title: Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay.
abstract: Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
authors.forename: Simone
authors.initials: S
authors.lastname: Riis
authors.affiliation: Thermo Fisher Scientific, Frederick, MD, USA.
authors.forename: Rhonda
authors.initials: R
authors.lastname: Newman
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
authors.forename: Hilal
authors.initials: H
authors.lastname: Ipek
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
authors.forename: Jens I
authors.initials: JI
authors.lastname: Andersen
authors.affiliation: Thermo Fisher Scientific, Frederick, MD, USA.
authors.forename: David
authors.initials: D
authors.lastname: Kuninger
authors.affiliation: Thermo Fisher Scientific, Frederick, MD, USA.
authors.forename: Shayne
authors.initials: S
authors.lastname: Boucher
authors.affiliation: Thermo Fisher Scientific, Frederick, MD, USA.
authors.forename: Mohan C
authors.initials: MC
authors.lastname: Vemuri
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
authors.forename: Cristian P
authors.initials: CP
authors.lastname: Pennisi
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Zachar
authors.affiliation: Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
authors.forename: Trine
authors.initials: T
authors.lastname: Fink
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism*; D002118:Calcium; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D014945:Wound Healing
chemical_list: D003470:Culture Media; D002118:Calcium