Expression of Pluripotent Markers L1CAM and SSEA-5, Common to Human Retinoblastomas, Xenografts, Teratomas, Embryonic Tumors and Induced Pluripotent Stem Cells

Abstract

 

Purpose

 

Cancer stem cells and therapeutic stem cells share common and divergent pathways involved in cell growth and differentiation. In this study, we sought to bridge the gap between retinoblastoma (RB) tumor cells and iPSCs undergoing retinal differentiation by testing the hypothesis that L1CAM and SSEA-5 could be used to evaluate pluripotency in both models.

 

Methods

 

Magnetically enriched stem-like ABCG2+ and non-stem like ABCG2- cells from the human RB143 retinoblastoma cell line underwent microarray analysis using the Agilent platform. We used ensemble based statistical models to predict differentially expressed genes of pluripotency. The larger list was condensed into genes with biological relevance to the behavior of cancer stem cells and iPSCs. One of these genes was L1CAM, a neural cell adhesion marker. We included SSEA-5 for analysis based on promising published data as a novel marker of pluripotency. We used immunohistochemistry to analyze RB tumors and cell lines, RB xenografts, human embryonic tumors, iPSC-derived teratomas, as well as iPS cells before and after previously established retinal differentiation protocols to determine whether these markers would be useful for monitoring pluripotency. The expression of L1CAM in these cells was also assessed by qRT-PCR.

 

Results

 

RB143 cells and xenografts were immunoreactive to L1CAM and SSEA-5. L1CAM was expressed in most human RB and human embryonic tumors examined. SSEA-5 was preferentially expressed in embryonic tumors and some RB tumors. In iPS-induced teratomas, we saw immunoreactivity to both markers, with some areas of co-localization. Undifferentiated iPSCs exhibited strong immunoreactivity to SSEA-5 and L1CAM, with a few positive cells remaining even after 20 days of retinal differentiating treatment. L1CAM showed little change by qRT-PCR, suggesting post-transcriptional regulation.

 

Conclusions

 

Both SSEA-5 and L1CAM are cell surface markers that show promise as indicators of pluripotency. Stem cell regulatory mechanisms common to RB cancer stem cells and retinal therapeutic stem cells may be useful in monitoring and controlling the growth and differentiation of iPSCs. Further studies may aid in growth regulatory strategies that are essential to the development of safe ocular stem cell replacement therapies.

 

 

Embryonal carcinoma: SSEA-5 (red), L1CAM (green)

 

Keywords: 703 retinoblastoma • 721 stem cells • 744 tumors