Pooled Human Serum Increases Regenerative Potential of In Vitro Expanded Stem Cells from Human Extracted Deciduous Teeth

  • Nazmul Haque
  • Noor Hayaty Abu KasimEmail author
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1083)


In regenerative therapy, in vitro expansion of stem cells is critical to obtain a significantly higher number of cells for successful engraftment after transplantation. However, stem cells lose its regenerative potential and enter senescence during in vitro expansion. In this study, the influence of foetal bovine serum (FBS) and pooled human serum (pHS) on the proliferation, morphology and migration of stem cells from human extracted deciduous teeth (SHED) was compared. SHED (n = 3) was expanded in KnockOut DMEM supplemented with either pHS (pHS-SM) or FBS (FBS-SM). pHS was prepared using peripheral blood serum of six healthy male adults, aged between 21 and 35 years old. The number of live SHED was significantly higher, from passage 5 to 7, when cultured in pHS-SM compared to those cultured in FBS-SM (p < 0.05). Number of cells having flattened morphology, characteristics of partially differentiated and senescent cells, was significantly lower (p < 0.05) in pHS-SM (3%) compared to those in FBS-SM (7%). Furthermore, migration of SHED in pHS-SM was found to be more directional. The presence of selected ten paracrine factors known for their proliferation and migration potential was detected in all six individual human sera, used to produce pHS, none of which were detected in FBS. Ingenuity Pathway Analysis showed the possible involvement of the ‘ephrin receptor signalling pathway’ to regulate the proliferation and migration of SHED in pHS-SM. In conclusion, pHS-SM showed significantly higher proliferation rate and could maintain significantly lower number of senescent cells and support directional migration of cells.


Engraftment Foetal bovine serum Morphology Paracrine factors Regenerative medicine 



bone marrow


epidermal growth factor


foetal bovine serum




fibroblast growth factor-2


flat spindle-shaped


granulocyte colony-stimulating factor


granulocyte macrophage colony-stimulating factor


hepatocyte growth factor


leukaemia inhibitory factor


mesenchymal stem cells


platelet-derived growth factor BB


pooled human serum


rapidly self-renewing


stem cell factor


stromal cell-derived factor-1α


stem cells from human extracted deciduous teeth




vascular endothelial growth factor



This work was supported by High Impact Research MOHE Grant UM.C/625/1/HIR/MOHE/DENT/01 from the Ministry of Higher Education Malaysia. The authors thank the staff nurses from Oral and Maxillofacial Sciences Department, Faculty of Dentistry, University of Malaya, for their support in the collection of blood from donors.

Conflicts of Interest

The authors deny any conflicts of interest related to this study.


  1. Ahn, H.-J., Lee, W.-J., Kwack, K., & Kwon, Y. D. (2009). FGF2 stimulates the proliferation of human mesenchymal stem cells through the transient activation of JNK signaling. FEBS Letters, 583(17), 2922–2926.CrossRefGoogle Scholar
  2. Arvanitis, D., & Davy, A. (2008). Eph/ephrin signaling: Networks. Genes & Development, 22(4), 416–429.CrossRefGoogle Scholar
  3. Bianchi, G., Banfi, A., Mastrogiacomo, M., Notaro, R., Luzzatto, L., Cancedda, R., & Quarto, R. (2003). Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Experimental Cell Research, 287(1), 98–105.CrossRefGoogle Scholar
  4. Bieback, K., Hecker, A., Kocaömer, A., Lannert, H., Schallmoser, K., Strunk, D., & Klüter, H. (2009). Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells, 27.CrossRefGoogle Scholar
  5. Blázquez-Prunera, A., Díez, J. M., Gajardo, R., & Grancha, S. (2017). Human mesenchymal stem cells maintain their phenotype, multipotentiality, and genetic stability when cultured using a defined xeno-free human plasma fraction. Stem Cell Research & Therapy, 8, 103.CrossRefGoogle Scholar
  6. Bonab, M. M., Alimoghaddam, K., Talebian, F., Ghaffari, S. H., Ghavamzadeh, A., & Nikbin, B. (2006). Aging of mesenchymal stem cell in vitro. BMC Cell Biology, 7(1), 14.CrossRefGoogle Scholar
  7. Boyd, A. W., Bartlett, P. F., & Lackmann, M. (2014). Therapeutic targeting of EPH receptors and their ligands. Nature Reviews. Drug Discovery, 13(1), 39–62.CrossRefGoogle Scholar
  8. Chase, L. G., Lakshmipathy, U., Solchaga, L. A., Rao, M. S., & Vemuri, M. C. (2010). A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem Cell Research & Therapy, 1(8), 1549–1553.Google Scholar
  9. Colter, D. C., Sekiya, I., & Prockop, D. J. (2001). Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proceedings of the National Academy of Sciences of the United States of America, 98(14), 7841–7845.CrossRefGoogle Scholar
  10. Cristofalo, V. J., Allen, R. G., Pignolo, R. J., Martin, B. G., & Beck, J. C. (1998). Relationship between donor age and the replicative lifespan of human cells in culture: A reevaluation. Proceedings of the National Academy of Sciences of the United States of America, 95(18), 10614–10619.CrossRefGoogle Scholar
  11. Díez, J. M., Bauman, E., Gajardo, R., & Jorquera, J. I. (2015). Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools. Stem Cell Research & Therapy, 6, 28.CrossRefGoogle Scholar
  12. Docheva, D., Padula, D., Popov, C., Mutschler, W., Clausen-Schaumann, H., & Schieker, M. (2008). Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy. Journal of Cellular and Molecular Medicine, 12(2), 537–552.CrossRefGoogle Scholar
  13. Dolley-Sonneville, P. J., Romeo, L. E., & Melkoumian, Z. K. (2013). Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions. PloS One, 8(8), e70263.CrossRefGoogle Scholar
  14. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D., & Horwitz, E. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8(4), 315–317.CrossRefGoogle Scholar
  15. Eggenhofer, E., Luk, F., Dahlke, M. H., & Hoogduijn, M. J. (2014). The life and fate of mesenchymal stem cells. Frontiers in Immunology, 5, 148.CrossRefGoogle Scholar
  16. Estrada, J. C., Albo, C., Benguria, A., Dopazo, A., Lopez-Romero, P., Carrera-Quintanar, L., Roche, E., Clemente, E. P., Enriquez, J. A., Bernad, A., & Samper, E. (2012). Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis. Cell Death and Differentiation, 19(5), 743–755.CrossRefGoogle Scholar
  17. Eubanks, E. J., Tarle, S. A., & Kaigler, D. (2014). Tooth storage, dental pulp stem cell isolation, and clinical scale expansion without animal serum. Journal of Endodontia, 40(5), 652–657.CrossRefGoogle Scholar
  18. Fierro, F., Illmer, T., Jing, D., Schleyer, E., Ehninger, G., Boxberger, S., & Bornhäuser, M. (2007). Inhibition of platelet-derived growth factor receptorβ by imatinib mesylate suppresses proliferation and alters differentiation of human mesenchymal stem cells in vitro. Cell Proliferation, 40(3), 355–366.CrossRefGoogle Scholar
  19. Forte, G., Minieri, M., Cossa, P., Antenucci, D., Sala, M., Gnocchi, V., Fiaccavento, R., Carotenuto, F., De Vito, P., Baldini, P.M., Prat, M., & Di Nardo, P. (2006). Hepatocyte growth factor effects on mesenchymal stem cells: proliferation, migration, and differentiation. Stem Cells, 24(1), 23–33.CrossRefGoogle Scholar
  20. Govindasamy, V., Abdullah, A. N., Ronald, V. S., Musa, S., Ab Aziz, Z. A., Zain, R. B., Totey, S., Bhonde, R. R., & Abu Kasim, N. H. (2010). Inherent differential propensity of dental pulp stem cells derived from human deciduous and permanent teeth. Journal of Endodontia, 36(9), 1504–1515.CrossRefGoogle Scholar
  21. Haasters, F., Prall, W. C., Anz, D., Bourquin, C., Pautke, C., Endres, S., Mutschler, W., Docheva, D., & Schieker, M. (2009). Morphological and immunocytochemical characteristics indicate the yield of early progenitors and represent a quality control for human mesenchymal stem cell culturing. Journal of Anatomy, 214(5), 759–767.CrossRefGoogle Scholar
  22. Handorf, A. M., & Li, W. J. (2011). Fibroblast growth factor-2 primes human mesenchymal stem cells for enhanced chondrogenesis. PloS One, 6(7), e22887.CrossRefGoogle Scholar
  23. Haque, N., Kasim, N. H., & Rahman, M. T. (2015). Optimization of pre-transplantation conditions to enhance the efficacy of mesenchymal stem cells. International Journal of Biological Sciences, 11(3), 324–334.CrossRefGoogle Scholar
  24. He, X., Ma, J., & Jabbari, E. (2010). Migration of marrow stromal cells in response to sustained release of stromal-derived factor-1alpha from poly(lactide ethylene oxide fumarate) hydrogels. International Journal of Pharmaceutics, 390(2), 107–116.CrossRefGoogle Scholar
  25. Heiskanen, A., Satomaa, T., Tiitinen, S., Laitinen, A., Mannelin, S., Impola, U., Mikkola, M., Olsson, C., Miller-Podraza, H., Blomqvist, M., Olonen, A., Salo, H., Lehenkari, P., Tuuri, T., Otonkoski, T., Natunen, J., Saarinen, J., & Laine, J. (2007). N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible. Stem Cells, 25(1), 197–202.CrossRefGoogle Scholar
  26. Honczarenko, M., Le, Y., Swierkowski, M., Ghiran, I., Glodek, A. M., & Silberstein, L. E. (2006). Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells, 24(4), 1030–1041.CrossRefGoogle Scholar
  27. Hu, F., Wang, X., Liang, G., Lv, L., Zhu, Y., Sun, B., & Xiao, Z. (2013). Effects of epidermal growth gactor and basic fibroblast growth factor on the proliferation and osteogenic and neural differentiation of adipose-derived stem cells. Cellular Reprogramming, 15(3), 224–232.CrossRefGoogle Scholar
  28. Kawada, H., Fujita, J., Kinjo, K., Matsuzaki, Y., Tsuma, M., Miyatake, H., Muguruma, Y., Tsuboi, K., Itabashi, Y., Ikeda, Y., Ogawa, S., Okano, H., Hotta, T., Ando, K., & Fukuda, K. (2004). Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood, 104(12), 3581–3587.CrossRefGoogle Scholar
  29. Khanna-Jain, R., Vanhatupa, S., Vuorinen, A., Sandor, G., Suuronen, R., Mannerstrom, B., & Miettinen, S. (2012). Growth and differentiation of human dental pulp stem cells maintained in fetal bovine serum, human serum and serum-free/Xeno-free culture media. Journal of Stem Cell Research & Therapy, 2, 4.CrossRefGoogle Scholar
  30. Kobayashi, T., Watanabe, H., Yanagawa, T., Tsutsumi, S., Kayakabe, M., Shinozaki, T., Higuchi, H., & Takagishi, K. (2005). Motility and growth of human bone-marrow mesenchymal stem cells during ex vivo expansion in autologous serum. Journal of Bone and Joint Surgery. British Volume (London), 87(10), 1426–1433.CrossRefGoogle Scholar
  31. Kolf, C. M., Cho, E., & Tuan, R. S. (2007). Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Research & Therapy, 9(1), 1–10.Google Scholar
  32. Komoda, H., Okura, H., Lee, C. M., Sougawa, N., Iwayama, T., Hashikawa, T., Saga, A., Yamamoto-Kakuta, A., Ichinose, A., Murakami, S., Sawa, Y., & Matsuyama, A. (2010). Reduction of N-Glycolylneuraminic acid xenoantigen on human adipose tissue-derived stromal cells/mesenchymal stem cells leads to safer and more useful cell sources for various stem cell therapies. Tissue Engineering – Part A, 16(4), 1143–1155.CrossRefGoogle Scholar
  33. Krausgrill, B., Vantler, M., Burst, V., Raths, M., Halbach, M., Frank, K., Schynkowski, S., Schenk, K., Hescheler, J., Rosenkranz, S., & Müller-Ehmsen, J. (2009). Influence of cell treatment with PDGF-BB and reperfusion on cardiac persistence of mononuclear and mesenchymal bone marrow cells after transplantation into acute myocardial infarction in rats. Cell Transplantation, 18(8), 847–853.CrossRefGoogle Scholar
  34. Lee, R. H., Hsu, S. C., Munoz, J., Jung, J. S., Lee, N. R., Pochampally, R., & Prockop, D. J. (2006). A subset of human rapidly self-renewing marrow stromal cells preferentially engraft in mice. Blood, 107(5), 2153–2161.CrossRefGoogle Scholar
  35. Lennartsson, J., & Rönnstrand, L. (2012). Stem cell factor receptor/c-Kit: From basic science to clinical implications. Physiological Reviews, 92(4), 1619–1649.CrossRefGoogle Scholar
  36. Li, X., Hou, J., Wu, B., Chen, T., & Luo, A. (2014). Effects of platelet-rich plasma and cell coculture on angiogenesis in human dental pulp stem cells and endothelial progenitor cells. Journal of Endodontia, 40(11), 1810–1814.CrossRefGoogle Scholar
  37. Li, C.-Y., Wu, X.-Y., Tong, J.-B., Yang, X.-X., Zhao, J.-L., Zheng, Q.-F., Zhao, G.-B., & Ma, Z.-J. (2015). Comparative analysis of human mesenchymal stem cells from bone marrow and adipose tissue under xeno-free conditions for cell therapy. Stem Cell Research & Therapy, 6(1), 1–13.CrossRefGoogle Scholar
  38. Matsuoka, F., Takeuchi, I., Agata, H., Kagami, H., Shiono, H., Kiyota, Y., Honda, H., & Kato, R. (2013). Morphology-based prediction of osteogenic differentiation potential of human mesenchymal stem cells. PloS One, 8(2), e55082.CrossRefGoogle Scholar
  39. Metcalf, D. (2003). The unsolved enigmas of leukemia inhibitory factor. Stem Cells, 21(1), 5–14.CrossRefGoogle Scholar
  40. Murakami, M., Horibe, H., Iohara, K., Hayashi, Y., Osako, Y., Takei, Y., Nakata, K., Motoyama, N., Kurita, K., & Nakashima, M. (2013). The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells with high regenerative potential. Biomaterials, 34(36), 9036–9047.Google Scholar
  41. Nakamura, S., Yamada, Y., Katagiri, W., Sugito, T., Ito, K., & Ueda, M. (2009). Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. Journal of Endodontia, 35(11), 1536–1542.CrossRefGoogle Scholar
  42. Oikonomopoulos, A., van Deen, W. K., Manansala, A. R., Lacey, P. N., Tomakili, T. A., Ziman, A., & Hommes, D. W. (2015). Optimization of human mesenchymal stem cell manufacturing: The effects of animal/xeno-free media. Scientific Reports, 5, 16570.CrossRefGoogle Scholar
  43. Pan, S., Dangaria, S., Gopinathan, G., Yan, X., Lu, X., Kolokythas, A., Niu, Y., & Luan, X. (2013). SCF promotes dental pulp progenitor migration, neovascularization, and collagen remodeling – potential applications as a homing factor in dental pulp regeneration. Stem Cell Reviews & Reports, 9(5), 655–667.CrossRefGoogle Scholar
  44. Paula, A. C. C., Martins, T. M. M., Zonari, A., Frade, S. P. P. J., Angelo, P. C., Gomes, D. A., & Goes, A. M. (2015). Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation. Stem Cell Research & Therapy, 6(1), 76.CrossRefGoogle Scholar
  45. Phadnis, S. M., Joglekar, M. V., Venkateshan, V., Ghaskadbi, S. M., Hardikar, A. A., & Bhonde, R. R. (2006). Human umbilical cord blood serum promotes growth, proliferation, as well as differentiation of human bone marrow-derived progenitor cells. In Vitro Cellular and Developmental Biology – Animal, 42(10), 283–286.PubMedGoogle Scholar
  46. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., & Marshak, D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science, 284(5411), 143–147.CrossRefGoogle Scholar
  47. Poloni, A., Maurizi, G., Rosini, V., Mondini, E., Mancini, S., Discepoli, G., Biasio, S., Battaglini, G., Felicetti, S., Berardinelli, E., Serrani, F., & Leoni, P. (2009). Selection of CD271(+) cells and human AB serum allows a large expansion of mesenchymal stromal cells from human bone marrow. Cytotherapy, 11(2), 153–162.CrossRefGoogle Scholar
  48. Pons, J., Huang, Y., Arakawa-Hoyt, J., Washko, D., Takagawa, J., Ye, J., Grossman, W., & Su, H. (2008). VEGF improves survival of mesenchymal stem cells in infarcted hearts. Biochemical and Biophysical Research Communications, 376(2), 419–422.CrossRefGoogle Scholar
  49. Prasanna, S. J., Gopalakrishnan, D., Shankar, S. R., & Vasandan, A. B. (2010). Pro-inflammatory cytokines, IFNgamma and TNFalpha, influence immune properties of human bone marrow and Wharton jelly mesenchymal stem cells differentially. PloS One, 5(2), e9016.CrossRefGoogle Scholar
  50. Prockop, D. J., Sekiya, I., & Colter, D. C. (2001). Isolation and characterization of rapidly self-renewing stem cells from cultures of human marrow stromal cells. Cytotherapy, 3(5), 393–396.CrossRefGoogle Scholar
  51. Rodrigues, M., Griffith, L., & Wells, A. (2010). Growth factor regulation of proliferation and survival of multipotential stromal cells. Stem Cell Research & Therapy, 1(4), 32.CrossRefGoogle Scholar
  52. Rojas, M., Xu, J., Woods, C. R., Mora, A. L., Spears, W., Roman, J., & Brigham, K. L. (2005). Bone marrow-derived mesenchymal stem cells in repair of the injured lung. American Journal of Respiratory Cell and Molecular Biology, 33(2), 145–152.CrossRefGoogle Scholar
  53. Saller, M. M., Prall, W. C., Docheva, D., Schonitzer, V., Popov, T., Anz, D., Clausen-Schaumann, H., Mutschler, W., Volkmer, E., Schieker, M., & Polzer, H. (2012). Increased stemness and migration of human mesenchymal stem cells in hypoxia is associated with altered integrin expression. Biochemical and Biophysical Research Communications, 423(2), 379–385.CrossRefGoogle Scholar
  54. Sethe, S., Scutt, A., & Stolzing, A. (2006). Aging of mesenchymal stem cells. Ageing Research Reviews, 5(1), 91–116.CrossRefGoogle Scholar
  55. Son, B. R., Marquez-Curtis, L. A., Kucia, M., Wysoczynski, M., Turner, A. R., Ratajczak, J., Ratajczak, M.Z., & Janowska-Wieczorek, A. (2006). Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells, 24(5), 1254–1264.CrossRefGoogle Scholar
  56. Strojny, C., Boyle, M., Bartholomew, A., Sundivakkam, P., & Alapati, S. (2015). Interferon gamma-treated dental pulp stem cells promote human mesenchymal stem cell migration in vitro. Journal of Endodontia, 41(8), 1259–1264.CrossRefGoogle Scholar
  57. Tamama, K., Fan, V. H., Griffith, L. G., Blair, H. C., & Wells, A. (2006). Epidermal growth factor as a candidate for ex vivo expansion of bone marrow–derived mesenchymal stem cells. Stem Cells, 24(3), 686–695.CrossRefGoogle Scholar
  58. Tamama, K., Kawasaki, H., & Wells, A. (2010). Epidermal growth factor (EGF) treatment on multipotential stromal cells (mscs). Possible enhancement of therapeutic potential of msc. Journal of Biomedicine and Biotechnology, 2010, 10.Google Scholar
  59. Tateishi, K., Ando, W., Higuchi, C., Hart, D. A., Hashimoto, J., Nakata, K., Yoshikawa, H., & Nakamura, N. (2008). Comparison of human serum with fetal bovine serum for expansion and differentiation of human synovial MSC: Potential feasibility for clinical applications. Cell Transplantation, 17(5), 549–557.CrossRefGoogle Scholar
  60. Wang, X., Sha, X. J., Li, G. H., Yang, F. S., Ji, K., Wen, L. Y., Liu, S. Y., Chen, L., Ding, Y., & Xuan, K. (2012). Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Archives of Oral Biology, 57(9), 1231–1240.CrossRefGoogle Scholar
  61. Werner, S., & Grose, R. (2003). Regulation of wound healing by growth factors and cytokines. Physiological Reviews, 83(3), 835–870.CrossRefGoogle Scholar
  62. Yanada, S., Ochi, M., Kojima, K., Sharman, P., Yasunaga, Y., & Hiyama, E. (2006). Possibility of selection of chondrogenic progenitor cells by telomere length in FGF-2-expanded mesenchymal stromal cells. Cell Proliferation, 39(6), 575–584.CrossRefGoogle Scholar
  63. Yu, Q., Liu, L., Lin, J., Wang, Y., Xuan, X., Guo, Y., & Hu, S. (2015). SDF-1α/CXCR4 axis mediates the migration of mesenchymal stem cells to the hypoxic-ischemic brain lesion in a rat model. Cell Journal (Yakhteh), 16(4), 440–447.Google Scholar

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© Springer International Publishing AG  2017

Authors and Affiliations

  1. 1.Department of Restorative Dentistry, Faculty of DentistryUniversity of MalayaKuala LumpurMalaysia
  2. 2.Regenerative Dentistry Research Group, Faculty of DentistryUniversity of MalayaKuala LumpurMalaysia

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