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A. Shahdadfar, K. Haug, O. K. Olstad, E. W. Gulliksen, M. C. Moe, L. Drolsum, B. Nicolaissen; Expansion of Human Corneal Limbal Epithelial Tissue (HCLETs) for Transplant Purposes: The Effect of Substrate and Medium on Expression of Factors Associated With Stemness. Invest. Ophthalmol. Vis. Sci. 2010;51(13):731.
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© ARVO (1962-2015); The Authors (2016-present)
Protocols using human amniotic membrane (HAM) and plastic (Lancet 1997;349, Transplantation 2004;77) as substrates, and culture medium containing FBS and complex mixtures of additives have been extensively used. As the presence of stem/progenitor characteristics ex vivo reportedly depends on culture conditions includning feeder cells (Indian J Med Res. 2008;128), we cultured HCLET on HAM or plastic inserts and separately on HAM in 2 different culture media. Our focus was on maintenance of stem/progenitor cell marker genes.
HCLET was cultured (n=3) either on HAM or on plastic inserts in DMEM/F12 with supplements, complex medium, CM (Stem Cells 2009;27). In another experiment, HCLET was cultured (n=3) on HAM in either CM, or DMEMF12 with 10% FBS (MFBS) containing antibiotics. All cultures were incubated for 3 weeks. Medium was changed every 2-3 days. Samples were harvested for microarray and qRT-PCR analysis.
Microarray and qRT-PCR analysis of cultured HCLET on either HAM or plastic inserts shows that only 46 genes are more than 2 fold differentially expressed. mRNA analysis of genes associated with cell stemness such as ABCG2, P63, OCT4, SOX2, KRT3, KERA, CX43, and OCLN by qRT-PCR confirms the microarray related genes which are equally expressed between these two conditions, but KRT4 gene is significantly down regulated in cells cultured on plastic inserts. HCLET cultured on HAM and in either CM, or MFBS showed that 375 genes are more than 2 fold differentially expressed between these two media. mRNA analysis of all above mentioned genes by qRT-PCR confirms the microarray related genes which are equally expressed.
Our preliminary data shows that plastic inserts as substrate, and culture medium containing 10% FBS compatibly maintain stemness characteristics of expanding HCLET cells.
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