title: Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS.
abstract: The aim of the present study was to investigate the molecular mechanisms underlying activation of cell death pathways using genome-wide transcriptional analysis in human limbal epithelial cell (HLEC) cultures following conventional hypothermic storage in Optisol-GS. Three-week HLEC cultures were stored in Optisol-GS for 2, 4, and 7 days at 4 °C. Partek Genomics Suite software v.6.15.0422, (Partec Inc., St. Louis, MO, USA) was used to identify genes that showed significantly different (P < 0.05) levels of expression following hypothermic storage compared to non-stored cell sheets. There were few changes in gene expression after 2 days of storage, but several genes were differently regulated following 4 and 7 days of storage. The histone-coding genes HIST1H3A and HIST4H4 were among the most upregulated genes following 4 and 7 days of hypothermic storage. Bioinformatic analysis suggested that these two genes are involved in a functional network highly associated with cell death, necrosis, and transcription of RNA. HDAC1, encoding histone deacetylase 1, was the most downregulated gene after 7 days of storage. Together with other downregulated genes, it is suggested that HDAC1 is involved in a regulating network significantly associated with cellular function and maintenance, differentiation of cells, and DNA repair. Our data suggest that the upregulated expression of histone-coding genes together with downregulated genes affecting cell differentiation and DNA repair may be responsible for increased cell death following hypothermic storage of cultured HLEC. In summary, our results demonstrated that a higher number of genes changed with increasing storage time. Moreover, in general, larger differences in absolute gene expression values were observed with increasing storage time. Further understanding of these molecular mechanisms is important for optimization of storage technology for limbal epithelial sheets.
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo 0407, Norway. utheim2@gmail.com.|Department of Ophthalmology, Drammen Hospital, Vestre Viken Hospital Trust, Drammen 3004, Norway. utheim2@gmail.com.|Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo 0372, Norway. utheim2@gmail.com.|The Norwegian Dry Eye Clinic, 0159 Oslo, Norway. utheim2@gmail.com.|Faculty of Health Sciences, University College of South East Norway, Kongsberg 3603, Norway. utheim2@gmail.com.
authors.forename: Tor Paaske
authors.initials: TP
authors.lastname: Utheim
authors.affiliation: Department of Ophthalmology, Drammen Hospital, Vestre Viken Hospital Trust, Drammen 3004, Norway. panagiotis.salvanos@icloud.com.|Faculty of Medicine, University of Oslo, Oslo 0372, Norway. panagiotis.salvanos@icloud.com.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Salvanos
authors.affiliation: Department of Ophthalmology, Oslo University Hospital, Oslo 0407, Norway. outheim@gmail.com.
authors.forename: Øygunn Aass
authors.initials: ØA
authors.lastname: Utheim
authors.affiliation: The Norwegian Dry Eye Clinic, 0159 Oslo, Norway. raeder.sten@gmail.com.
authors.forename: Sten
authors.initials: S
authors.lastname: Ræder
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo 0407, Norway. lara.pasovic@gmail.com.|Faculty of Medicine, University of Oslo, Oslo 0372, Norway. lara.pasovic@gmail.com.
authors.forename: Lara
authors.initials: L
authors.lastname: Pasovic
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo 0407, Norway. o.k.olstad@medisin.uio.no.
authors.forename: Ole Kristoffer
authors.initials: OK
authors.lastname: Olstad
authors.affiliation: El centro de Oftalmología Barraquer, Universitari Barraquer/Universitat Autonoma de Barcelona, Barcelona 08021, Spain. mpaz@barraquer.com.
authors.forename: Maria Fideliz
authors.initials: MF
authors.lastname: de la Paz
authors.affiliation: Department of Ophthalmology, Drammen Hospital, Vestre Viken Hospital Trust, Drammen 3004, Norway. amer.sehic@odont.uio.no.
authors.forename: Amer
authors.initials: A
authors.lastname: Sehic
keywords: ex vivo expanded human limbal epithelial cells; gene expression; histone-coding genes; hypothermic storage; limbal stem cell deficiency