp.149~155 (2004)
- Morsczeck C, "Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth", Matrix Biol, 24, pp.155~165 (2005)
- Sonoyama W, "Mesenchymal stem cell-mediated functional tooth regeneration in swine", PLoS One, 1, pp.e79~ (2006)
- Miura M, "SHED: stem cells from human exfoliated deciduous teeth", Proc Natl Acad Sci U S A, 100, pp.5807~5812 (2003)
- Huang WH, "Mesiodens in the primary dentition stage: a radiographic study", ASDC J Dent Child, 59, pp.186~189 (1992)
- Huang AHC, "Isolation and characterization of dental pulp stem cells from a supernumerary tooth", J Oral Pathol Med, 37, pp.571~574 (2008)
- Hayflick L, "The serial cultivation of human diploid cell strains", Exp Cell Res, 25, pp.585~621 (1961)
- Till JE, "A direct measurement of the radiation sensitivity of normal mouse bone marrow cells", Radiat Res, 14, pp.213~222 (1961)
- Wagers AJ, "Plasticity of adult stem cells", Cell, 116, pp.639~648 (2004)
- Phinney DG, "Donor variation in the growth properties and osteogenic potential of human marrow stromal cells", J Cell Biochem, 75, pp.424~436 (1999)
- Ryu E, "Identification of senescenceassociated genes in human bone marrow mesenchymal stem cells", Biochem Biophys Res Commun, 371, pp.431~436 (2008)
- Wall ME, "Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells", Tissue Eng, 13, pp.1291~1298 (2007)
- Park BW, "Osteogenic phenotypes and mineralization of cultured human periosteal-derived cells", Arch Oral Biol, 52, pp.983~989 (2007)
- Rho GJ, "Porcine mesenchymal stem cells-current technological status and future perspective", Front Biosci (Landmark Ed), 14, pp.3942~3961 (2009)
- Huang GJ, "Mesenchymal stem cells derived from dental tissues vs. those from other sources:their biology and role in regenerative medicine", J Dent Res, 88, pp.792~806 (2009)
- Graziano A, "Dental pulp stem cells: a promising tool for bone regeneration", Stem Cell Rev, 4, pp.21~26 (2008)
- Stenderup K, "Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells", Bone, 33, pp.919~926 (2003)
- Huang AH, "Isolation and characterization of dental pulp stem cells from a supernumerary tooth", J Oral Pathol Med, 37, pp.571~574 (2008)
- Lindroos B, "Characterisation of human dental stem cells and buccal mucosa fibroblasts", Biochem Biophys Res Commun, 368, pp.329~335 (2008)
- Karaoz E, "Isolation and in vitro characterisation of dental pulp stem cells from natal teeth", Histochem Cell Biol, 133, pp.95~112 (2010)
- Berchem G, "Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis", Oncogene, 21, pp.5951~ (2002)
- Mets T, "In vitro aging of human bone marrow derived stromal cells", Mech Ageing Dev, 16, pp.81~89 (1981)
- Min JH, "Dentinogenic potential of human adult dental pulp cells during the extended primary culture", Hum Cell, 24, pp.43~50 (2011)
- 박세아, "탯줄유래 줄기세포의 계대배양에 따른 특성 변화의 분석", Clinical and Experimental Reproductive Medicine, 36(1), pp.23~34 (2009), 대한생식의학회
- Izadpanah R, "properties of mesenchymal stem cells derived from bone marrow and adipose tissue", J Cell Biochem, 99, pp.1285~1297 (2006)
- Conget PA, "Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells", J Cell Physiol, 181, pp.67~73 (1999)
- Deans RJ, "Mesenchymal stem cells: biology and potential clinical uses", Exp Hematol, 28, pp.875~884 (2000)