Animals were sacrificed and perfusion-fixed at 4 weeks posttransplantation and the recovered grafts then sectioned. Microscopic inspection revealed that the grafts had been maintained as a largely intact and continuous cell monolayer. A positive pan-cytokeratin (pCK) signal, an established RPE marker, was confirmed for fetal and adult hRPE monolayers (Figures 3A and 3C). Additional pCK reactivity was seen “underneath” the cell carrier, likely from host RPE. Moreover, fetal and adult hRPE on PET carriers stained positively for the human-specific marker SC121, confirming survival of human RPE for 1 month as a monolayer (Figure 3). Costaining of SC121 with an antibody to MCT1 and Ezrin, both apical membrane markers, further confirmed that the RPE were (still) polarized (Figure 3). SC121+ RPE transplants were negative for the expression of the cell-cycle marker ki67, the proliferation marker phosphohistone H3, and for the apoptotic marker caspase-3 (Figures 3F–3H), indicating absence of proliferation and apoptosis. We estimated the total human RPE cell survival to be approximately 95% after 1 month by using the SC121 positivity; we measured the total length of the carrier and the length of SC121 stain and calculated the percent coverage of SC121 over the total carrier length. On transmission electron micrography (TEM), polarized RPE cells were observed on the PET carriers from both fetal and adult transplants (Figures 3I and 3J). These results confirm survival of polarized human RPE from fetal and adult donors xenografted into rabbit SRS over 4 weeks.
Animals
sacrificed and perfusion-fixed
4 weeks
weeks
human RPE
survival
1 month
month
human RPE cell
survival
approximately 95%
%
human RPE cell
survival
after 1 month
month
polarized human RPE
survival
over 4 weeks
weeks