Release of an active form of BMP4 by nestin-positive MSCs. (A)RT-PCR and quantitative RT-PCR were performed on npMSCs and nnMSCs. The results of quantitative RT-PCR are expressed as percent of gene expression in npMSCs compared to nnMSCs (arbitrarily as 100%) after normalisation with the GAPDH house-keeping gene expression. (B) Western blotting analysis of conditioned media by npMSCs, nnMSCs and NSC. The mature and biologically-active form of BMP4 (27 kDa) is only observed in medium conditioned by npMSCs and the 57 kDa immature and non biologically-active form can be seen in nnMSC and in neural stem cells conditioned medium. This high MW form is not present in npMSC conditioned medium, indicating a complete cleavage and activation of BMP4. (C) Effect of anti-BMP4 antibodies in differentiating NSCs cultivated in DEM/F12 or npMSCs conditioned medium. Anti-BMP4 antibodies inhibit the npMSCs effect on astroglial and oligodendroglial cell numbers but not the effect on neurons number (47.13 ± 1.53% of GFAP-positive cells, 9.98 ± 0.92% of Tuj1-positive cells and 5.67 ± 0.45% of O4-positive cells with anti-BMP4 compared to respectively 77.5 ± 2.5%, 4.30 ± 0.95% and 1.47± 0.60% in npMSC conditioned-medium). Tuj1-positive cells remains significantly lower (***student T test, p < 0.001, n = 5).