Fig. 1. Single neural stem cells can differentiate into neurons, glia and myocytes. A single retrovirally labeled E14 CD31^- CD35^- NSC was expanded in suspension in MB-media for 6 days then transferred to an adherent surface and allowed to differentiate. Differentiated progeny were identified by immunostaining 14 days post-plating. (A-C) Epifluorescent images of an expanding EGFP⁺ NSC. The resulting sphere was dissociated and grown as an adherent culture for 14 days to complete the differentiation process. (D) Presented are epifluorescent images of EGFP⁺ cells (green) and sk-MHC immunoreactive cells (red). Arrowheads indicate a phenotypic EGFP⁺ neuron in a cluster of EGFP⁺ sk-MHC⁺ myocytes. Scale bar: 80 µm. (E-H) The observed multilineage differentiation covered many combinations of differentiation possibilities. (E,F) Confocal z-sections of differentiated clones containing BAG5⁺ cardiac myocytes (red) and MAP2 immunoreactive neurons (green); (G,H) BAG5 cardiac myocytes (red) and GFAP⁺ glial cells (green). To further verify that differentiated clones arose from a single EGFP⁺ cell, Southern analysis was performed on random differentiated progeny; (I) Southern blot. Clone 15-1 is the founder clone and clone 15-2 represents the secondary clone arising from the primary clone 15-1. Controls 1 and 2 are mixtures of two or more individual clones.