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Comparative analysis of ABCG2+ and ABCG2- retinoblastoma cells in three-dimensional culture
Linda Cassidy1, Robert Diaz2, Ruby Yanru Chen-Tsai3, Gail M Seigel4
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.

Article ID: 100005OCYLC2014
doi:10.5348/ocy-2014-5-OA-1

Address correspondence to:
Gail M. Seigel
PhD, University at Buffalo
Center for Hearing & Deafness SUNY Eye Institute
3435 Main Street, Cary 137, Buffalo
NY, USA. 14214
Phone: 011-1-716-829-2588
Fax: 011-1-716-829-2980
Email: gseigel@frontiernet.net

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How to cite this article
Cassidy L, Diaz R, Chen-Tsai RY, Seigel GM. Comparative analysis of ABCG2+ and ABCG2- retinoblastoma cells in three-dimensional culture. Oncocytology 2014;4:1–7.


Abstract
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 ABCG2- subpopulations, particularly in three-dimensional culture. The aim of this study was to compare ABCG2+ (stem-like) populations with ABCG2- cells in retinoblastoma in three-dimensional, tumor-like populations.
Methods: We compared aggregates of ABCG2+ and ABCG2- Rb cells to test the hypothesis that ABCG2+ cells would preferentially exhibit stem cell markers and less mature retinal marker expression as compared with ABCG2- cells. Retinoblastma cells (Y79 and WERI-RB27) were immunomagnetically enriched into ABCG2+ and ABCG2- populations and grown as three-dimensional aggregates. Enriched populations were examined as aggregates, along with embryoid bodies formed by iPSCs.
Results: Immuno staining revealed that both ABCG2+ and ABCG2- cell 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 ABCG2- cells.
Conclusion: ABCG2+ aggregates express more stem cell markers as compared with ABCG2-Rb 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.

Keywords: ABCG2, In vitro tumor assay, Stem cells, Retinoblastoma

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Author Contributions:
Linda Cassidy – Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Robert Diaz – Design and acquisition of embryoid body and cell aggregate data. Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Ruby Yanru Chen-Tsai – Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Gail M Seigel – Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Guarantor of submission
The corresponding author is the guarantor of submission.
Source of support
None
Conflict of interest
Authors declare no conflict of interest.
Copyright
© 2014 Fairuz Nazri Abd Rahman et al. This article is distributed under the terms of Creative Commons Attribution License which permits unrestricted use, distribution and reproduction in any medium provided the original author(s) and original publisher are properly credited. Please see the copyright policy on the journal website for more information.



About The Authors

Linda Cassidy MS was a lab technician in the Center for Hearing and Deafness at Suny Buffalo.



Robert Diaz is Senior Scientist at Applied Stem Cell, Inc., Menlo Park, CA. His focus includes the development of stem cell models of human diseases and stem cell based assays to efficiently characterize stem cells and their differentiated cell types.



Ruby Yanru Chen-Tsai is Chief Scientific Officer of Applied Stem Cell, Inc., Menlo Park, CA. Her area of interest includes developing stem cell and animal models of human diseases using gene-editing technologies and their applications in cell based bioassays and stem cell therapies. She earned her PhD degree from Cornell University and did her post-doc research at Stanford University.



Gail M. Seigel is a faculty member in the Center for Hearing and Deafness at the State University of New York at Buffalo and a member of the Suny Eye Institute. She earned her B.S. degree from Rutgers University and her PhD from Albany Medical College. She is a fellow of the Association for Research in vision and ophthalmology. Her lab studies growth and differentiation of stem cells in retinoblastoma.