Abstract
Purpose:
Retinal pigment epithelium (RPE) is essential for a variety of processes in the outer retina including nourishing photoreceptors, regeneration of visual pigments, phagocytosis/degradation of outer segments, and absorption of excess incoming light. Maintaining these processes is difficult under culture conditions. However, using optimized conditions, the human RPE cell line ARPE-19 can acquire epithelial morphology and express mRNA for visual cycle genes RPE65 and RDH5 after 8 weeks in culture. We wished to examine the protein and gene expression of secreted proteins in differentiated ARPE-19.
Methods:
ARPE-19 cells cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose, 1% FBS, 1 mM sodium pyruvate, and antibiotics and grown for either 4 days or 4 months were incubated with serum-free media for 16 hours and the conditioned media (CM) collected. CM was qualitatively and quantitatively assessed for secreted protein by mass spectrometry (MS) and ELISA, respectively. CM was also analyzed by immunoblots probed for RPE specific proteins. Gene expression in ARPE-19 cells was assessed by RT-PCR.
Results:
ARPE-19 cells cultured in DMEM with pyruvate for 4 months exhibited the classic native RPE phenotype. MALDI TOF/TOF MS was used to analyze the secreted proteins from ARPE-19 cells grown in culture for 4 days or 4 months. Proteins known to be abundantly expressed in RPE were detected in CM from 4-month cultures. However, they were either absent or negligible from 4-day cultures. Cystatin C is among the most highly expressed proteins in native RPE and was detected in the medium from 4-month cultures at a concentration of 218 ± 5 ng/ml. Cystatin C mRNA expression in ARPE-19 cells grown 4 months is about 58-fold greater than in 4-day cells. PEDF was also found to be abundantly secreted by 4-month cultures, but not by 4-day cultures.
Conclusions:
ARPE-19 cells exhibit native RPE characteristics after 4 months in culture and express genes/proteins preferentially expressed in RPE. We identified several proteins known to be abundantly expressed by native RPE, in particular, cystatin C and PEDF. MS and immunoblot analysis shows that cystatin C and PEDF are robustly expressed and secreted into the medium by ARPE-19 cells grown for 4 months; however, none was detected in the 4-day cultures. Our results further establish that ARPE-19 can be differentiated to a native-like phenotype using optimized conditions.