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.

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Self-organized patterning and architecture construction studying is a priority goal for fundamental developmental and stem cell biology. To study the spatiotemporal patterning of pluripotent stem cells of different origins, we developed a three-dimensional embryoid body (EB) differentiation model quantifying volumetric parameters and investigated how the EB architecture formation, patterning, and scaling depend on the proliferation, cavitation, and differentiation dynamics, external environmental factors, and cell numbers. We identified three similar spatiotemporal patterns in the EB architectures, regardless of cell origin, which constitute the EB archetype and mimick the pre-gastrulation embryonic patterns. We found that the EB patterning depends strongly on cellular positional information, culture media factor/morphogen content, and free diffusion from the external environment and between EB cell layers. However, the EB archetype formation is independent of the EB size and initial cell numbers forming EBs; therefore, it is capable of scaling invariance and patterning regulation. Our findings indicate that the underlying principles of reaction-diffusion and positional information concepts can serve as the basis for EB architecture construction, patterning, and scaling. Thus, the 3D EB differentiation model represents a highly reproducible and reliable platform for experimental and theoretical research on developmental and stem cell biology issues.