Sains Malaysiana 48(9)(2019): 1855–1865

http://dx.doi.org/10.17576/jsm-2019-4809-06 

 

Pengkulturan dan Pencirian Penanda Molekul Sel Stem Manusia daripada Pulpa Gigi Susu (SHED) dan Gigi Kekal (DPSC)

(Culture and Molecular Markers Characterization of Stem Cells from Human Deciduous (SHED) and Permanent (DPSC) Teeth Pulp)

 

SHAHRUL HISHAM ZAINAL ARIFFIN1*, THANALETCHUMI MANOGARAN1, INTAN ZARINA ZAINOL ABIDIN2, ZAIDAH ZAINAL ARIFFIN3 & ROHAYA MEGAT ABDUL WAHAB4

 

1Pusat Bioteknologi dan Makanan Berfungsi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, Malaysia

 

2Centre for Research and Postgraduate Studies, Cyberjaya University College of Medical Sciences, 63000 Cyberjaya, Selangor Darul Ehsan, Malaysia

 

3Pusat Pengajian Biologi, Fakulti Sains Gunaan, Universiti Teknologi MARA, 40450 UiTM Shah Alam, Selangor Darul Ehsan, Malaysia

 

4Unit Ortodontik, Pusat Kesihatan Pergigian Keluarga, Fakulti Pergigian, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Wilayah Persekutuan, Malaysia

 

Received: 25 February 2019/Accepted: 25 June 2019

 

ABSTRAK

Sel stem pulpa gigi manusia yang dipencilkan daripada tisu pulpa gigi merupakan sel stem dewasa bersifat multipoten. Objektif kajian ini adalah untuk mengenal pasti teknik pengkulturan in vitro sel stem pulpa gigi susu (SHED) dan gigi kekal (DPSC) melalui penentuan pasaj, kesan eraman tripsin-EDTA dan potensi proliferasi serta untuk mencirikan kedua-dua sel ini melalui profil penanda molekul. Kaedah pencernaan enzim digunakan pada tisu pulpa gigi susu dan gigi kekal masing-masing untuk pemencilan sel SHED dan DPSC. Kedua-dua sel dikulturkan daripada pasaj 1 hingga 5 dan pewarnaan tripan biru digunakan untuk memperoleh lengkuk pertumbuhan bagi menentukan masa penggandaan populasi sel (PDT) pada setiap pasaj. Kesan tripsin-EDTA terhadap kedua-dua sel dikaji menggunakan pewarnaan Alamar biru untuk menentukan masa eraman yang optimum semasa proses pengsubkulturan. Potensi proliferasi in vitro bagi kedua-dua sel selama 21 hari ditentukan melalui asai 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromida (MTT). Pencirian kedua-dua sel melalui pengekspresan penanda biologi molekul ditentukan melalui pendekatan RT-PCR. Morfologi kedua-dua sel didapati menyerupai sel fibroblas pada kesemua pasaj. Sel SHED dan DPSC pada pasaj 3 menunjukkan PDT terendah iaitu masing-masing 43 ± 2.3 dan 63 ± 3.1 jam. Pendedahan SHED terhadap tripsin-EDTA menunjukkan penurunan peratus sel viabel berbanding DPSC. Pertumbuhan sel SHED didapati ~2.3 kali ganda lebih tinggi berbanding DPSC. Pencirian molekul kedua-dua sel menunjukkan pengekspresan penanda sel stem mesekima dan bukannya penanda sel stem hematopoietik. Kesimpulannya, morfologi sel yang homogenus dan nilai PDT terendah yang ditunjukkan oleh sel daripada pasaj 3 menjadikannya pasaj terbaik untuk menentukan potensi proliferasi sel dan pengekpresan penanda molekul. SHED didapati mampu berproliferasi dengan lebih baik berbanding DPSC. Walau bagaimanapun, DPSC lebih rentan terhadap tripsin-EDTA berbanding SHED. Pencirian molekul pula mendapati kedua-dua sel merupakan sel stem jenis mesenkima.

 

Kata kunci: Kesan tripsin-EDTA; masa penggandaan sel; potensi proliferasi; profil penanda biologi molekul; sel stem pulpa gigi manusia

 ABSTRACT

Human dental pulp stem cells are adult multipotent stem cells isolated from dental pulp tissue. Our objective was to determine in vitro culture technique for stem cells from deciduous tooth (SHED) and permanent tooth (DPSC) through cell passage identification, the effect of trypsin-EDTA and proliferation potential, and to characterize both cells using molecular markers profile. Enzyme digestion method was used on dental pulp tissue from deciduous and permanent tooth for SHED and DPSC isolation, respectively. Both cells were cultured at passage 1 until 5 and trypan blue assay was used to obtain growth curve in determining cell population doubling time (PDT) for each passage. Effect of trypsin-EDTA on both cells were studied using Alamar blue assay to determine optimum incubation time for subculturing process. Cell proliferation potential for both cells within 21 days was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell characterization through molecular biology markers was performed using RT-PCR. Both cells at all passages appeared fibroblast-like. SHED and DPSC at passage 3 exhibited the lowest PDT with 43 ± 2.3 and 63 ± 3.1 h, respectively. SHED exposed to trypsin-EDTA showed decrease in cell viability percentage compared to DPSC. Cell growth for SHED was ~2.3-fold higher than DPSC. Both cells expressed mesenchymal stem cell markers and not hematopoietic stem cell markers. In conclusion, homogenous morphology and lowest PDT value indicated that cells at passage 3 are the best to determine proliferation potential and molecular markers expression. SHED proliferated better than DPSC. However, DPSC was more resistant to trypsin-EDTA than SHED. Based on molecular marker profile, both cells are mesenchymal stem cells.

 

Keywords: Cell doubling time; human dental pulp stem cells; molecular biology markers profile; proliferation potential; trypsin-EDTA effect

 

REFERENCES

Ab Kadir, R., Zainal Ariffin, S.H., Megat Abdul Wahab, R., Kermani, S. & Senafi, S. 2012a. Characterization of mononucleated human peripheral blood cells. The Scientific World Journal 2012: 843843.

Ab Kadir, R., Zainal Ariffin, S.H., Megat Abdul Wahab, R. & Senafi, S. 2012b. Molecular characterisation of human peripheral blood stem cells. South African Journal of Science 108(5-6): 67-73.

Chowdhury, S.R., Aminuddin, B.S. & Ruszymah, I. 2012. Effect of supplementation of dermal fibroblasts conditioned medium on expansion of keratinocytes through enhancing attachment. Indian Journal of Experimental Biology 50(5): 332-339.

Chowdhury, S.R., Izzah, A.G., Manira, M., Lokanathan, Y., Aminuddin, B.S. & Ruszymah, I. 2015. Dermal fibroblast conditioned medium for skin wound healing. Tissue Engineering Part A 21(S1): S158.

Coughlin, S., Noviski, M., Mueller, J.L., Chuwonpad, A., Raschke, W.C., Weiss, A. & Zikherman, J. 2015. An extracatalytic function of CD45 in B cells is mediated by CD22. Proceedings of the National Academy of Science of the United States of America 112(47): E6515-E6524.

Fan, W., Li, J., Wang, Y., Pan, J., Li, S., Zhu, L., Guo, C. & Yan, Z. 2016. CD105 promotes chondrogenesis of synovium-derived mesenchymal stem cells through Smad2 signaling. Biochemical and Biophysical Research Communications 474(2): 338-344.

Farges, J.C., Keller, J.F., Carrouel, F., Durand, S.H., Romeas, A., Bleicher, F., Lebecque, S. & Staquet, M.J. 2009. Odontoblasts in the dental pulp immune response. Journal of Experimental Zoology, Part B, Molecular and Developmental Evolution 312(5): 425-436.

Fong, D., Nicholas, D. & Hoemann, C.D. 2017. Mesenchymal stem cell detachment with trace trypsin is superior to EDTA for in vitro chemotaxis and adhesion assays. Biochemical and Biophysical Research Communications 484(3): 656-661.

Galle, J., Preziosi, L. & Tosin, A. 2009. Contact inhibition of growth described using multiphase model and an individual cell based model. Applied Mathematics Letters 22(10): 1483-1490.

Gounaris, E., Erdman, S.E., Restaino, C., Gurish, M.F., Friend, D.S., Gounari, F., Lee, D.M., Zhang, G., Glickman, J.N., Shin, K., Rao, V.P., Poutahidis, T., Weissleder, R., McNagny, K.M. & Khazaie, K. 2007. Mast cells are an essential hematopoietic component for polyp development. Proceedings of the National Academy of Science of the United States of America 104(50): 19977-19982.

Grayson, W.L., Bunnell, B.A., Martin, E., Frazier, T., Hung, B.P. & Gimble, J.M. 2015. Stromal cells and stem cells in clinical bone regeneration. Nature Reviews Endocrinology 11(3): 140-150.

Huang, Z., Ruan, H.B., Xian, L., Chen, W., Jiang, S., Song, A., Wang, Q., Shi, P., Gu, X. & Gao, X. 2014. The stem cell factor/kit signalling pathway regulates mitochondrial function and energy expenditure. Nature Communications 5: Article No. 4282.

Idrus, R., Rameli, M.A., Low, K.C., Law, J.X., Chua, K.H., Abdul Latiff, M. & Saim, A. 2014. Full-thickness skin wound healing using autologous keratinocytes and dermal fibroblasts with fibrin: Bilayered versus single-layered substitute. Advances in Skin and Wound Care 27(4): 171-180.

Intan Zarina, Z.A., Shahrul Hisham, Z.A., Zaidah, Z.A. & Rohaya, M.A.W. 2010. Keupayaan pembezaan tiga jenis sel primitif daripada hasil perbezaan tempoh proliferasi darah mencit. Sains Malaysiana 39(2): 305-313.

Intan Zarina, Z.A., Shahrul Hisham, Z.A., Rohaya, M.A.W., Sahidan, S. & Zaidah, Z.A. 2008. Osteoclast and osteoblast development of Mus musculus haemopoietic mononucleated cells. Journal of Biological Sciences 8(3): 506-516.

Ip, J.E., Wu, Y.J., Huang, J., Zhang, L., Pratt, R.E. & Dzau, V.J. 2007. Mesenchymal stem cells use integrin β1 not CXC chemokine receptor 4 for myocardial migration and engraftment. Molecular Biology of the Cell 18(8): 2873-2882.

Ishiy, F.A.A., Fanganiello, R.D., Kobayashi, G.S., Kague, E., Kuriki, P.S. & Passos-Bueno, M.R. 2018. CD105 is regulated by hsa-miR-1287 and its expression is inversely correlated with osteopotential in SHED. Bone 106: 112-120.

Jeon, M., Song, J.S., Choi, B.J., Shin, D.M., Jung, H.S. & Kim, S.O. 2014. In vitro and in vivo characteristics of stem cells from human exfoliated deciduous teeth obtained by enzymatic disaggregation and outgrowth. Archives of Oral Biology 59(10): 1013-1023.

Kanafi, M.M., Pal, R. & Gupta, P.K. 2013. Phenotypic and functional comparison of optimum culture conditions for upscaling of dental pulp stem cells. Cell Biology International 37(2): 126-136.

Karamzadeh, R., Eslaminejad, M.B. & Aflatoonian, R. 2012. Isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods. Journal of Visual Experiments: JoVE 69: 4372.

Kirches, E., Steffen, T., Waldt, N., Hebert, E., Pachow, D., Wilisch-Neumann, A., Keilhof, G., Schneider, T., Braunsdorf, W.E.K., Warnke, J.P. & Mawrin, C. 2018. The expression of the MSC-marker CD73 and of NF2/Merlin are correlated in meningiomas. Journal of Neuro-Oncology 138(2): 251-259.

Kunimatsu, R., Nakajima, K., Awada, T., Tsuka, Y., Abe, T., Ando, K., Hiraki, T., Kimura, A. & Tanimoto, K. 2018. Comparative characterization of stem cells from human exfoliated deciduous teeth, dental pulp, and bone marrow-derived mesenchymal stem cells. Biochemistry and Biophysical Research Communications 501(1): 193-198.

Law, J.X., Chowdhury, S.R., Saim, A. & Idrus, R. 2015. Concentration-dependent effect of platelet-rich plasma on keratinocyte and fibroblast wound healing. Cytotherapy 17(3): 293-300.

Lennartsson, J. & Ronnstrand, L. 2012. Stem cell factor receptor/c-Kit: From basic science to clinical implications. Physiological Reviews 92(4): 1619-1649.

Miura, M., Gronthos, S., Zhao, M., Lu, B., Fisher, L.W., Robey, P.G. & Shi, S. 2003. SHED: Stem cells from human exfoliated deciduous teeth. Proceedings of the National Academy of Science of the United States of America 100(10): 5807-5812.

Monguio-Tortajada, M., Roura, S., Galvez-Monton, C., Franquesa, M., Bayes-Genis, A. & Borras, F.E. 2017. Mesenchymal stem cells induce expression of CD73 in human monocytes in vitro and in a swine model of myocardial infarction in vivo. Frontiers in Immunology 8: 1577.

Ode, A., Kopf, J., Kurtz, A., Schmidt-Bleek, K., Schrade, P., Kolar, P., Buttgereit, F., Lehmann, K., Hutmacher, D.W., Duda, G.N. & Kasper, G. 2011. CD73 and CD29 concurrently mediate the mechanically induced decrease of migratory capacity of mesenchymal stromal cells. European Cell Material 22: 26-42.

Rohaina, C.M., Yogeswaran, L., Rabiatul Adawiyah, R., Chowdhury, S.R., Aminuddin, B.S. & Ruszymah, H.I. 2014. The effect of nasal fibroblast conditioned medium on in vitro wound healing model. Regenerative Research 3(2): 87-88.

Rohaya, M.A.W., Nur Akmal, M.R., Sahidan, S., Intan Zarina, Z.A., Zaidah, Z.A. & Shahrul Hisham, Z.A. 2017. Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells. PeerJ 5: e3180.

Salzig, D., Schmiermund, A., Grace, P.P., Elseberg, C., Weber, C. & Czermak, P. 2013. Enzymatic detachment of therapeutic mesenchymal stromal cells grown on glass carriers in a bioreactor. The Open Biomedical Enginering Journal 7: 147-158.

Shahrul Hisham, Z.A., Rus Dina, R.D., Zulham, Y., Ikmal, M.J., Sahidan, S. & Rohaya, M.A.W. 2017. Penyerapan akar gigi apeks luaran hasil rawatan ortodontik pada enam dan 12 bulan. Sains Malaysiana 46(8): 1299-1307.

Shahrul Hisham, Z.A., Thanaletchumi, M., Intan Zarina, Z.A., Sahidan, S. & Rohaya, M.A.W. 2016. Isolation and morphology of stem cells from deciduous tooth (SHED) and human dental pulp stem cell (hDPSC). AIP Conference Proceedings 1784 2016: 020008.

Shahrul Hisham, Z.A., Zulham, Y., Intan Zarina, Z.A., Rohaya, M.A.W. & Zaidah, Z.A. 2011. Cellular and molecular changes in orthodontic tooth movement. The Scientific World Journal 11(1): 1-16.

Shahrul Hisham, Z.A., Intan Zarina, Z.A., Muhamad Dain, Y. & Rohaya, M.A.W. 2010. Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus. Cell Communication and Signaling 8: 29. doi: 10.1186/1478-811X-8-29.

Shahrul-Hisham, Z.A., Rohaya, M.A.W., Ismanizan, I., Nor- Muhammad, M. & Zaidah, Z.A. 2005. Stem cells, cytokines and their receptors. Asia Pacific Journal Molecular Biology and Biotechnology 13(1): 1-13.

Souza, L.M., Bittar, J.D., Silva, I.C.R., Toledo, O.A., Brigido, M.M. & Pocas-Fonseca, M.J. 2015. Comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp. Brazilian Journal of Oral Science 9(4): 427-433.

Sorrentino, A., Ferracin, M., Castelli, G., Biffoni, M., Tomaselli, G., Baiocchi, M., Fatica, A., Negrini, M., Peschle, C. & Valtieri, M. 2008. Isolation and characterization of CD146+ multipotent mesenchymal stromal cells. Experimental Hematology 36(8): 1035-1046.

Ting, L., Chen, L., Zhang, C. & Zhao, J. 2015. Effect of accutase or trypsin dissociation on the apoptosis of human striatum-derived neural stem cells. Acta Academiae Medicinae Sinicae 37(2): 185-194.

Tsuji, K., Ojima, M., Otabe, K., Horie, M., Koga, H., Sekiya, I. & Muneta, T. 2017. Effects of different cell-detaching methods on the viability and cell surface antigen expression of synovial mesenchymal stem cells. Cell Transplantation 26(6): 1089-1102.

Umemoto, T., Yamato, M., Ishihara, J., Shiratsuchi, Y., Utsumi, M., Morita, Y., Tsukui, H., Terasawa, M., Shibata, T., Nishida, K., Kobayashi, Y., Petrich, B.G., Nakauchi, H., Eto, K. & Okane, T. 2012. Integrin-alphavbeta3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells. Blood 119(1): 83-94.

Wachs, F., Couillard-Despres, S., Engelhardt, M., Wilhelm, D., Ploetz, S., Vroemen, M., Kaesbauer, J., Uyanik, G., Klucken, J., Karl, C., Tebbing, J., Svendsen, C., Weidner, N., Kuhn, H., Winkler, J. & Aigner, L. 2003. High efficacy of clonal growth and expansion of adult neural stem cells. Laboratory Investigation 83(7): 949-962.

Wagner, W., Bork, S., Horn, P., Krunic, D., Walenda, T., Diehlmann, A., Benes, V., Blake, J., Huber, F.X., Eckstein, V., Boukamp, P. & Ho, A.D. 2009. Aging and replicative senescence have related effects on human stem and progenitor cells. PLoS ONE 4(6): p. e5846.

Wan Haifa Haryani, W.O., Shahrul Hisham, Z.A., Zaidah, Z.A., Muhd Fauzi, S., Sahidan, S. & Rohaya, M.A.W. 2010. Anticancer screening of ethanol extract from selected piperaceae family and its determination via trypan blue staining. Sains Malaysiana 39(6): 941-949.

Xu, J., Wang, W., Kapila, Y., Lotz, J. & Kapila, S. 2009. Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells. Stem Cells Development 18(3): 487-496.

Yazid, F., Luchman, N.A., Wahab, R.M.A., Ariffin, S.H.Z. & Senafi, S. 2018a. Proliferation and osteoblast differentiation mice dental pulp stem cells between enzyme digestion and outgrowth method. Sains Malaysiana 47(4): 649-656.

Yazid, F., Ng, A.N.M.N., Wong, Y.Q., Luchman, N.A., Zainal Ariffin, S.H. & Megat Abdul Wahab, R. 2018b. Osteogenic performance of MC3T3-E1 cells on granular hydroxyapatite scaffold. Asian Journal of Medicine and Biomedicine 3: OP20.

Yazid, M.D., Shahrul Hisham, Z.A., Sahidan, S., Zaidah, Z.A. & Rohaya, M.A.W. 2011. Stem cell heterogeneity of mononucleated cells from murine peripheral blood: Molecular analysis. The Scientific World Journal 2011(11): 2150-2159.

Yazid, F., Gnanasegaran, N., Kunasekaran, W., Govindasamy, V. & Musa, S. 2014. Comparison of immunomodulatory properties of dental pulp stem cells derived from healthy and inflamed teeth. Clinical Oral Investigation 18(9): 2103-2112.

 

*Corresponding author; email: hisham@ukm.edu.my

 

 

 

 

previous