Embryonic stem (ES) cells represent a valuable resource for transplantation and tissue engineering applications. For derivation of neural cells, a five-stage differentiation protocol has been widely applied, which involves the propagation of ES cells, formation of embryoid bodies (EBs), selection of neural stem cells (NSCs), expansion of NSCs, and further maturation of NSCs to neurons. During the expansion stage (the fourth stage), two types of cells with distinct morphologies normally emerge, with one type being monolayer cells and the other sphere-like aggregates growing on top of the monolayer cells. In this study, we focus on how the monolayer cells may affect different aspects of aggregate cells, which may have important implications for regenerative medicine. We find that monolayer cells can support the proliferation and decrease the apoptosis rate of sphere cells, as well as facilitate the production of Tuj1-positive cells from sphere cells. In addition, transplantation of monolayer cells into nude mice does not result in tumor formation nor affects the tumorigenicity of sphere cells, when grafted together with monolayer cells.
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