After approximately 15 passages the RPE cells entered senescence without evidence of cell death. Optimal proliferation of RPE cells occurred when W3 was added prior to passage 6. Cytogenic analysis of passage 10 RPE cells did not show karyotype aberrations. Additionally, when the RPE cells were allowed to reach confluence, they differentiated into a cobblestone monolayer and became pigmented, and cell staining showed the presence of tight junctions (Figure). RPE cells are one of the most active phagocytic cells known and this activity is required to recycle the discs containing visual pigments at outer photoreceptor segments.
The W3-expanded and differentiated RPE cells were found to be phagocytic as measured by the internalization of oposin (Figure). The target of W3 in β cells is Erb3 binding protein 1 (Ebp1), a tyrosine kinase related to the epidermal growth factor receptor (EGFR) receptor tyrosine kinase family. Pull-down experiments using a biotinylated analog of W3 supports that Ebp1 is also a primary target of W3 in RPE cells.
To demonstrate that the W3-expanded RPE cells are functional in vivo, the authors transplanted these cells into rats harboring a mutation that inactivates photoreceptor outer segment phagocytosis resulting in a decrease in visual acuity with age. The RPE grafted eyes showed improved luminance threshold values and optokinetic responses. Additionally, P90 rats showed four to six layers of photoreceptors compared to only one found in untreated rats. The fact that W3 acts as proliferative molecule in both β cells and RPE cells with a common target suggest that this could be useful for the expansion of other primary cell types.