SSEA-4 can be used to prospectively isolate human MSCs. (A) A representative FACS profile of time course analyses of SSEA-4 expression in fresh unsorted human BM (D0) and adherent cells at 2, 4, 7, 14, and 21 days in culture. Fluorescence intensity of FITC-labeled SSEA-4 is indicated on the x-axis. Percentages represent the fraction of cells that express SSEA-4 (background is subtracted). The y-axis represents PE, which provides a measure of autofluorescence. (B) Fresh BM was sorted based on SSEA-4 expression. SSEA4⁺ and SSEA-4⁻ cells were plated at the same cell density (3 × 10⁴ cells/cm²). SSEA-4⁻ cells failed to grow (left), whereas SSEA4⁺ cells demonstrated extensive growth (right). (C) Growth curve of SSEA-4⁺ cells sorted on day 0 and plated at 6.3 × 10⁴ cells. (D) SSEA-4 and CD45 expression on SSEA-4⁺–sorted cells at passage 2. (E) After appropriate induction, day-0 sorted SSEA4⁺cells were capable of differentiating along fat (left), cartilage (middle), and bone (right) lineages, as observed by Oil Red O, Alcian Blue, and Alizarin Red S staining, respectively (top row), as well as by relevant gene expression analyses (bottom row). Gene expression for unrelated lineages (controls) is shown below. Original magnification for panels B and E, 10×/0.3 NA Ph1.