title: Rho kinase inhibitor Y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.
abstract: The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes.
authors.affiliation: Department of Dermatology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. e.vandenbogaard@derma.umcn.nl
authors.forename: Ellen H
authors.initials: EH
authors.lastname: van den Bogaard
authors.forename: Diana
authors.initials: D
authors.lastname: Rodijk-Olthuis
authors.forename: Patrick A M
authors.initials: PA
authors.lastname: Jansen
authors.forename: Ivonne M J J
authors.initials: IM
authors.lastname: van Vlijmen-Willems
authors.forename: Piet E
authors.initials: PE
authors.lastname: van Erp
authors.forename: Irma
authors.initials: I
authors.lastname: Joosten
authors.forename: Patrick L J M
authors.initials: PL
authors.lastname: Zeeuwen
authors.forename: Joost
authors.initials: J
authors.lastname: Schalkwijk
mesh_terms: D016475:3T3 Cells; D000328:Adult; D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects*; D003001:Cloning, Molecular; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008954:Models, Biological; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011565:Psoriasis / Q000473:pathology; D011725:Pyridines / Q000494:pharmacology*; D012867:Skin / Q000187:drug effects / Q000473:pathology; D017768:Skin, Artificial; D014161:Transduction, Genetic; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000577:Amides; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases