Characterization of MSC clone B9. (A) Growth curve of B9 cells obtained by single-cell sorting from the bulk population at 33 cell doublings. (B) Clonal cells have similar morphology to the parental population and stained positive for X-Gal. (C) Flow cytometric analysis of B9 cells at passage 4. Histogram profiles are gated on forward and side scatters, and PI negative (live) cells are shown in the first 2 panels. Plots show isotype control staining profile (thin line) versus specific antibody staining profile (thick line). Percentages represent the fraction of cells that express a given surface antigen. (D) Multilineage differentiation of clone B9 at passage 18 for fat (left) demonstrated by Oil Red O staining (top left) and expression of fat-specific markers in a time-course study (bottom left); for cartilage (middle) shown by Alcian Blue staining (top middle) and expression of cartilage markers (bottom middle); and for bone (right) demonstrated by Alizarin Red S staining (top right) and expression of bone-specific genes (bottom right). For panels B and D, original magnification was 10×/0.3 NA Ph1.