title: Human epithelial stem cells persist within tissue-engineered skin produced by the self-assembly approach.
abstract: To adequately and permanently restore organ function after grafting, human tissue-engineered skin substitutes (TESs) must ultimately contain and preserve functional epithelial stem cells (SCs). It is therefore essential that a maximum of SCs be preserved during each in vitro step leading to the production of TESs such as the culture process and the elaboration of a skin cell bank by cryopreservation. To investigate the presence and functionality of epithelial SCs within the human TESs made by the self-assembly approach, slow-cycling cells were identified using 5'-bromo-2'-deoxyuridine (BrdU) in the three-dimensional construct. A subset of basal epithelial cells retained the BrdU label and was positive for the SC-associated marker keratin 19 within TESs after a chase of 21 days in culture post-BrdU labeling. Moreover, keratinocytes harvested from TESs gave rise to SC-like colonies in secondary monolayer subcultures, indicating that SCs were preserved within TESs. To evaluate the effect of cryopreservation with dimethyl sulfoxide and storage in liquid nitrogen on SCs, human epithelial cells were extracted from skin samples, amplified in culture, and used to produce TESs, before cryopreservation as well as after thawing. We found that the proportion and the growth potential of epithelial SCs in monolayer culture and in TESs remained constant before and after cryopreservation. Further, the functionality of these substitutes was demonstrated by successfully grafting human TESs on athymic mice for 6 months. We conclude that human epithelial skin SCs are adequately preserved upon human tissue reconstruction. Thus, these TESs produced by the self-assembly approach are suitable for clinical applications.
authors.affiliation: LOEX Centre of Université Laval, Tissue Engineering and Regenerative Medicine: LOEX-FRQS Research Center of CHU de Québec and Department of Surgery, Faculty of Medicine, Université Laval, Quebec, QC, Canada.
authors.forename: Amélie
authors.initials: A
authors.lastname: Lavoie
authors.forename: Claudia
authors.initials: C
authors.lastname: Fugère
authors.forename: Annie
authors.initials: A
authors.lastname: Beauparlant
authors.forename: Benjamin
authors.initials: B
authors.lastname: Goyer
authors.forename: Danielle
authors.initials: D
authors.lastname: Larouche
authors.forename: Claudie
authors.initials: C
authors.lastname: Paquet
authors.forename: Maxime
authors.initials: M
authors.lastname: Desgagné
authors.forename: Sarah
authors.initials: S
authors.lastname: Sauvé
authors.forename: Hubert
authors.initials: H
authors.lastname: Robitaille
authors.forename: Martine
authors.initials: M
authors.lastname: Dunnwald
authors.forename: Dong Hyun
authors.initials: DH
authors.lastname: Kim
authors.forename: Roxane
authors.initials: R
authors.lastname: Pouliot
authors.forename: Julie
authors.initials: J
authors.lastname: Fradette
authors.forename: Lucie
authors.initials: L
authors.lastname: Germain
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D015925:Cryopreservation; D004847:Epithelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D053539:Keratin-19 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D017768:Skin, Artificial; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*; D055815:Young Adult
chemical_list: D053539:Keratin-19; D001973:Bromodeoxyuridine