Fig. 4. LIF promotes, whereas BMP4 inhibits, a GFAP-expressing multipotential stem cell fate and neuron production. (A) Experimental paradigm for the cytokine induction, rGFAP promoter and FACS selection, and stem cell analysis of GFAP⁺ cells. Neurospheres were expanded, infected with the rGFAPp-EGFP retrovirus, further expanded, plated for 5 days to differentiate with or without cytokines and then sorted on the basis of eGFP expression (green). Sorted cells were plated in a neurosphere-forming assay to assess self-renewal and subsequent spheres were plated for differentiation to assess the ability to form neurons (red), astrocytes (purple) and oligodendrocytes (blue). (B,G) In the neurosphere-forming assay, 2% of control GFAP⁺ cells self-renew (green indicates cells that are GFP positive). (C-E,G) LIF and noggin treatments each increase the percentage of GFAP⁺ cells that form neurospheres (green). LIF treatment also increases neurosphere size, which is further increased by additional BMP inhibition. (F,G) BMP4 treatment prevents the self-renewal of GFAP⁺ cells. (H) A single GFP-positive neurosphere plated for differentiation gives rise to oligodendrocytes (O4, blue), neurons (ß-tubulin, red) and astrocytes (GFAP, pink), demonstrating multipotentiality. (I-M) GFP-expressing neurosphere populations were dissociated to assess neuronal progeny. (I,M) GFAP-expressing (GFP positive, green) cells produce few neurons (red). (J,M) Noggin treatment alone does not significantly change neuron production. (K,M) LIF treatment significantly increases the number of neurons generated. (L,M) LIF treatment with BMP inhibition further increases neuron production. (G) ^*P<0.05, ^**P<0.01; (M) ^*P<0.01, ^**P<0.005 ANOVA. Scale bars: 100 µm in B-F; 20 µm in H-L.