Abstract
PurposeTo evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC).MethodsLimbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67.ResultsMorphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM.ConclusionLimbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67.
Highlights
IntroductionCorneal epithelium is renewed by stem cells (SC) located in the basal layer of the limbal epithelium (LE) in a special supporting microenvironment known as the limbal SC niche
Limbal cells cultivated on amniotic membrane (AM) exhibited a lower expression profile of limbal mesenchymal (LMSC) and CXCR4 markers as limbal cells cultivated on plastic culture plates
Corneal epithelium is renewed by stem cells (SC) located in the basal layer of the limbal epithelium (LE) in a special supporting microenvironment known as the limbal SC niche
Summary
Corneal epithelium is renewed by stem cells (SC) located in the basal layer of the limbal epithelium (LE) in a special supporting microenvironment known as the limbal SC niche. The niche plays an important role in the maintenance of limbal epithelial SC (LESC) properties and is tightly regulated by factors from the surrounding tissue [1]. When the limbal SC containing niche is partially or totally damaged, a blinding and painful disease of limbal stem cell deficiency (LSCD) ensues [2]. Transplantation of LESCs is necessary to restore vision [3,4]. In 1997, Pellegrini and colleagues first described transplantation of ex vivo expanded—cultured LE sheets containing LESCs (Cultivated Limbal Epihelial Transplanation) from a small amount of limbal tissue biopsy [5,6]. A variety of culturing techniques have been developed to optimise and standardise the ex vivo expansion of LE sheets on appropriate carrier substrates [6]
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