Human embryonic and neuronal stem cell markers in retinoblastoma.
OBJECTIVE
Retinoblastoma (RB) is the most common intraocular tumor of early childhood. The early onset of RB, coupled with our previous findings of cancer stem cell characteristics in RB, led us to hypothesize that subpopulations of RB tumors harbor markers and behaviors characteristic of embryonic and neuronal origin.


METHODS
Our RB sources included: human pathological tissues, and the human RB cell lines Y79 and WERI-RB27. Microarray screening, single and dual-label immunocytochemistry and RT-PCR were performed to detect embryonic and neuronal stem cell markers, such as Oct3/4, Nanog, CD133, and Musashi-1. To test for functional evidence of stem cell behavior, we examined RB cells for their ability to form neurospheres and retain BrdU label as indicators of self-renewal and slow cell cycling, respectively.


RESULTS
Microarray comparisons of human RB tumors with normal retinal tissue detected upregulation of a number of genes involved in embryonic development that were also present in Y79 cells, including Oct3/4, Nanog, Musashi-1 and Musashi-2, prominin-1 (CD133), Jagged-2, Reelin, Thy-1, nestin, Meis-1,NCAM, Patched, and Notch4. Expression of Musashi-1, Oct3/4 and Nanog was confirmed by immunostaining and RT-PCR analyses of RB tumors and RB cell lines. CD133 expression was confirmed by PCR analysis. Y79 and WERI-RB27 contained populations of Hoechst-dim/ABCG2-positive cells that co-localized with embryonic stem cell markers Oct3/4-ABCG2 and Nanog-ABCG2. Subpopulations of Y79 and WERI-RB27 cells were label-retaining (as seen by BrdU incorporation) and were able to generate neurospheres, both hallmarks of a stem cell phenotype.


CONCLUSIONS
Small subpopulation(s) of RB cells express human embryonic and neuronal stem cell markers. There are also subpopulations that demonstrate functional behavior (label retention and self-renewal) consistent with cancer stem cells. These findings support the hypothesis that RB is a heterogeneous tumor comprised of subpopulation(s) with stem cell-like properties.
Seigel
Gail M
GM
Ross Eye Institute, University of Buffalo, Buffalo, NY 14214, USA. gseigel@frontiernet.net
Hackam
Abigail S
AS
Ganguly
Arupa
A
Mandell
Lorrie M
LM
Gonzalez-Fernandez
Federico
F
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism*; D052138:Genes, Neoplasm; D018398:Homeodomain Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D051379:Mice; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000091042:Reelin Protein; D012160:Retina / Q000378:metabolism / Q000473:pathology; D012175:Retinoblastoma / Q000276:immunology / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000235:genetics
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C491297:MSI1 protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016601:RNA-Binding Proteins; D000091042:Reelin Protein; C000717127:RELN protein, human; C000717128:Reln protein, mouse