Induction of Differentiation by Pyruvate and DMEM in the Human Retinal Pigment Epithelium Cell Line ARPE-19
Induction of Differentiation by Pyruvate and DMEM in the Human Retinal Pigment Epithelium Cell Line ARPE-19
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DOI：
10.1167/iovs.10-6374
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发表时间：
2011-09-01
影响因子：
4.4
通讯作者：
Coffey, Peter J.
中科院分区：
文献类型：
--
作者：
Ahmado, Ahmad;Carr, Amanda-Jayne;Coffey, Peter J.
目的.培养的视网膜色素上皮细胞（RPE）可能成为视网膜疾病移植的治疗选择。然而，在体外维持天然RPE表型已被证明具有挑战性。人RPE细胞系ARPE-19被广泛用作原代RPE的替代物。它在DMEM/F12培养基中生长作为标准，但其表型取决于培养条件，并且通常不存在许多分化标记。本研究的目的是检查ARPE-19的这种敏感表型如何通过含有或不含有代谢产物丙酮酸的生长培养基来调节，以阐明更好的RPE生长条件。将p22至p28代的ARPE-19细胞在过滤器上在含或不含丙酮酸盐和1%胎牛血清的DMEM/F12或DMEM培养基中培养长达3个月。采用色素沉着、免疫细胞化学、蛋白/mRNA表达、跨上皮阻力、VEGF分泌和超微结构评估分化。丙酮酸与DMEM组合诱导深色色素沉着并促进分化标记物如CRALBP和MerTK。重要的是，RPE 65蛋白通过蛋白质印迹法检测，并通过丙酮酸盐、高糖和DMEM增强。ARPE-19细胞在此培养基中也能吞噬人光感受器外节（POS）。VEGF分泌在DMEM培养物中更大，受葡萄糖的影响，但不受丙酮酸的影响。DMEM/F12无色素沉着。这项研究证明了重要的分化标志物，包括色素沉着和蛋白质印迹的RPE 65蛋白，并显示人POS吞噬ARPE-19文化使用一个简单的分化协议。结果有利于使用高葡萄糖DMEM与丙酮酸盐的未来RPE分化研究。（Invest Ophthalmol维斯科学。2011;52：7148-7159）DOI：10.1167/iovs.10-6374
PURPOSE. Cultured retinal pigment epithelium (RPE) may become a therapeutic option for transplantation in retinal disease. However maintaining a native RPE phenotype in vitro has proven challenging. The human RPE cell-line ARPE-19 is used widely as an alternative to primary RPE. It is grown in DMEM/F12 medium as standard, but its phenotype is dependent on culture conditions, and many differentiation markers are usually absent. The purpose of this study was to examine how this sensitive phenotype of ARPE-19 can be modulated by growth media with or without the metabolite pyruvate to elucidate better RPE growth conditions.METHODS. ARPE-19 cells at passages p22 to p28 were cultured on filters for up to 3 months in DMEM/F12 or DMEM media with or without pyruvate and 1% fetal calf serum. Assessment of differentiation was performed using pigmentation, immunocytochemistry, protein/mRNA expression, transepithelial resistance, VEGF secretion, and ultrastructure.RESULTS. Pyruvate, in combination with DMEM, induced dark pigmentation and promoted differentiation markers such as CRALBP and MerTK. Importantly, RPE65 protein was detected by Western blotting and was enhanced by pyruvate, high glucose, and DMEM. ARPE-19 cells maintained in this medium could also phagocytose human photoreceptor outer segments (POS). VEGF secretion was greater in DMEM cultures and was affected by glucose but not by pyruvate. Pigmentation never occurred in DMEM/F12.CONCLUSIONS. This study demonstrated important differentiation markers, including pigmentation and Western blots of RPE65 protein, and showed human POS phagocytosis in ARPE-19 cultures using a simple differentiation protocol. The results favor the use of high-glucose DMEM with pyruvate for future RPE differentiation studies. (Invest Ophthalmol Vis Sci. 2011;52:7148-7159) DOI:10.1167/iovs.10-6374