Cultured CD271⁺/CD45⁻/CD146^−/low and CD271⁺/CD45⁻/CD146⁺ BM-MNCs give rise to standard mesenchymal stromal cell cultures. Morphology, differentiation capacity, and FACS profile of cultured CD271⁺/CD45⁻/CD146^−/low and CD271⁺/CD45⁻/CD146⁺ cells were compared. (A) Fibroblastic colonies derived from CD146^−/low cells and CD146⁺ cells on day 14 shown with crystal violet staining. Scale bars for the colonies indicate 400 μm; scale bars for the close-ups indicate 50 μm. (B) Mean diameter of CD271⁺/CD45⁻/CD146⁺- and CD271⁺/CD45⁻/CD146^−/low-initiated colonies (day 14). Every dot represents one colony (CD146⁺ n = 46; CD146^−/lown = 51) of 2 independent experiments. The lines represent the mean diameter of the colonies in millimeters. Error bars represent SEM. (C) In vitro differentiation capacity of cultures generated from a single CD271⁺/CD45⁻/CD146^−/low–sorted cell (top row) and a single CD271⁺/CD45⁻/CD146⁺–sorted cell (bottom row) from the same bone marrow sample. Clonal cultures were differentiated toward the osteoblastic, adipogenic, and chondrogenic lineage. Osteoblasts were stained with Alizarin Red (left), and adipocytes were stained with Oil Red O (second from left). Small inserted photographs show Alizarin Red and Oil Red O staining on undifferentiated cells. Original magnifications were 40× and 200×, respectively. Chondrocyte pellets were stained with an anti-aggrecan plus secondary antibody (third from right). Control sections were stained with the secondary antibody only (small inserted photograph). In addition, chondrocyte sections were stained with toluidine blue (second from right) for proteoglycans and Alcian blue (far right) for metachromasia. Scale bars indicate 200 μm. (D) Representative FACS profile of single-cell–derived cultured stromal cells. Cells were measured in the fourth and second passages, when sufficient amounts were available. Cultures were initiated with either a CD271⁺/CD45⁻/CD146⁺ (red tinted histograms) or a CD271⁺/CD45⁻/CD146^−/low (gray tinted histograms) FACS-sorted bone marrow cell. Cultured cells were stained for typical MSC markers and analyzed by flow cytometry. Black open histograms represent the corresponding isotype controls.