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Influence of Sex on Gene Expression in Human Corneal Epithelial Cells

ABSTRACT: Objectives. Sex-associated differences have been identified in the anatomy, physiology and pathophysiology of the human cornea. We hypothesize that many of these differences are due to fundamental variations in gene expression. Our objective in this study was to determine whether such differences do exist in human corneal epithelial cells both in vivo and in vitro. Methods. Human corneal epithelial cells were isolated from the corneoscleral rims of male and female donors. Cells were processed either directly for RNA extraction, or first cultured in phenol red-free keratinocyte serum free media. The RNA samples were examined for differentially expressed mRNAs by using of CodeLink Bioarrays and Affymetrix GeneChips. Data were analyzed with GeneSifter.Net software. Results. Our results demonstrate that sex significantly influences the expression of over 400 genes in human corneal epithelial cells in vivo. These genes are involved a broad spectrum of biological processes, molecular functions and cellular components, such as metabolic processes, DNA replication, cell migration, RNA binding, oxidoreductase activity and nucleoli. We also identified significant, sex-related effects on gene expression in human corneal epithelial cells in vitro. However, with few exceptions (e.g. X- and Y-linked genes), these sex-related differences in gene expression in vitro were typically different than those in vivo. Conclusions. Our findings support our hypothesis that sex-related differences exist in the gene expression of human corneal epithelial cells. Keywords: Female vesus Male.

ORGANISM(S): Homo sapiens

PROVIDER: GSE14621 | GEO | 2009/11/13

SECONDARY ACCESSION(S): PRJNA112269

REPOSITORIES: GEO

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Project description:Corneal epithelial cells derived from hPSCs provides an important cells source for the construction of the in vitro preclinical models aimed for ophthalmic drugs tests. However, the recent differentiation protocols lack the optimal culturing conditions that hinder the robustness and the quality of cells as well as the scale-up application of cells production. Here we introduce a simplified, yet efficient small molecules-based corneal induction method (SSM-CI) for the generation of corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimization the cells culturing time and steps using only two defined xenobiotic-free and serum-free culturing mediums in combination with the TGFß pathway, Wnt/ß-catenin pathway signaling chemical inhibitors, and human bFGF growth factor for a period of 25 days. Compared to both conventional human corneal epithelial cell line (HCE-T) as well as the human primary corneal epithelial cells (hPCEpC), human embryonic stem cells derived corneal epithelial cells generated by SSM-CI has highly expressed major differentiation as well as maturation markers such as PAX6 and CK12. RNA-seq analysis indicated the genuine diversion of hPSCs into the corneal epithelium lineage where corneal progenitor and adult corneal epithelial phenotypes were significantly upregulated. Furthermore, despite the inhibition of TGF-β and Wnt/β-catenin at the early stage of differentiation, an upregulation of the TGF-β and Wnt/β-catenin pathways related transcripts we noticed in the late stage which indicated the necessity of these pathways in the generation of mature corneal epithelial cells. Moreover, there was a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells where a decrease of glycolysis related transcripts and an increase in fatty acid oxidation related one was noticed. That was also corresponded by the overexpression of metabolic enzymes and transporters related transcripts that were mainly responsible for the metabolism of fatty acids. Thus SSM-CI provide a comprehensive method for the generation of functional corneal epithelial cells that has the potential for the employment in future preclinical models.

Project description:Purpose. Simian virus 40 (SV40)-immortalized human corneal epithelial (HCE-T) cells have been widely used as an in vitro model of human corneal epithelial cells. To better understand the nature of this cell line, we assessed it for genomic aberrations and cellular heterogeneity. Methods. For the quantitative measurement of genomic aberrations, array based comparative genomic hybridization (CGH) analysis was performed. For identification of cellular heterogeneity, cell morphology, growth kinetics, trans-epithelial electrical resistance and transcriptional efficiency were analyzed. We also carried out real-time PCR and chromosomal fluorescent in situ hybridization (cFISH) against some gained or lost loci in order to assess genomic heterogeneity. To assess differences in the gene expression profiles between HCE-T cells and normal corneal epithelial cells, we collected expressed sequence tags (ESTs) for this cell line. Southern blotting and inverse PCR analyses were used to determine the genomic integration site of the SV40 large T antigen gene (LTAG). Results. Array CGH analysis indicated that the genomic content of HCE-T cells is different from the normal healthy genome. Our results from cellular functional assays, real-time PCR and cFISH strongly showed that HCE-T cells consist of a not insignificant number of heterogeneous cell populations. The genomic integration site of the SV40 large T antigen was at p22.1 of chromosome 9. Conclusions. Our results indicate that HCE-T cells have an altered genomic content and that they are composed of heterogeneous cell populations. This should be considered when conducting experiments or interpreting the results of studies which use this cell line. Keywords: comparative genomic hybridization Genomic DNAs from HCE-T cells, immortalized cell line of human corneal epithelial cells and normal female duploid cells were subjected to array CGH analysis to identify genomic alterations in the HCE-T cells. These DNAs were labeled with Cy5 and Cy3, respectively.

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Influence of Sex on Gene Expression in Human Corneal Epithelial Cells

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