The biological roles of cancer-testis antigens of the Melanoma antigen (Mage) family in mammalian development, stem cell differentiation and carcinogenesis are largely unknown. In order to understand the involvement of the Mage family genes in maintenance of normal and cancer stem cells, the expression patterns of Mage-a, Mage-b, Mage-d, Mage-e, Mage-h and Mage-l gene subfamilies were analyzed during the self-renewal and differentiation of mouse pluripotent stem and teratocarcinoma cells. Clustering analysis based on the gene expression profiles of undifferentiated and differentiating cell populations revealed strong correlations between Mage expression patterns and differentiation and malignant states. Gene co-expression analysis disclosed the potential contributions of Mage family members in self-renewal and differentiation of pluripotent stem and teratocarcinoma cells. Two gene clusters including Mage-a4 and Mage-a8, Mageb1, Mage-d1, Mage-d2, Mage-e1, Mage-l2 were identified as functional antagonists with opposing roles in the regulation of proliferation and differentiation of mouse pluripotent stem and teratocarcinoma cells. The identified aberrant expression patterns of Mage-a2, Mage-a6, Mage-b4, Mageb-16 and Mage-h1 in teratocarcinoma cells can be considered as specific teratocarcinoma biomarkers promoted the malignant phenotype. Our study first provides a model for the involvement of Mage family members in regulatory networks during the self-renewal and early differentiation of normal and cancerous stem cells for further research of the predicted functional modules and the development of new cancer treatment strategies.

A significant challenge for the development of safe pluripotent stem cell-based therapies is the incomplete in vitro differentiation of the pluripotent stem cells and the presence of residual undifferentiated cells initiating teratoma development after transplantation in recipients. To understand the mechanisms of incomplete differentiation, a comparative study of retinoic acid-induced differentiation of mouse embryonic stem (ES) and teratocarcinoma (EC) cells was conducted. The present study identified differences in proliferative activity, differentiation, and tumorigenic potentials between ES and EC cells. Higher expression of Nanog and Mvh, as well as Activin A and BMP4, was found in undifferentiated ES cells than in EC cells. However, the expression levels of Activin A and BMP4 increased more sharply in the EC cells during retinoic acid-induced differentiation. Stimulation of the Activin/Nodal and BMP signaling cascades and inhibition of the MEK/ERK and PI3K/Act signaling pathways resulted in a significant decrease in the number of Oct4-expressing ES cells and a loss of tumorigenicity, similar to retinoic acid-stimulated EC cells. Thus, this study demonstrates that a differentiation strategy that modulates prodifferentiation and antiproliferative signaling in ES cells may be effective for eliminating tumorigenic cells and may represent a valuable tool for the development of safe stem cell therapeutics.

Pluripotent stem cells can differentiate into various lineages but undergo genetic and epigenetic changes during long-term cultivation and, therefore, require regular monitoring. The expression patterns of cancer-testis antigens (CTAs) MAGE-A2, -A3, -A4, -A6, -A8, -B2, and GAGE were examined in undifferentiated human embryonic stem (hES) cells, their differentiated derivatives, teratocarcinoma (hEC) cells, and cancer cell lines of neuroectodermal and mesodermal origin. Undifferentiated hES cells and embryoid body cells expressed MAGE-A3, -A6, -A4, -A8, and GAGEs while later differentiated derivatives expressed only MAGE-A8 or MAGE-A4. Likewise, mouse pluripotent stem cells also express CTAs of Magea but not Mageb family. Despite similarity of the hES and hEC cell expression patterns, MAGE-A2 and MAGE-B2 were detected only in hEC cells but not in hES cells. Moreover, our analysis has shown that CTAs are aberrantly expressed in cancer cell lines and display low tissue specificity. The identification of CTA expression patterns in pluripotent stem cells and their derivatives may be useful for isolation of abnormally CTA-expressing cells to improve the safety of stem-cell based therapy.