Notara, M; Haddow, DB; MacNeilss, S; Daniels, JT; (2007) A xenobiotic-free culture system for human limbal epithelial stem cells. REGEN MED , 2 (6) 919 - 927. 10.2217/17460722.214.171.1249.
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Aims: Murine 3T3 feeder cells are commonly used for stem cell expansion. Although 'feeder-free' systems are being developed for a variety of stem cells including embryonic, the use of feeder cells currently remains optimal for the expansion of epithelial stem cells. In this study, MRC-5, a human embryonic fibroblast cell line, has been investigated for its potential use as a feeder layer in human limbal epithelial (HLE) cell expansion under serum-free conditions, with the aim of developing a xenobiotic-free culture system for therapeutic corneal regeneration applications. Materials and Methods: MRC-5 feeder cells were compared with J2 3T3 mouse fibroblasts, in both serum-supplemented and serum-free conditions, in terms of their relative ability to support HLE cell metabolic activity, expression of the putative stem cell markers ABCG2 and P63 alpha, cell differentiation using the cornea-specific cytokeratin 3 antibody and colony-forming efficiency. Results: The proportion of HLE stem cells maintained was determined by functional colony-forming efficiency assays. The metabolic activity results showed that HLE cells cultured on MRC-5 fibroblasts under serum-free conditions proliferated as well as cells cultured on J2 cells under serum-free conditions. Moreover the HLE cultured on MRC-5 fibroblasts under serum-free conditions expressed high levels of putative stem cell markers ABCG2 and P63 alpha and low levels of the differentiation marker CK3, indicating that they retained poorly differentiated 'stem cell-like' characteristics under those culture conditions. Clonal analysis of HLE cells cultured on growth-arrested feeder layers of J2 and MRC-5 fibroblasts showed that cells expanded on MRC-5 and J2 fibroblasts in serum-free conditions had a colony-forming efficiency of approximately 1.5%, indicating the maintenance of stem cells. Conclusions: These results demonstrate feasibility of expanding HLE cells for clinical purposes by using a human fibroblast cell line as a feeder layer, avoiding the use of bovine serum, while preserving the proliferative potential and stem cell characteristics of HLE cells.
|Title:||A xenobiotic-free culture system for human limbal epithelial stem cells|
|Keywords:||cell transplantation, feeder layer, human fibroblasts, limbal epithelium, stem cells, xenobiotic system, HUMAN OCULAR SURFACE, CORNEAL EPITHELIUM, CLONAL GROWTH, POPULATION, MRC-5, DIFFERENTIATION, KERATINOCYTES, EXPRESSION, ABCG2, NICHE|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Genetics, Evolution and Environment|
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