Abstract
Background: Cancer stem cell (CSC) theory proposes that tumours are composed of a heterogeneous mixture of undifferentiated CSCs and their differentiated progeny. While undifferentiated CSCs and differentiated counterparts have been studied in isolation, little is known of their interaction in vivo. The aim of this study was to assess whether undifferentiated CSCs and the differentiated cells they produce interact when grown together. Design: Co-culture experiments involved undifferentiated Pluripotent NTera2 human Embryonal Carcinoma cells and NTera2 cells differentiated by retinoic acid treatment for 7 days. Conditioned-media experiments involved incubation of NTera2 cells in undifferentiated (‘undiff-conn’) or differentiated (‘diff-conn’) conditioned media for 7 days. Differentiation status was confirmed by loss of expression of SSEA4 (flow cytometry) and Oct4-Sox2-Nanog (qPCR). Results: Undifferentiated and differentiated cells were co-cultured in a 1:9 ratio, which arrested the growth of undifferentiated cells after only 7 days. Flow cytometry (SSEA4) indicated that NTera2 cells had not differentiated but had stopped proliferating. This data led us to hypothesise that differentiated cells secrete factors into their environment that can regulate the growth of undifferentiated CSCs. Confirming this, treatment with undiff-conn media increased the expression of SSEA4, suggesting an enhancement of the undifferentiated state. Furthermore, treatment with diff-conn media forced all cells to differentiate. A further validation, qPCR analysis showed that Oct4-Sox2-Nanog levels were increased in undiff-conn treated cells and dramatically lost in diff-conn treated cells. Conclusions: Our data indicate that undifferentiated CSCs secrete factors to promote their undifferentiated state. In parallel, differentiated cells secrete factors that can arrest CSC proliferation in co-culture and force-differentiate CSCs in conditioned media conditions. These effects are achieved through a standard Oct4-Sox2-Nanog mechanism. These results are striking when it is noted that forcing differentiation upon CSCs removes their tumorigenic potential. As such, if the factors secreted by differentiated NTera2 cells can be identified in future work, they may be useful as a potential anti-cancer force-differentiation treatment.