Human corneal epithelial subpopulations: oxygen dependent ex vivo expansion and transcriptional profiling

Abstract

Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defective or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity. Since the first successful treatment of LSCD by transplantation of ex vivo expanded LESCs in 1997, many attempts have been carried out to optimize culture conditions to improve the outcome of surgery. To date, progress in this field of bioengineering is substantially hindered by both the lack of specific biomarkers of LESCs and the lack of a precise molecular characterization of in situ epithelial subpopulations. The aim of this dissertation was to optimize culture systems with regard to the environmental oxygen concentration for selective ex vivo expansion of LESCs and to analyse in situ subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum-free EpiLife medium, using a range of physiologically relevant oxygen concentrations (2%, 5%, 10%, 15% and 20%). Using immunocytochemistry and advanced fluorescence microscopy, cells were characterized regarding growth, cell cycle distribution, colony-forming efficiency (CFE), phenotypes and cytomorphometry. Limbal epithelial cells expanded in 2% O2 exhibited slow growth, low fraction of cells in S/G2 , high CFE, high expression of stem cell markers ABCG2 and p63α, and low fraction of differentiation marker CK3 resembling a LESC phenotype. The effect of hypoxia to maintain LESCs in culture was not dependent on the system used for propagation (Bath et al. 2013a). Laser capture microdissection was used to isolate cellular subpopulations in situ from the spatially defined differentiation pathway in human corneal epithelium according to an optimized protocol for maintenance of expression profiles. Isolated total RNA from basal limbal crypts (BLCs), superficial limbal crypts (SLCs), paracentral/central cornea and limbal stroma was amplified and converted to fragmented cDNA libraries for use in deep paired-end next-generation sequencing. Global transcriptional profiling was carried out using bioinformatics. The location of primitive cells in BLCs, migratory and activated cells in SLCs and differentiated cells in paracentral/central cornea was evident from mapping of significantly upregulated genes in each compartment to the gene ontology (GO). Interestingly, many GO terms in BLCs were also involved in neurogenic processes, whereas many GO terms in SLCs were related to vasculature. Mapping upregulated genes in BLCs to pathway annotations in Kyoto Encyclopedia of Genes and Genomes described many active pathways as signalling and cancer-associated pathways. We supply extensive information on possible novel biomarkers, reveal insight into both active pathways and novel regulators of LESCs such as Lrig1 and SOX9 and provide an immense amount of data for future exploration (Bath et al. 2013b). Selective ex vivo expansion of LESCs in hypoxia and the comprehensive molecular characterization of corneal epithelial subpopulations in situ are expected to be beneficial for the future treatment of LSCD by cultured limbal epithelial transplantation.