Self-assembling 3D spheroid cultures of human neonatal keratinocytes have enhanced regenerative properties

Stem Cell Research

Kim E. Creek¹  Diego Altomare²  Lucia Pirisi³  Yvon Woappi⁴ 

[1] Corresponding author at: Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.;Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA;Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC 29208, USA;Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29208, USA;

关键词: Anoikis;    Epidermal stem cells;    Spheroids;    3-D culture;    Human keratinocytes;    Cell-based model;   

DOI  :  

来源: DOAJ

【 摘 要 】

Relative to conventional two-dimensional (2-D) culture, three-dimensional (3-D) suspension culture of epithelial cells more closely mimics the in vivo cell microenvironment regarding cell architecture, cell to matrix interaction, and osmosis exchange. However, primary normal human keratinocytes (NHKc) rapidly undergo terminal differentiation and detachment-induced cell death (anoikis) upon disconnection from the basement membrane, thus greatly constraining their use in 3-D suspension culture models. Here, we examined the 3-D anchorage-free growth potential of NHKc isolated from neonatal skin explants of 59 different individuals. We found that 40% of all isolates naturally self-assembled into multicellular spheroids within 24 h in anchorage-free culture, while 60% did not. Placing a single spheroid back into 2-D monolayer culture yielded proliferating cells that expressed elevated levels of nuclear P63 and basal cytokeratin 14. These cells also displayed prolonged keratinocyte renewal and a gene expression profile corresponding to cellular heterogeneity, quiescence, and de-differentiation. Notably, spheroid-derived (SD) NHKc were enriched for a P63/K14 double-positive population that formed holoclonal colonies and reassembled into multicellular spheroids during 3-D suspension subculture. This study reveals marked phenotypic differences in neonatal keratinocyte suspension cultures isolated from different individuals and present a model system that can be readily employed to study epithelial cell behavior, along with a variety of dermatological diseases.

【 授权许可】

Unknown