Neural cells are isolated from forebrain of 14.5 days old mouse embryos. In selective conditions these cells cluster forming sphere-like structures neurospheres (NSCs). NSCs are heterogeneous structures containing only 25% of stem cells and progenitors being reportedly capable to transdifferentiate. Initially, aim of this study is to establish optimal culture conditions prior to investigation of transdifferentiation capacity of transplanted NSCs in kidney and adrenal glands. It was reported low reproducibility of such experiments but the age of transplanted NSCs was not considered as significant issue. In fact, it is generally assumed that progenitors and stem cells remain intact in long-term culture and therefore convenient for potential transplantation. In spite, we hypothesized that potential ageing effects of overall sphere certainly will impact the fraction of stem cells and progenitors. This assumption was tested exploring distinct aspects of ageing in long-term NSC culture. Potential alterations that might occur due to ageing were monitored within 116 weeks of culturing. Initially, tremendous structural and numerous chromosomal aberrations were observed upon 16 weeks of culturing. This was accompanied with p53 up regulation. Moreover, p53 engagement may indicate higher apoptotic rate as self-defense mechanism in order to prevent further DNA burden. Telomere shortening was considered as senescence indication what may imply reduced number of cell divisions from certain time point. Telomere length measurement revealed decrease after 5 and 16 weeks. Although with jeopardized overall homeostasis, NSC unexpectedly displayed gradually elevated capacity to form spheres even in seeded low cell density up to 16 weeks culturing. Increased capacity to form spheres is accompanied with decreasing ability to differentiate into neural lineages. Genetic instability and diminished differentiation capacity seem to be a consequence of long term culturing implying potential transformation.
03 - 07 May 2008
European Society of Endocrinology