Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications

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.

Cultivation and characterization of pterygium as an ex vivo study model for disease and therapy

“…The pterygium graft-containing cultures showed 2.5 times less positivity for CXCR4 compared to multi-layered outgrowing cell cultures. Vimentin, a crucial cytoskeletal mediator for the repair of cellular function during woundhealing from lens epithelial cells [26] and in LESCs cultivated in longterm cultures [18], was found highly expressed by the pterygium-graft containing and the stratified outgrowing cells. Similarly, ΔNp63 was expressed at a steady and similar extent in both cell cultures, the protein being known for its role in activation or inhibition of apoptosis in a cell-and tissue-type specific manner.…”

“…using viscoelastic material), therefore, providing both cell types an equal chance to expand, without the use of any growth factors. The outgrowing cells expressed CK8-18 −a typical epithelial cell marker also expressed by other human corneal cells such as epithelial and endothelial cells [15,16], as well as the LESC marker CK19 [17,18].…”

Limbal epithelial stem cells in regeneration of corneal epithelium – clinical and molecular aspects

“…Potwierdzono natomiast, że komórki warstwy epitelialnej rogówki wykazują ekspresję cytokeratyny 3 (CK3) oraz cytokeratyny 12 (CK12) [26,27,28,29]. Nie stwierdzono obecności tych białek w komórkach epitelialnych pochodzących z rąbka rogówki [23,30,31]. Co więcej, markerami negatywnymi dla rąbko-wych komórek macierzystych oprócz wymienionych cytokeratyn mogą być: nestyna, E-kadheryna, koneksyna 43 i inwolukryna [16,23].…”

Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue

“…Limbal stem cell deficiency may cause recurrent corneal epithelial erosions, inflammatory reactions and ingrowth of fibro-vascular tissue onto the corneal surface. Removal of the diseased tissue and transplantation of ex vivo engineered epithelium may restore the integrity of the corneal surface (Pellegrini et al 1997;Nakamura et al 2004;Sangwan et al 2011;Pathak et al 2013;Romero et al 2014;Zakaria et al 2014;Ljubimov & Saghizadeh 2015;Szab o et al 2015;Etxebarria et al 2017;Liu et al 2017). Removal of the diseased tissue and transplantation of ex vivo engineered epithelium may restore the integrity of the corneal surface (Pellegrini et al 1997;Nakamura et al 2004;Sangwan et al 2011;Pathak et al 2013;Romero et al 2014;Zakaria et al 2014;Ljubimov & Saghizadeh 2015;Szab o et al 2015;Etxebarria et al 2017;Liu et al 2017).…”