Comparative analysis of ABCG2+ and ABCG2- retinoblastoma cells in three-dimensional culture

Aims: Retinoblastoma (Rb) is the most common intraocular malignancy of early childhood. Subpopulations of Rb cells express the stem cell marker ABCG2; yet little is known regarding the differences between ABCG2+ and ABCG2subpopulations, particularly in threedimensional culture. The aim of this study was to compare ABCG2+ (stem-like) populations with ABCG2cells in retinoblastoma in threedimensional, tumor-like populations. Methods: We compared aggregates of ABCG2+ and ABCG2Rb cells to test the hypothesis that ABCG2+ cells would preferentially exhibit stem cell markers and less mature retinal marker expression as compared with ABCG2cells. Retinoblastma cells (Y79 and WERI-Rb27) were immunomagnetically enriched into ABCG2+ and ABCG2populations and grown as threedimensional aggregates. Enriched populations were examined as aggregates, along with embryoid bodies formed by iPSCs. Results: Immunostaining revealed that both ABCG2+ and Linda Cassidy1, Robert Diaz2, Ruby Yanru Chen-Tsai3, Gail M Seigel4 Affiliations: 1MS, Lab Technician, Center for Hearing & Deafness, University at Buffalo, Buffalo, NY, USA; 2PhD, Applied Stem Cell, Inc., Menlo Park, CA, USA; 3PhD, Applied Stem Cell, Inc., Menlo Park, CA, USA; 4PhD, Research Assistant Professor, Center for Hearing & Deafness, SUNY Eye Institute, University at Buffalo, Buffalo, NY, USA. Corresponding Author: Gail M. Seigel, PhD, University at Buffalo, Center for Hearing & Deafness SUNY Eye Institute, 3435 Main Street, Cary 137, Buffalo, NY, USA. 14214; Ph: 011-1-716-829-2588, Fax No: 011-1-716-829-2980; Email: [email protected] Received: 29 January 2014 Accepted: 21 February 2014 Published: 15 March 2014 ABCG2cell aggregates were immunoreactive to tubulin III (ectoderm), but immunonegative for smooth muscle actin (mesoderm) and alpha-fetoprotein (endoderm), typical human embryonic markers. ABCG2+ aggregates also 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 ABCG2cells. Conclusion: ABCG2+ aggregates express more stem cell markers as compared with ABCG2Rb cells, but both appear to be restricted to an ectodermal lineage. These results aid in our understanding of ABCG2+ subpopulations of cells in Rb, with possible implications for disease development and chemoresistance.