Age-related macular degeneration (AMD) is one of the most common causes of blindness, and results from damage to the macula at the back of the eye. The adult retinal pigmented epithelial-19 (ARPE-19) cell line is currently being used to assist in developing therapies for AMD as these cells display many ‘RPE-like’ characteristics, including a pigmentation-specific gene expression profile similar to that observed in primary human RPE cells. Generation of these differentiated phenotypes require extended periods of culture, but specific culture conditions can result in more rapid cell maturation compared with those in standard culture media.
To characterise the molecular events associated with maturation, ARPE-19 cells were cultured in different media (DMEM-based media or X-VIVOTM 10), and serial sampling of cell material and extracellular culture media was conducted over two time-course experiments at regular intervals. Metabolomics (1H quantitative NMR spectroscopy), transcriptomics (RNA-Seq) and proteomics (LC-MS/MS) were used to generate complementary omics data for collected materials. Significant differences between conditions over the experimental culture time course were derived and included in pathway analysis.
Visual pigmentation was observed from week three with cells cultured in X-VIVOTM 10 based media compared to those cultured in DMEM-based media. The rate of change of 24 media metabolites by 1H NMR spectroscopy was evaluated and revealed the rate of tyrosine uptake was higher with cells maturing in X-VIVOTM 10 media, consistent with melanogenesis. RNA-Seq analysis showed significant transcriptional changes in the X-VIVOTM 10 cell system including upregulation of key pathways: Wnt and ERK/ MAPK signalling cascade; transcription factor MITF; tyrosine-metabolism pathway genes melanogenic enzymes tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase.
This study provides a novel complementary omic approach in altered melanogenesis/pigmentation pathways associated with ARPE-19 cell maturation and demonstrates the potential use of non-invasive 1H NMR quantitative profiling methods in the monitoring of cell maturation/differentiation in vitro using extracellular media.