Fig. 5. BMP-treated CNS stem cells efficiently differentiate into smooth muscle and co-express Sox9, SMMHC1+2 and calponin. (A) Differentiation paradigm 1 (for panels B-F,M) and paradigm 2 (panels G-L,N-V). Cortical stem cells were seeded at low density after first passage. (B-F) Cells differentiated by (B) FGF2-withdrawal or (C) 8 ng/ml TGFß1 co-treatment are all SMA^–. By contrast, 20 ng/ml of BMP2, BMP 4 or BMP 7 efficiently generate SMA⁺ cells. (M) Quantitation of B-F. Notice that the passage 5 experiment varies from paradigm 1 by using a 7-day co-treatment and 7-day withdrawal. (G-L) Dose-response assay showing no or few SMA⁺ cells in (G) FGF2-expanded or (H,I) low-dose BMP2 co-treated cells. (J) Percentage of SMA⁺ cells increases at 10 ng/ml BMP2 but yields immature-looking cells, and plateaus to almost 100% at 20 ng/ml BMP2, even during continued mitogenic expansion (K,L, quantitation in N). (O) Initial transient exposure to 20 ng/ml BMP2 during FGF2 expansion is sufficient to induce SMA⁺ differentiation by 8 days. (P-V) Cells co-treated with 10 ng/ml FGF2 (F) and 20 ng/ml BMP2 (B) nearly all co-express SMA and Sox9 (Q-R), SMMHC (S-T) and calponin (U-V). Graphs show mean ± s.e.m. (n=3). Bars 40 µm (B-L); 20 µm (Q-V).