Abstract
Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.
Highlights
Cornea epithelial regeneration is essential for maintaining its transparency and normal vision
The epithelial differentiation marker, CK3, which has been reportedly negative in limbal crypts in vivo [1], revealed similar lack of expression in our limbal explants and the outgrowing cells, consistent with the findings by others that, in culture, either the limbal epithelial stem cells (LESCs) or the side population expresses CK3, which is gradually lost over time [12]
Cadaveric limbal samples can have superficial CK3 expression, but not in the basal layers [3]. These results demonstrate that either a CK3 negative population is selected by the depletion of differentiated epithelial cells or differentiated cells revert and lose this marker
Summary
Cornea epithelial regeneration is essential for maintaining its transparency and normal vision. The LESCs possess self-renewal capacity, being able to regenerate the whole corneal epithelium within 12–24 hours time [3]. Loss of LESCs and/or function due to disease or injury can result in impaired corneal function, neovascularization, conjunctival. Long-Term 3D Cultures of Human Cornea Limbal Explants ingrowth and loss of vision. LESC deficiency (LESCD) [4]—partial or total, can be treated by restoring the limbal area using biopsies from the patient’s healthy eye or transplanting LESCs harvested from autologous or cadaver donor tissue, cultured and expanded ex vivo [5, 6]
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