Mineralization of spinal cord cells. A: Alizarin red S stainings performed on spinal cord cells and on human fetal brain neural stem cells (cultured for 3 passages in serum-containing spinal cord cell medium). Cells were incubated for 14 days in control (left-hand wells) or in osteogenic medium (right-hand wells). High-magnification photographs of corresponding wells are presented on the right-hand side. Scale bars = 10 μm. B: QPCR detection of alkaline phosphatase mRNA in spinal cord cells after 0 and 14 days of differentiation in osteogenic media. Y axis represents the ratio between alkaline phosphatase mRNA (PA) and GAPDH mRNA. This experiment is representative of three independent experiments. C: Detection of alkaline phosphatase activity with naphtol ASBI phosphate/Na3-fast red staining in spinal cord cells and human fetal neural stem cells placed in osteogenic media for 14 days. No staining is detected in neural precursors. D: Characterisation of one clone, representative of 34, derived from the primary human spinal cord culture. Nkx6.1 is detected in the nuclei of most cells. Cells placed in osteogenic differentiation medium for 21 days stained with Alizarine red S. Scale bars = 10 μm.