Research reportCharacterisation and functional features of a spontaneously immortalised human corneal epithelial cell line with progenitor-like characteristics
Introduction
The cornea is the transparent front part of the eye that covers the pupil, iris, and the anterior chamber. Together with the lens, the cornea refracts light, and aids the eye to focus. The corneal epithelium is a rapidly regenerating squamous epithelium. During normal epithelial turnover as well as following an injury, the maintenance of the corneal epithelial cell mass is attained by a population of unipotent limbal epithelial stem cells (LESC) believed to exist in the basal epithelium of the vascularised natural border between cornea and conjunctiva, namely the limbus [24], [8], [5], [26]. Primary cultures of limbal and corneal epithelial cells can often be maintained only for a few passages due to the rapidly differentiating nature of the cells. Therefore, immortalized cell lines are a readily available source of corneal epithelial cells and can provide a useful research tool for long term observation.
Current cell lines have either been purposefully or spontaneously transformed. Examples of cell lines established by the incorporation of oncogenes into their genome have been popular for in vitro models for corneal epithelial studies [11], [1]. Telomerase immortalized cell lines can overcome replicative senescence induced by telomere length reduction by the introduction of the human telomerase reverse transcriptase subunit (hTERT) which would facilitate the stability of telomere length and therefore increase the number of cell cycles [21]. The establishment of a spontaneously transformed cell line has been successful for rabbit corneal epithelial cells [3] whereas there are limited reports on spontaneously derived human corneal epithelial cell lines [16], [17].
In this study, the spontaneous generation of a corneal epithelial cell line, which emerged from a single primary limbal fibroblast cell culture, is described. This unusual phenomenon of a dormant corneal epithelial population emerging from a fibroblastic population after five culture passages has not been previously reported. The cell line, namely HCE-S, after being clonally isolated and passaged until reaching over 100 cell duplications, was characterized. Functional features of HCE-S such as its EGF response, have also been investigated.
Section snippets
Isolation and culture of human limbal fibroblasts
After removing the epithelium for culture, as described before [18], [19], the scleral and corneal part of the de-epithelialised stroma were cut off and were dissected in to fragments of approximately 2 mm × 2 mm. These pieces were then placed onto the surface of a 25 cm2 flask (three per flask). The tissue was allowed to dry in a class II hood for about 15 min to aid adherence of the explants on to the tissue culture plastic. Then, 5 ml of DMEM (Invitrogen) plus 10% FBS and 1% penicillin–streptomycin
Metabolic activity
For investigation of the response of HCE-S metabolic activity as an indicator of proliferation in response to an EGF concentration gradient, cells were plated at a seeding density of 8 × 103 cells into 96-well plates. The cells were left to adhere overnight and then they were serum starved for 24 h using DMEM containing antibiotics and 0.1% Bovine Serum Albumin from Sigma (baseline media). After serum starvation, the cells were exposed to the six treatment groups: DMEM containing antibiotics and
HCE-S cells emerging from HLF cultures.
The human limbal fibroblast (HLF) primary cultures derived from cadaveric tissue were maintained as described and passaged regularly. The cells exhibited normal fibroblastic bipolar morphology (Fig. 1a). After passage 5, colonies of epithelial-like cells were observed in the cultures (Fig. 1b, indicated by arrow), which were persistent and grew in size with every passage (Fig. 1b and c, indicated by arrows), gradually outcompeting the HLFs which reached senescence around passage 10. The
Discussion
This study describes the characterisation of a transformed human corneal epithelial cell line, namely HCE-S, which was derived spontaneously from a single primary human limbal fibroblast culture. This cell population appeared in distinct colonies in several of the flasks of human limbal fibroblast cultures probably after being carried between passages in a non-proliferative state in the simple fibroblast medium, or by low contamination with limbal epithelial progenitor cells which had the
Conflict of interest
The authors declare that they have no competing financial interests.
Funding
MRC (MN), NIHR BMRC for Ophthalmology, Moorfields Eye Hospital & the UCL Institute of Ophthalmology (JTD)
Acknowledgments
The authors gratefully acknowledge the Medical Research Council (MRC) and also the Special Trustees of Moorfields Eye Hospital for their support and funding. The National Institute for Health Research Biomedical Research Centre (NIHR BMRC) for Ophthalmology, Moorfields Eye Hospital & the UCL Institute of Ophthalmology is also acknowledged for part-funding.
References (27)
Corneal epithelial cell cultures as a tool for research, drug screening and testing
Experimental Eye Research
(2008)Partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type IV adhesiveness
Experimental Eye Research
(2005)Differential expression of matrix metalloproteinases 2 and 9 by glial Muller cells – response to soluble and extracellular matrix-bound tumor necrosis factor-alpha
American Journal of Pathology
(2002)Establishment of a corneal epithelial cell line spontaneously derived from human limbal cells
Experimental Eye Research
(2007)TGF beta stimulated re-epithelialisation is regulated by CTGF and Ras/MEK/ERK signalling
Experimental Cell Research
(2008)- et al.
The corneal epithelial stem cell niche
Ocular Surface
(2005) An Sv40-immortalized human corneal epithelial-cell line and its characterization
Investigative Ophthalmology & Visual Science
(1995)- et al.
Three clonal types of keratinocyte with different capacities for multiplication
Development of a spontaneous permanent cell-line of rabbit corneal epithelial-cells that undergoes sequential stages of differentiation in cell-culture
Journal of Cell Science
(1994)Corneal stem cells in review
Wound Repair and Regeneration
(2001)