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Apoptosis Gene Expression Profiling of Ex Vivo Expanded Human Limbal Epithelial Cells Sten Raeder,1 Tor Paaske Utheim,1 Øygunn Aass Utheim,1 Savita Prabhakar,5 Yiqing Cai,2 Borghild Roald,3 Kristiane Haug,1 Anders Kvalheim,1 Edvard Berger Messelt,2 Hewen Zhang, 5 Liv Drolsum,1 Bjørn Nicolaissen,1 Torstein Lyberg,4; 1Center for Eye Research, University of Oslo, Department of Ophthalmology, Ullevål University Hospital, Norway, 2Department of Oral Biology, Faculty of Dentistry, University of Oslo, Norway, 3Department of Pathology, Ullevål University Hospital, University of Oslo, Norway. 4Center for Clinical Research, University of Oslo, Center for Eye research Ulleval University Kirkeveien, Oslo, 0407, Norway, 5SuperArray Bioscience Corporation, 7320 Executive Way, Suite 101, 101, Frederick MD, 21704. Introduction Histology and Immunostaining Apoptosis Gene Expression Profiling using RT2Profiler™ Profiler™ PCR Array Transplantation of ex vivo expanded human limbal epithelial cells (HLEC) is used as a therapy therapy for limbal stem cell deficiency (LSCD). HLEC may be cultured ex vivo by a variety of expansion protocols. Although these protocols have shown good clinical outcomes, limbal epithelial epithelial stem cell therapy still faces challenges regarding surgery logistics, tissue sterility, tissue transportation, transportation, and availability of tissue. Our laboratory was the first to report a method for shortshort-term organ culture (OC) eye bank storage of cultured HLEC which may be beneficial in limbal epithelial stem cell therapy.1 This study was conducted to investigate whether conventional OC storage and OptisolOptisol-GS storage were applicable to cultured HLEC and to evaluate the extent of cell death due to apoptosis after eye bank storage of cultured HLEC. HLEC were expanded on intact amniotic membranes for 21 days and stored for one week at 31º 31ºC and 23º 23ºC in OC medium or at 5º 5ºC in OptisolOptisol-GS. Cultures were fixed in formaldehyde and embedded in paraffin. paraffin. Low labeling indices were observed under different storage conditions as assessed by immunostaining immunostaining with caspasecaspase-3 (range 0.0%0.0%1.2%) and a DNA fragmentation assay (TUNEL, range 0.0%0.0%-2.3%). Cellular results were confirmed at the gene expression level using the Human Apoptosis RT2Profiler™ Profiler™ PCR Array from SuperArray Bioscience, which profiles the expression of key apoptosis genes. The results results showed that propro-apoptotic genes were significantly downdown-regulated and antianti-apoptotic genes were significantly upup-regulated in all three storage conditions. This study demonstrates that eye bank storage of cultured cultured HLEC is associated with minor cell death due to apoptosis at both the cellular level and the gene expression expression level. This work was supported in part by the Eastern Norway Regional Health Authority, the Norwegian Norwegian Association of the Blind and Partially Sighted and the Blindmission IL. Figure 2: Organ Culture Storage of Cultured HLEC at Ambient Temperature is Superior to OC Storage at 31° 31°C and OptisolOptisol-GS Storage at 5° 5°C. Figure 4: Anti- and Pro-Apoptotic Genes with Greater than Three-Fold Change in Expression (p<0.05) in Cultured Human Limbal Epithelial Cells following 1-week Storage at Three Different Temperatures Experimental Design Hypothesis We hypothesized that OC storage at 31°C and Optisol-GS hypothermic storage may preserve the characteristics of cultured HLEC. Accordingly, we compared these conventional storage methods with the novel storage method. Furthermore, because cell death due to apoptosis has been reported in human corneal epithelium after OC culture storage2 and hypothermic storage3, we studied expression of apoptosis-regulatory genes and examined apoptosis markers in cultured HLEC following eye bank storage. Functional Gene Grouping TNF Ligand Family TNF Receptor Family Bcl-2 Family Gene Symbol 1-week organ culture storage at 31ºC Fold-Change FASLG 14.25 TNF -18.24 TNFSF10 -4.21 TNFRSF9 -4.09 BAG4 BAK1 18.87 -6.13 -3.01 -3.13 -32.41 -22.96 -27.47 -16.64 -8.96 BIRC1 5.2 -6.69 APAF1 4.48 6.14 3.42 CARD8 30.16 10.36 CASP5 17.81 11.53 -4.05 -5.67 RIPK2 p53 & DNA Damage 9.44 FADD 16.72 FAS 4.93 TRADD -9.11 -4.25 23.86 11.75 15.21 17.79 -12.23 -8.85 CIDEB 10.21 ABL1 10.46 CASP6 15.78 GADD45A Culture Conditions 3-week HLEC (n=48) cultures were either organ-cultured at 31°C (n=12) or 23°C (n=12) or stored in Optisol-GS at 5°C (n=12) in a closed container for one week. Figure 1 explains in brief as to how the human limbal tissue from human donor eyes were cultured on intact amniotic membranes and stored in the eye bank. 7.58 CARD6 CASP9 CIDE Domain Family 11.58 HRK PYCARD Figure 3: Quantification of Apoptotic Cells A 11.61 13.27 BIRC8 Death Domain Family -4.98 8.72 4.7 BIRC6 CARD Family -5.07 BNIP3L MCL1 IAP Family -14.3 -7.5 BCL2 BCL2A1 BNIP2 1-week Optisol-GS storage at 5ºC Fold-Change 7.65 -7.44 BCL2L11 HLEC cultures were fixed in neutral-buffered 4% formaldehyde and embedded in paraffin. Serial sections of 5 µm were stained with haematoxylin and eosin (H&E). Immunohistochemistry was performed with a panel of antibodies for markers of human ocular surface epithelia. Histological evaluation and semiquantitative immunohistochemical localization of the epithelial markers were carried out by two independent investigators using a microscope at a magnification of 400X. Results: The ultrastructure was preserved at 23ºC, while storage at 31ºC and 5ºC was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLEC remained undifferentiated under all storage conditions. 1-week organ culture storage at 23ºC Fold-Change 10.8 Figure 4. RNA was isolated from the formalin-fixed paraffin-embedded (FFPE) tissue using SuperArray’s ArrayGradeTM FFPE RNA Isolation Kit (GA-023). RNA (40 ng)) was amplified and reverse transcribed using a modified version of the TrueLabeling PicoAMP Kit (GA-130) and the RT2 PCR Array First Strand Kit (C-02) from SuperArray Bioscience. The RT2Profiler™ Human Apoptosis PCR Array (APHS-012) from SuperArray Bioscience was used to analyze the mRNA levels of 84 key genes involved in apoptosis, in a 384-well format. Three biological replicates were included from each experimental group. Relative changes in gene expression were calculated using the ΔΔCt method. Results: Following storage at 23ºC and 5ºC, down-regulation of BCL2A1 and BIRC1, and reduced expression of TNF receptor signaling components (TNF and TRADD) was revealed suggesting a reduction in nuclear factor-κB activity. Under all storage conditions, expression of BNIP2 was up-regulated, whereas MCL1 expression was down-regulated. FIGURE 1. Eye Bank Storage of Cultured Human Limbal Epithelial Cells (HLEC) B Apoptotic and labeling index (%) 4 3,5 3 H&E apoptotic index Caspase-3 labeling index TUNEL labeling index Conclusions 2,5 2 1,5 1 0,5 0 3-week HLEC cultures at 37ºC 1-week OC storage 1-week OC storage at 31ºC at 23ºC 1-week Optisol-GS storage at 5ºC Figure 3A demonstrates H&E staining (top), cleaved caspase-3 immunohistochemistry (middle), and TUNEL staining (bottom) of cultured human limbal epithelial cells after one week’s organ culture storage at 23ºC. H&E staining demonstrates an apoptotic epithelial cell with circular nuclear fragments (arrow). Cleaved caspase-3positive surface cells have cytoplasmic immunoreactivity and well defined nuclear membranes (arrowheads). A TUNEL positive surface cell (arrowhead) is also observed. (Original magnification: 400X) Figure 3B contains a histogram demonstrating the H&E apoptotic index, caspase-3 labeling index, and TUNEL labeling index in cultured human limbal epithelial cells after three weeks’ culture and one week’s storage at the three different temperatures. Results are expressed as mean percent of the apoptotic or labeling index in the individual experimental groups. Error bars denote 1 SE. Results:: No significant increase in cleaved caspase-3 or TUNEL staining was observed in response to eye bank storage under any of the three storage conditions. Our results indicate that OC storage of cultured HLEC at ambient temperature is superior to OC storage at 31°C and Optisol-GS storage at 5°C as the original layered structure of cultured HLEC is preserved at 23°C storage. Apoptosis is minimal following eye bank storage of cultured HLEC, though multi-gene profiling revealed interesting alterations in gene expression in cultured HLEC. Eye bank storage of cultured HLEC may provide a reliable source of tissue for treating limbal stem cell deficiency, although its feasibility for clinical use has to be evaluated further. References 1. Utheim TP, Raeder S, Utheim OA, et al. A novel method for preserving preserving cultured limbal epithelial cells. Br J Ophthalmol Published Online First: 23 November 2006.doi:10.1136/bjo.2006.103218. 2006.doi:10.1136/bjo.2006.103218. 2. Crewe JM, Armitage WJ. Integrity of epithelium and endothelium in in organorgan-cultured human corneas. Investigative Ophthalmology & Visual Science. 2001;42:17572001;42:1757-1761. 3. Komuro A, Hodge DO, Gores GJ, Bourne WM. Cell death during corneal corneal storage at 4 degrees C. Invest Ophthalmol Vis Sci. 1999;40:28271999;40:2827-2832.