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Aboulghassem Shahdadfar, Kristiane Haug, Ole K. Olstad, Eli W. Gulliksen, Liv Drolsum, Bjørn Nicolaissen; Expansion of Human Corneal Limbal Epithelial Tissue (HCLET) for Tissue Engineering Purposes : The Effect of Substrate on Expression of Factors Associated with Stemness and Proliferation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5132.
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© ARVO (1962-2015); The Authors (2016-present)
Protocols using human amniotic membrane (HAM) and plastic (1, 2) as substrates have been extensively used. As the presence of stem/progenitor characteristics ex vivo reportedly depends on culture conditions including feeder cells (3), we cultured HCLET on HAM or plastic inserts. Our focus was on proliferation and maintenance of stem/progenitor cell marker genes in these two culture conditions.
HCLET was cultured in two steps, in first step either on HAM or on plastic inserts in complex medium, CM (DMEM/F12 supplemented with 5% FBS, 0.5% DMSO, 2 ng/ ml human EGF, 5 µg/ ml insulin, 5 µg/ ml transferrin, 5 ng/ ml selenium, 3 ng/ ml hydrocortisone, 30 ng/ ml cholera toxin, 50 µg/ ml gentamicin and 1.25 µg/ ml amphotericin B) for 3 weeks and in second step, expanded cells in each condition were cross-cultured to opposite condition for more 3 weeks. All cultures were incubated at 37º C with 5% humidified CO2 and medium was changed every 2-3 days. Samples were harvested for microarray, qRT-PCR, immunohistochemistry and EM analysis.
In the first step, 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, and OCLN by qRT-PCR confirms the microarray related genes which are equally expressed between these two conditions, but KI 67 gene is significantly up regulated and KRT4 gene is down regulated in cells cultured on plastic inserts. In second step, where expanded cells on plastic insert with higer proliferation capacitiy cross-cultured on HAM, the expression of Ki 67 gene keeps constantly up regulated.
Our data show that culture of HCLET on plastic inserts compatibly maintain stemness characteristics and keeps its higher proliferation capacity although cross-cultured on HAM.
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