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Gail M Seigel, Linda Cassidy, Robert Diaz, Ruby Y Tsai; Comparative Analysis Of ABCG2+ Stem-Like Retinoblastoma Cells And Induced Pluripotent Stem Cells As Three-Dimensional Aggregates. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3068.
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© ARVO (1962-2015); The Authors (2016-present)
Retinoblastoma (RB), an intraocular malignancy of early childhood, expresses a number of stem cell markers including ABCG2. In this study, we compared ABCG2+ and ABCG2- RB cells with induced pluripotent stem cell (iPSC)-derived embryoid bodies to assess their potential for pluripotency as three-dimensional aggregates. We tested the hypothesis that both iPSC-derived embryoid bodies and ABCG2+ cells would both preferentially exhibit pluripotent stem cell markers and less mature retinal marker expression as compared with ABCG2- cells.
Immunomagnetic enrichment of WERI-RB27 and Y79 retinoblastoma cells created populations that were ABCG2+ or ABCG2-. Enriched ABCG2+ and ABCG2- populations were examined as aggregates in three-dimensional culture and compared with embryoid bodies formed by iPSCs. Resulting cell aggregates were assessed by immunohistochemistry for a variety of stem cell and mature markers.
ABCG2+ and ABCG2- cells formed aggregates that differed morphologically from iPSC-derived embryoid bodies. Immunostaining demonstrated that both ABCG2+ and ABCG2- cell aggregates were immunoreactive to tubulin III (ectoderm), but immunonegative for smooth muscle actin (mesoderm) and alpha-feto protein (endoderm). However, ABCG2+ aggregates exhibited greater immunoreactivity to stem cell markers (ABCG2, ALDH1A1 and CD164), but less immunoreactivity to mature markers (MAP-2 and S-Antigen) as compared with ABCG2- RB cells. In contrast, iPSC-induced embryoid bodies contained cells that were immunoreactive for primitive markers including Nestin, PAX6, CD164 and ALDH1A1.
Aggregate cultures of enriched ABCG2+ RB cells possess a higher degree of stem-like features as compared with ABCG2- RB cells. Both populations express Tubulin III, a marker of embryonic ectoderm, but neither alpha feto-protein (endoderm) nor smooth muscle actin (mesoderm) as compared with pluripotent iPSC-derived embryoid bodies. This suggests a restriction to ectodermal lineage for both ABCG2+ and ABCG2- RB cells. These results may have implications for RB tumor development, as well as the potential to lead to novel therapeutic approaches for tumor eradication in RB.
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