Figure 1.

Maturation of RPE in vitro is associated with increased mitochondrial mass. (A) Transepithelial resistance of ARPE-19 matured on Transwells for up to 4 weeks (n = 3 for each time point). (B) Gene expression of RPE-specific genes measured by qPCR during ARPE-19 maturation (n = 3 for each time point). (C) MitoTracker CMTMRos staining demonstrates increased mitochondrial network with maturation time. Images were acquired with variable exposure times using a ×40 objective. Scale bar: 20 μm. (D) Quantification of image area covered by MitoTracker fluorescence as a measure of mitochondrial network size. Area covered is significantly greater at 4 weeks compared with 70% confluent and 1 week confluent (n = 9 for 70% and 1 week and n = 6 for 4 weeks). (E) MitoTracker staining images acquired with identical exposure times using a ×20 objective demonstrates increased fluorescence at longer maturation times. Values shown indicate MFI per microgram of protein. Scale bar: 50 μm. (F) Quantification of MFI per microgram of protein and (G) MFI per cell nucleus. Significantly greater MFI per microgram and MFI/nucleus is observed with longer maturation times (n = 9 for 70% and 1 week and n = 6 for 4 weeks). (H) Measure of maximal citrate synthase activity showed a significant increase at 4 weeks (n = 4 per time point). (I) Relative expression of genes involved in mitochondrial dynamics (MFN1, MFN2, FIS1) and mitochondrial DNA replication and transcription (POLG, TFAM) during RPE maturation in vitro. Significant increases in FIS1 and MFN1 occur with RPE maturation (n = 3 per time point). Data were analyzed by ANOVA followed by Tukey's multiple comparison test.