The influence of ageing and culture conditions on limbal epithelial cells

Abstract

Introduction. The use of limbal stem cells as a cellular therapy has expanded over the last decade. Currently, the success rate of limbal stem cell transplantations is around 76% with advanced donor age being a possible reason for this figure. Medium and oxygen concentration could also have a detrimental effect on the growth of these cells for transplantation. With age the limbus undergoes a topological change which could alter the stem cells surroundings. A mouse model of corneal ageing was also used to complement the human study and avoid culturing and tissue storage artefacts. Aims. The main aim of this thesis was to assess the effect of age on corneal epithelial progenitor cells. This then led to the question; is the mouse cornea affected by age and can interventions mediate these outcomes? And finally do culture conditions affect the growth of corneal epithelial stem cell containing cultures, and how does this relate to changes to the niche with age? Methods. Limbal derived progenitor cells were extracted from human corneosclearal disks donated for research. Cells were cultured either in LEM or DKSFM and either in hypoxia (3%) or normoxia (21%). Immunofluorescence, qPCR, TRAP assay, morphological and clonal analysis where used to assess progenitor composition. The mice used were strain B6.129S-Tert, tm1Yjc/J. Old/AL and DR mice were 22 months old, young were 3 months old. Rapamycin treated mice were 16 months old and were treated for four months, starting at 12 months of age. Dietary restriction was implemented for a period of 16 months from 6 months of age, additionally in the mouse eye a DNA damage marker was also assessed, both telomeric and non-telomericly. Results and Conclusions. Age has a detrimental effect on the ability to culture a limbally derived clonal population. However age did not affect the levels of positive and negative gene expression markers or ΔNp63 protein level. Interestingly the time that corneal tissue is stored for did not affect the ability to isolate these progenitor cells. The growth of cells in hypoxia decreased negative marker KRT3 and senescence marker p21 regardless of culture medium. In the mouse model, age had a detrimental effect on the cornea with decreased TAp63, increased ɣH2A.X and TAFs. DR however, tended to have a beneficial effect on the mouse cornea. Interestingly rapamycin seems to be detrimental to the mouse cornea, with similarities with the effects of age.