Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells

摘要

Background: The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype.ududMethods: Primary human corneal stroma-derived stem cells (CSSC) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukaemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult™; serum-free keratinocyte medium (K-SFM); and StemPro®-34. Effect on proliferation, morphology, protein and mRNA expression were evaluated.ududResults: All media supported proliferation of CSSC with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stem cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6.ud udDiscussion: Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we considerate to be the most appropriate for development as a clinical grade medium for the production of CSSC phenotypes suitable for cell therapy.