Fig. 5. LIF maintains GFAP-expressing multipotent progenitors. (A) LIF and low (1-2 ng/ml) EGF propagate neural progenitor cells as a monolayer for at least 10 passages. (B) At higher passages (p7 is shown), GFAP-expressing cells proliferate, as shown by BrdU incorporation (green, 2-hour pulse), and exhibit the progenitor marker LeX (red). (C) Higher passage cultures are still able to generate neurons (green), whereas nearby GFAP-expressing cells remain in cell cycle, as demonstrated by Ki67 expression (red). (D,E) To directly test the ability of GFAP-expressing cells for self-renewal and multipotentiality, higher passage cells were infected with the rGFAPp-EGFP retrovirus, selected by FACS, plated in a neurosphere-forming assay to assess self-renewal (in the absence of high mitogen), and subsequent spheres plated for differentiation to assess the ability to form neurons (red) and astrocytes (blue). (D) GFAP-expressing cells are able to self-renew at high passages, as shown by neurosphere formation (green, GFP). (E) Spheres formed from high passage GFAP-expressing cells retain the ability to produce neurons (red; blue indicates astrocytes; green, GFP). Scale bars: 20 µm in A-C,E; 100 µm in D.