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Production of progenitor cells from primary human epithelial cell monolayer cultures

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Abstract

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70–80% confluence, they begin to float up into the overlying medium and are called “epithelial pop-up keratinocytes (ePUKs)” allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.

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Acknowledgements

We would like to thank Dr. Cynthia Marcelo for suggestions of our experiments. We also thank Eve Bingham and James Washington for their critical editing of this manuscript. The author, A.M., would like to thank Eve for teaching her skin keratinocyte culture methodology. This study was supported by NIH grants R01 DE 019431 and R01 DE 013417 to SEF.

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Authors and Affiliations

  1. Department of Oral and Maxillofacial Surgery, School of Dentistry, University of Michigan, Ann Arbor, MI, USA

    Atsuko Miyazawa, Shiuhyang Kuo & Stephen E. Feinberg

  2. Department of Oral and Maxillofacial Surgery, School of Life dentistry at Tokyo, The Nippon Dental University, Tokyo, Japan

    Atsuko Miyazawa

  3. Department of Surgery, Medical School, University of Michigan, 1150 W Medical Center Drive, MSRB 2, A560B, Ann Arbor, MI, 48109, USA

    Stephen E. Feinberg

Authors
  1. Atsuko Miyazawa
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  2. Shiuhyang Kuo
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  3. Stephen E. Feinberg
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Corresponding author

Correspondence to Stephen E. Feinberg.

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The Institutional Review Board at the University of Michigan approved the use of the skin, and donors provided informed consent for research use.

Conflict of interest

The authors declare that they have no conflict of interest.

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Editor: Tetsuji Okamoto

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Miyazawa, A., Kuo, S. & Feinberg, S.E. Production of progenitor cells from primary human epithelial cell monolayer cultures. In Vitro Cell.Dev.Biol.-Animal 54, 413–422 (2018). https://doi.org/10.1007/s11626-018-0259-1

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  • DOI: https://doi.org/10.1007/s11626-018-0259-1

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