For a more detailed examination, we performed immunohistochemistry with Cx43, AQP1, and AQP4, which are expressed on the CE.
5,10 In the mouse eye, Cx43-positive staining was seen in the lens epithelium, RPE and CE, and it was especially evident in the gap junction between PE and NPE (
Figs. 1A,
1E). Cx43 immunoreactivity was not seen in the NR. The lens fiber staining was suspected to be nonspecific.
10 AQP1-positive staining was seen in the NPE and iris, especially in the anterior pars plicata (
Figs. 1B,
1C). AQP4-positive staining was seen in the NPE, especially in the pars plana (
Fig. 1D). In optic cup–like aggregates from mouse ES/iPS cells, there were several differences as compared with the mouse eye. First, AQP1-positive staining was seen in PE/RPE. In previous studies, AQP1 protein was not detected in adult rat or human RPE preparations, possibly because of technical difficulties associated with the presence of melanin and lipofuscin in RPE cells.
11–13 Stamer et al.
14 reported that AQP1 protein was detected in human RPE in situ and in cultures of human adult and fetal RPE cells; the expression of AQP1 by RPE in vivo probably contributes to the efficient transepithelial water transport across RPE, maintains retinal attachment, and prevents subretinal edema. Therefore, AQP1-positive staining in our immature PE/RPE is consistent with previous findings. Second, there was an important difference that, although there were positive regions for ciliary markers, these regions were slightly different from the positive regions in the mouse eye. Cx43- and AQP1-positive staining was seen in the outer layer as we suspected, and these markers were not expressed in the inner layer of optic cup–like aggregates (
Figs. 1K–M). Moreover, AQP1-positive staining was also observed in the same outer region on day 18, which is supposedly a more mature state (
Fig. 1M). Therefore, it was suggested that the peripheral structure of optic cup–like aggregate from mouse ES/iPS cells did not have original opposed CE structures.