Cornea Differentially Expressed Genes Associated with Human Limbal Epithelial Phenotypes: New Molecules That Potentially Facilitate Selection of Stem Cell-Enriched Populations Lili Taka´cs,1 Eniko˝To´th,1,2 Gergely Losonczy,1 Attila Szanto,3 Tomi Ba¨hr-Ivacevic,4 Vladimir Benes,4 Andra´s Berta,1 and Gyo¨rgy Vereb2 PURPOSE. The aim of this study was to identify differentially to limbal stem cell deficiency (LSCD), conjunctivalization of expressed genes in the human limbal epithelium by microarray the corneal surface, and severe visual loss. In recent years, analysis. transplantation of cultured limbal epithelial stem cells resulted 4,5 METHODS. Total RNA isolates of human limbal and central in a breakthrough in the therapy of LSCD. However, many corneal epithelia were used after transcription for hybridiza- aspects of the ocular surface reestablishment after corneal tion on whole human genome expression microarrays. A set of stem cell transplantation are unclear. A better knowledge of differentially expressed genes detected by both microarrays the molecules expressed by limbal epithelial cells could help was established. In the case of eight selected molecules, mi- our understanding on stem cell functions, increase our com- croarray results were confirmed by qRT-PCR, and protein ex- prehension of the mechanisms involved in ocular surface re- pression in the cornea was examined by confocal immunoflu- generation, and, finally, improve the success rate of limbal orescence microscopy. Colocalization with the putative stem stem cell transplantation.6 cell marker C/EBP␦ was also examined. The spatial separation of limbal stem cells from transient RESULTS. The authors established a database of 126 limbal amplifying and differentiated cells in the central cornea allows 7 overexpressed genes. qRT-PCR confirmed microarray results in for the preparation of a stem cell-containing population. Tran- all examined cases (SPON1, IFITM1, ITM2A, PHLDA1, CXCR4, scriptional profiling of stem cell-enriched populations has FZD7, DCT, DKK4). Limbal localization of the protein product proven to be effective in determining a stem cell signature in of SPON1, IFITM1, ITM2A, CXCR4, and DKK4 was shown with the epidermis.8,9 Recently, microarray analysis of limbal epi- confocal immunofluorescence microscopy. SPON1, IFITM1, thelial cells has led to the identification of several new mole- and ITM2A signals mostly colocalized with C/EBP␦-positive cules that are preferentially expressed in the limbal epithe- putative resting limbal stem cells. lium.10,11 Despite intensive research, a definitive limbal epithelial CONCLUSIONS. By detecting several new differentially expressed genes in the human corneal limbus, this study further expands stem cell marker is still lacking. ABCG2, a xenobiotic trans- current knowledge on the molecular signature of limbal epi- porter molecule, has been associated with stem cells in several 12–14 thelial stem cells. Plasma membrane localization of IFITM1 and tissues, including the limbal epithelium. One isoform of ITM2A suggests their potential usefulness as targets to select the p63 transcription factor, ⌬Np63␣, is specific for activated, 15,16 stem cell-enriched populations from the limbal epithelium. highly proliferative corneal epithelial stem cells. After cor- (Invest Ophthalmol Vis Sci. 2011;52:1252–1260) DOI: neal wounding and activation of limbal stem cells, ⌬Np63␣- 10.1167/iovs.10-5242 positive cells may appear in multiple layers of the limbal epi- thelium and even in the peripheral cornea.16 In a recent study, xperimental evidence and clinical observations indicate Bmi-1, a polycomb complex protein found in several adult that corneal epithelial stem cells reside in the corneal stem cell types, was proposed as a limbal epithelial stem cell E 17 limbus,1 in the specific epithelial structures designated as lim- marker based on mRNA expression analysis. Another study bal palisades of Vogt.2,3 Damage to the limbal tissues may lead showed that CCAAT enhancer binding protein C/EBP␦ and Bmi-1 colocalized with a subpopulation of ⌬Np63␣-positive limbal basal epithelial cells and identified quiescent cells that formed holoclones in culture.18 It is considered now that From the Departments of 1Ophthalmology, 2Biophysics and Cell ⌬ ␣ ␦ 3 Np63 sustains the proliferative potential, whereas C/EBP Biology, and Biochemistry and Molecular Biology, Medical and Health regulates the self-renewal and mitotic rate of limbal stem Science Center, University of Debrecen, Debrecen, Hungary; and 18,19 4EMBL Genecore, Heidelberg, Germany. cells. Supported by Hungarian National Research Fund Grants OTKA The aim of the present study was to explore differentially F046321, K75752, K68616, and TAMOP-4.2.1/B-09/1/KONV-2010- expressed genes in the human limbal epithelium to character- 0007. ize the limbal epithelial phenotype and reveal new molecules Submitted for publication January 20, 2010; revised May 7 and potentially associated with the limbal epithelial stem cells. September 19, 2010; accepted October 10, 2010. Several previously unreported genes were found to be upregu- Disclosure: L. Taka´cs, None; E. To´th, None; G. Losonczy, None; lated in limbal samples, and differential expression of selected A. Szanto, None; T. Ba¨hr-Ivacevic, None; V. Benes, None; A. Berta, molecules was confirmed by qRT-PCR and immunofluores- None; G. Vereb, None ␦ Corresponding author: Lili Taka´cs, Department of Ophthalmol- cence staining. Colocalization with C/EBP was used to exam- ogy, Medical and Health Science Center, University of Debrecen, Nagy- ine the relationship of the newly revealed molecules to puta- erdei krt. 98, Debrecen H-4012, Hungary; [email protected]. tive quiescent limbal epithelial stem cells. Purposefully, several Investigative Ophthalmology & Visual Science, March 2011, Vol. 52, No. 3 1252 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from iovs.arvojournals.org on 09/24/2021 IOVS, March 2011, Vol. 52, No. 3 Differentially Expressed Corneal Limbal Genes 1253 membrane-bound molecules were chosen for validation stud- ferentially expressed genes created using these platforms, it was nec- ies to identify potential selection targets for flow cytometry. essary to uncover targets representing identical genes on the different microarrays. Sequence-based comparison between these two plat- forms was not feasible because oligonucleotide sequence information MATERIALS AND METHODS was available only for the Affymetrix microarray. Available sequence identifiers (GenBank and RefSeq) were used to search Entrez, Ensembl, Corneal Samples and Total RNA Isolation and Swissprot databases, and targets with at least one matching iden- All examinations and sample collections were conducted according to tifier were considered identical. the tenets of the Declaration of Helsinki and followed the guidelines of DEGs identified by both microarrays were further analyzed to point our Institutional Ethics Committee. Corneas of nine eyes enucleated out molecules that are possibly associated with the limbal stem cell for uveal melanoma were used for mRNA isolation. In three cases, phenotype. We looked for DEGs that were absent or marginal in the central and limbal regions of the same cornea were used (these sam- central cornea with at least threefold higher expression in the limbal ples are referred to as matching pairs in the rest of this article). In six epithelium, showing little variation between individual samples (P Ͻ other cases, the central 7-mm corneal button was excised for corneal 0.005). In addition, a PubMed search was performed for each DEG, and transplantation before mRNA isolation; in these cases, only the limbal those involved in stem cell biology or corneal development were region was used. Central corneal epithelia removed during photore- chosen for further studies. Several membrane-bound molecules were fractive keratectomy (PRK) were also collected from 17 patients. From selected to identify possible selection markers. the three whole corneas of enucleated eyes, the central 7-mm corneal button was excised with a trephine. Conjunctival remnants were cut qRT-PCR with scissors from the limbal rims. The epithelium was scraped from Nine limbal and 20 central RNA samples were reverse transcribed with the cornea and the limbus was scraped with a hockey knife under an random hexamers (cDNA High Capacity Archive Kit; Applied Biosys- operating microscope. To avoid expression changes during tissue tems, Foster City, CA). cDNAs were analyzed with quantitative real- culture or mRNA damage because of repeated freeze-thaw cycles, all time PCR using verified assays (TaqMan; Applied Biosystems) for the epithelial samples were scraped and placed into lysis buffer (Qiagen, individual genes and master mix (TaqMan; Applied Biosystems). All Valencia, CA) complemented with ␤-mercaptoethanol within 2 hours samples were analyzed in triplicates on a real-time PCR machine after enucleation or immediately after removal during PRK. Total RNA (ABI7900; Applied Biosystems). Template-free and reverse transcripta- was then isolated from the samples using silica-membrane columns se–free controls were used to determine nonspecific signals. Ct values (RNeasy Mini Kit; Qiagen) according to the manufacturer’s instruc- for each well were determined (Sequence Detection Software; Applied tions. RNA quality and quantity were checked (Bioanalyzer Nano Ϫ Biosystems), and the expression value was calculated as [238.5 Ct(gene)]. Chips; Agilent Technologies, Santa Clara, CA). The six