Previous studies have demonstrated that several types of human stem cells of non-dental origin can be induced to differentiate into enamel-secreting ameloblasts after recombined with mouse embryonic dental mesenchyme. However, the successful rate of ameloblastic differentiation is about rather low, which presents a major obstacle for future stem cell-based whole tooth bioengineering. Previous studies have shown that cultures at reduced temperature could improve the differentiation capability of stem cells in tissue engineering. In this study, we systematically investigated the effects of low temperature on the viability, proliferation and stemness of human keratinocytes stem cells (hKSCs) in cell culture and further examined ameloblastic differentiation of the hKSCs in human–mouse recombinant chimeric tooth germs. Our results demonstrated that low temperature indeed reduces growth rate and maintains healthy undifferentiated morphology of hKSCs without any effects on cell viability. Moreover, examination of stemness makers revealed improved stemness of hKSCs cultured at low temperature with increased expression of stemness markers K15, CD29 and p63 and decreased expression differentiation marker K10, as compared to those cultured at 37 °C. These low temperature treated hKSCs, when recombined with mouse embryonic dental mesenchyme, exhibited significantly increased rate (40%) of ameloblastic differentiation, as compared to that (17%) in tissue recombinants with those hKSCs treated at standard temperature. Our studies demonstrate that low temperature cell culture improves the stemness and plasticity of hKSCs, which in turn enhances ameloblastic differentiation capability of the stem cells in bioengineered teeth.
Tooth regeneration Human keratinocyte stem cells Ameloblasts Low temperature
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This study was supported by grants from the National Nature Science Foundation of China [Grant Numbers 81771034, 81870739 and 81200761] and Natural Science Foundation of Fujian Province [Grant Number 2016J01144].
Compliance with ethical standards
Conflict of interest
We declare there is no conflict of interest and no competing financial, personal or other relationships with other people or organizations.
Use of mice was approved by the Animal Ethical and Welfare Committee of Fujian Normal University. hKSCs were harvested from circumcised human foreskins from children 6–12 years old whose parents gave informed consent and were approved by the Ethics Committee of Fujian Normal University for the study.
Abiko Y, Nishimura M, Arai J, Kuraguchi J, Saitoh M, Kaku T (1996) Apoptosis in the reduced enamel epithelium just after tooth emergence in rats. Med Electron Microsc 29:84–89CrossRefGoogle Scholar
Barrandon Y, Grasset N, Zaffalon A et al (2012) Capturing epidermal stemness for regenerative medicine. Semin Cell Dev Biol 23:937–944CrossRefGoogle Scholar
Belinsky GS, Antic SD (2013) Mild hypothermia inhibits differentiation of human embryonic and induced pluripotent stem cells. Biotechniques 55:79–82CrossRefGoogle Scholar
Borowiec AS, Delcourt P, Dewailly E, Bidaux G (2013) Optimal differentiation of in vitro keratinocytes requires multifactorial external control. PLoS ONE 8:e77507CrossRefGoogle Scholar
Bose A, Teh MT, Mackenzie IC, Waseem A (2013) Keratin K15 as a biomarker of epidermal stem cells. Int J Mol Sci 14:19385–19398CrossRefGoogle Scholar
Cai J, Zhang Y, Liu P et al (2013) Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells. Cell Regen (Lond) 2:6Google Scholar
De Paiva CS, Pflugfelder SC, Li DQ (2006) Cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Stem Cells 24:368–375CrossRefGoogle Scholar
Dunnwald M, Tomanek-Chalkley A, Alexandrunas D, Fishbaugh J, Bickenbach JR (2001) Isolating a pure population of epidermal stem cells for use in tissue engineering. Exp Dermatol 10:45–54CrossRefGoogle Scholar
Hu X, Lee J, Zheng X et al (2018) Efficient induction of functional ameloblasts from human keratinocyte stem cells. Stem Cell Res Ther 9:126CrossRefGoogle Scholar
Hughes D, Song B (2016) Dental and nondental stem cell based regeneration of the craniofacial region: a tissue based approach. Stem Cells Int 2016:8307195CrossRefGoogle Scholar
Hunt L, Hacker DL, Grosjean F, De Jesus M, Uebersax L, Jordan M, Wurm FM (2005) Low-temperature pausing of cultivated mammalian cells. Biotechnol Bioeng 89:157–163CrossRefGoogle Scholar
Ikeda E, Morita R, Nakao K et al (2009) Fully functional bioengineered tooth replacement as an organ replacement therapy. Proc Natl Acad Sci USA 106:13475–13480CrossRefGoogle Scholar
Islam R, Jackson C, Eidet JR et al (2015) Effect of storage temperature on structure and function of cultured human oral keratinocytes. PLoS ONE 10:e0128306CrossRefGoogle Scholar
Morrison SJ, Spradling AC (2008) Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 132:598–611CrossRefGoogle Scholar
Nakao K, Morita R, Saji Y et al (2007) The development of a bioengineered organ germ method. Nat Methods 4:227–230CrossRefGoogle Scholar
Oshima M, Mizuno M, Imamura A et al (2011) Functional tooth regeneration using a bioengineered tooth unit as a mature organ replacement regenerative therapy. PLoS ONE 6:e21531CrossRefGoogle Scholar
Pellegrini G, Dellambra E, Golisano O et al (2001) p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98:3156–3161CrossRefGoogle Scholar
Ponec M, Gibbs S, Weerheim A, Kempenaar J, Mulder A, Mommaas AM (1997) Epidermal growth factor and temperature regulate keratinocyte differentiation. Arch Dermatol Res 289:317–326CrossRefGoogle Scholar
Poumay Y, Pittelkow MR (1995) Cell density and culture factors regulate keratinocyte commitment to differentiation and expression of suprabasal K1/K10 keratins. J Invest Dermatol 104:271–276CrossRefGoogle Scholar
Rumman M, Dhawan J, Kassem M (2015) Concise review: quiescence in adult stem cells—biological significance and relevance to tissue regeneration. Stem Cells 33:2903–2912CrossRefGoogle Scholar
Saito K, Fukuda N, Matsumoto T et al (2010) Moderate low temperature preserves the stemness of neural stem cells and suppresses apoptosis of the cells via activation of the cold-inducible RNA binding protein. Brain Res 1358:20–29CrossRefGoogle Scholar
Shea KL, Xiang W, LaPorta VS, Licht JD, Keller C, Basson MA, Brack AS (2010) Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration. Cell Stem Cell 6:117–129CrossRefGoogle Scholar
Shima A, Pham J, Blanco E, Barton ER, Sweeney HL, Matsuda R (2011) IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures. Exp Cell Res 317:356–366CrossRefGoogle Scholar
Sun XY, Fu XB, Han WD, Zhao YL, Liu HL, Sheng ZY (2011) Dedifferentiation of human terminally differentiating keratinocytes into their precursor cells induced by basic fibroblast growth factor. Biol Pharm Bull 34:1037–1045CrossRefGoogle Scholar
Utheim TP, Islam R, Fostad IG et al (2016) Storage temperature alters the expression of differentiation-related genes in cultured oral keratinocytes. PLoS ONE 11:e0152526CrossRefGoogle Scholar
Viano M, Alotto D, Aillon A, Castagnoli C, Silvagno F (2017) A thermal gradient modulates the oxidative metabolism and growth of human keratinocytes. FEBS Open Bio 7:1843–1853CrossRefGoogle Scholar
Volponi AA, Kawasaki M, Sharpe PT (2013) Adult human gingival epithelial cells as a source for whole-tooth bioengineering. J Dent Res 92:329–334CrossRefGoogle Scholar
Wang BM, Li LW, Du SR, Liu C, Lin X, Chen YP, Zhang YD (2010) Induction of human keratinocytes into enamel-secreting ameloblasts. Dev Biol 344:795–799CrossRefGoogle Scholar
Zaproudina N, Varmavuo V, Airaksinen O, Narhi M (2008) Reproducibility of infrared thermography measurements in healthy individuals. Physiol Meas 29:515–524CrossRefGoogle Scholar