9a6c6596eaca54dd19cdf6371e46a650 pmc_26901233.pdf abed69f0a5d86cd8b3f7aec119bbe5534304c98f pmc_26901233.pdf 0732e7501236d86c7e8f08f283b0a6c34dc6c0cf66c03ae0c015cb6b1fbde169 pmc_26901233.pdf Title: Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS Subject: 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. Keywords: ex vivo expanded human limbal epithelial cells; gene expression; histone-coding genes; hypothermic storage; limbal stem cell deficiency Author: Tor Paaske Utheim, Panagiotis Salvanos, T1O ygunn Aass Utheim, Sten RT1ae der, Lara Pasovic, Ole Kristoffer Olstad, Maria Fideliz de la Paz and Amer Sehic Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.14 CreationDate: Thu Feb 18 09:32:53 2016 CET ModDate: Thu Feb 18 09:32:53 2016 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1769903 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CUBOSZ+URWPalladioL-Ital Type 1 Custom yes yes no 66 0 GPFMUO+URWPalladioL-Bold Type 1 Custom yes yes no 67 0 ONDBGL+URWPalladioL-Roma Type 1 Custom yes yes no 68 0 [none] Type 3 Custom yes no no 69 0 ITYPRM+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 73 0 SGHEOH+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 74 0 DHQDGN+URWPalladioL-BoldItal Type 1 Custom yes yes no 128 0 [none] Type 3 Custom yes no no 129 0 LIOJCP+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 133 0 LIOKJB+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 134 0 LIOJMM+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 135 0 LIOJMN+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 136 0 LIOJMO+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 137 0 LIOJNP+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 138 0 LIOJOA+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 139 0 LIOKEP+TimesNewRomanPSMT TrueType WinAnsi yes yes no 140 0 [none] Type 3 Custom yes no no 192 0 [none] Type 3 Custom yes no no 193 0 LIOJCP+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 196 0 LIOKIA+TimesNewRomanPS-BoldMT TrueType WinAnsi yes yes no 197 0 LIOJMM+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 198 0 LIOJMN+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 199 0 LIOJMO+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 200 0 LIOJNP+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 201 0 LIOJOA+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 202 0 LIOKEP+TimesNewRomanPSMT TrueType WinAnsi yes yes no 203 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 293 0 [none] Type 3 Custom yes no no 294 0 LIOJCP+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 303 0 LIOJMM+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 304 0 LIOJMN+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 305 0 LIOJMO+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 306 0 LIOJNP+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 307 0 LIOJOA+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 308 0 LIOKEP+TimesNewRomanPSMT TrueType WinAnsi yes yes no 309 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2020-08-10 13:41:00 CEST RepresentationInformation: pmc_26901233.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2018-02-06 09:09:06 CET Size: 1769903 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 475 FreeObjects: 3 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: None Outlines: Item: Title: Introduction Item: Title: Results Children: Item: Title: Genes Exhibiting Higher Levels of Expression Following 2, 4, and 7 Days of Hypothermic Storage Compared to Control Item: Title: Genes Exhibiting Lower Levels of Expression Following 2, 4, and 7 Days of Hypothermic Storage Compared to Control Item: Title: Discussion Item: Title: Experimental Section Children: Item: Title: Human Tissue Preparation Item: Title: Human Limbal Explant Cultures on Intact Amniotic Membranes Item: Title: Hypothermic Storage of Cultured Human Limbal Epithelial Cells in Optisol-GS Item: Title: RNA Isolation Item: Title: Microarray Analysis Item: Title: Bioinformatic Analysis Info: Title: Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS Author: Tor Paaske Utheim, Panagiotis Salvanos, 1 ygunn Aass Utheim, Sten R1e der, Lara Pasovic, Ole Kristoffer Olstad, Maria Fideliz de la Paz and Amer Sehic Subject: 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. 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