CD133+ Tumor-initiating Cells Isolated from Embryonic Retinal Tumors in Transgenic Mice

Abstract

Purpose: : Human retinoblastoma occurs during the proliferative phase of retinal development and is caused by mutations in Rb1 that result in functionally defective or absent Rb1 protein. Similar retinal tumors occur in mice only when multiple Rb gene family members are absent. In one transgenic mouse strain, retinal tumors that develop in the post-proliferative stage have been shown to arise from dedifferentiated terminally differentiated retinal horizontal cells leading to speculation that human retinoblastomas may develop similarly. Here we test the hypothesis that a tumor-initiating cell can be isolated from tumors formed in a developing murine retina.

Methods: : A transgenic mouse model of retinoblastoma was developed by expressing SV40 T-antigen using the early eye development promoter Pax6. T-antigen, which sequesters all Rb family members and p53, is expressed in the retina and lens on E10 and tumors are observed on E11.5 prior to retinal differentiation. A cell line was developed from retinal tumor cells isolated on P7.

Results: : The derived cell line adheres in serum-containing media and forms neurospheres in supplemented serum-free media. All cultured cells express T-antigen confirming that they are derived from the original tumors. 1.5% of attached cells transferred to serum-free medium form neurospheres. 0.5% of adherent cells are CD133+. When FACS sorted cells are cultured in serum-free medium, 3-fold more neurospheres form in the CD133+ cultures than in the CD133- cultures (p < 0.001). In vivo, 6/7 mice injected with CD133+ cells and 1/7 mice injected with CD133- cells form tumors during the 6 month observation period (p=0.007). Unlike the primary adherent cells, the cultured neurospheres and the primary and secondary tumors heterogeneously express synaptophysin and GFAP suggesting differentiation similar to human retinoblastoma.

Conclusions: : There exist CD133+ cells within the developing, proliferating murine retina that can initiate retinal tumors.

Keywords: retinoblastoma • proliferation • tumors