Laura E Sidney1, Matthew J Branch2, Harminder S Dua2, Andrew Hopkinson2. 1. Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham, Nottingham, United Kingdom. Electronic address: laura.sidney@nottingham.ac.uk. 2. Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham, Nottingham, United Kingdom.
Abstract
BACKGROUND AIMS: 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. METHODS: Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. RESULTS: All media supported proliferation of CSSCs 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 stromal 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. CONCLUSIONS: Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy.
BACKGROUND AIMS: 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. METHODS: Primary humancorneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. RESULTS: All media supported proliferation of CSSCs 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 stromal 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. CONCLUSIONS: Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy.
Authors: Elisabetta Prina; Pritesh Mistry; Laura E Sidney; Jing Yang; Ricky D Wildman; Marina Bertolin; Claudia Breda; Barbara Ferrari; Vanessa Barbaro; Andrew Hopkinson; Harminder S Dua; Stefano Ferrari; Felicity R A J Rose Journal: Stem Cell Rev Rep Date: 2017-06 Impact factor: 5.739
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Authors: Mariana Lizeth Orozco Morales; Nagi M Marsit; Owen D McIntosh; Andrew Hopkinson; Laura E Sidney Journal: World J Stem Cells Date: 2019-02-26 Impact factor: 5.326
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Authors: Rosemond A Mensah; Seung Bin Jo; Hoon Kim; Sung-Min Park; Kapil D Patel; Kyong J Cho; Michael T Cook; Stewart B Kirton; Victoria Hutter; Laura E Sidney; Decio Alves-Lima; Hungyen Lin; Jung-Hwan Lee; Hae-Won Kim; David Ys Chau Journal: J Biomater Appl Date: 2021-06-18 Impact factor: 2.646