Low temperature culture enhances ameloblastic differentiation of human keratinocyte stem cells

  • Yingnan Song
  • Bingmei Wang
  • Hua Li
  • Xiaoxiao Hu
  • Xin Lin
  • Xuefeng Hu
  • Yanding ZhangEmail author
Original Paper


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 



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.

Ethical approval

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.

Supplementary material

10735_2019_9837_MOESM1_ESM.docx (15 kb)
Supplementary material 1 (DOCX 15 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Yingnan Song
    • 1
  • Bingmei Wang
    • 1
  • Hua Li
    • 1
  • Xiaoxiao Hu
    • 1
  • Xin Lin
    • 1
  • Xuefeng Hu
    • 1
  • Yanding Zhang
    • 1
    Email author
  1. 1.Southern Center for Biomedical Research, Fujian Key Laboratory of Developmental and Neural Biology, College of Life SciencesFujian Normal UniversityFuzhouPeople’s Republic of China

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