To evaluate whether editing of cystatin C gene had any effect on polarized secretion and TER, cells were cultured on transwell inserts allowing for separate collection of media conditioned to the apical or basolateral side of the monolayer. The human ARPE19 spontaneously immortalized RPE cell line was cultured in a similar manner and used for comparison. As seen in
Figure 3A, ARPE19 TER quickly reached its highest point of approximately 50 Ω × cm
2 after just 1 week in culture, a value consistent with previous studies,
24,34 whereas the iPS-derived RPE cells slowly matured toward significantly higher levels. CST3 B/B cells reached their maximum levels of approximately 150–200 Ω × cm
2 after 4 weeks in culture, whereas non-edited cells displayed maximum values approaching 300 Ω × cm
2 after 6 weeks in culture. Although these levels are lower than those observed for
in vitro cultures of primary RPE cells,
34–36 they are a clear improvement over the ARPE19 model cell line, prompting us to investigate whether any polarization of secreted proteins could be observed. Western blot analysis of proteins secreted from the apical and basolateral side of the monolayer showed a clear polarization of cystatin C secretion from both WT and CST3 B/B cells (
Fig. 3B). To demonstrate that this predominant basolateral secretion was not a global feature of the cells, we also probed the membranes against PEDF, a protein previously shown to be predominantly secreted apically from RPE cells, where the abundance was not significantly different between the two sides (
Fig. 3B). TER is known to be a function of tight junction strands
37 comprised in RPE of proteins such as ZO1, ZO2, claudins, and occludin, prompting us to investigate whether CST3 B/B cells displayed reduced expression of these proteins. Western blot analysis of iPS-RPE whole cell lysates showed no significant difference in expression of tight junction proteins ZO1 or ZO2; however, claudin-3 expression was significantly reduced in CST3 B/B cells (
Figs. 3C–D).