Spontaneous Fusion and Non-clonal Growth of Adult Neural Stem Cells

¹ Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California

^* To whom correspondence should be addressed. E-mail: gage{at}salk.edu.

	Abstract

Multipotent neural stem cells (NSCs) can be isolated from various regions of the adult brain and propagated in vitro. Recent reports have suggested spontaneous fusion events among NSCs when grown as free-floating neurospheres that may affect the genetic composition of NSC cultures. We used adult NSCs expressing either red fluorescent protein (RFP) or green fluorescent protein (GFP) to analyze the fusion frequency of rat and mouse NSCs. Fluorescence activated cell sorting (FACS) revealed, that under proliferating conditions approximately 0.2% of rat and mouse NSCs co-expressed RFP and GFP, irrespective of whether the cells were grown as neurospheres (mouse NSCs) or as attached monolayers (rat and mouse NSCs). Fused cells did not proliferate and could not be propagated, suggesting that aberrantly fused cells are not viable. Furthermore, we found that neither neurospheres nor monolayers grew clonally, as even very low-density cultures had spheres containing both GFP- and RFP-expressing cells and monolayer patches with GFP- and RFP-expressing cells in close proximity. The non-clonal growth between distinct NSC populations strongly suggests the use of careful and precise culture conditions, such as single cell assays, to characterize potency and growth of NSCs in vitro.