FIG. 6.

Proliferative potential of MSCs sorted based on scatter properties and surface marker expression. Heterogeneous parental MSCs (P2) were sorted by FACS to select for low scatter (Sc^LO) MSCs with either the highest 10% (HI) or lowest 10% (LO) of positive fluorescence intensities from anti-NG2 APC and/or anti-CD146 FITC. Representative histograms (A–H) from FACS analysis of 10,000 cells. (A) Parental MSCs were gated based on viability to eliminate debris (black) and sorted to select viable Sc^LO cells (green) from cells with higher scatter (red). (B) Sc^LO gate (shown) was selected to include ∼95% of rapidly dividing MSCs (green) labeled with CellTrace Violet as in Figure 5. Surface expression of NG2 (C), CD146 (E), and both markers (G) was determined for Sc^LO MSCs (white) relative to isotype controls (black). Purity of sorted populations for NG2 (D), CD146 (F), and both markers (H) was evaluated by reanalysis of the high (green) and low (red) expressing cells. (I) Colony-forming efficiency of sorted cells (n=3). (J) Cell division was measured as the MFI ratio of sorted MSCs immediately upon labeling with CellTrace Violet divided by the corresponding value 4 days after labeling (n=6). (K–N) Survival of sorted MSCs 24 h after plating at clonogenic levels. Fluorescence (K) and phase-contrast (L) micrographs of one sorted MSC/well labeled with CellTracker Green upon inoculating 96-well microplates. (M) Adherent cells after 24 h stained with Crystal Violet. Scale bars (K–M): 50 μm. (N) Cell survival for each MSC population (n=4). Data are expressed as the mean±standard deviation relative to the sorted parental control with the following phenotype: 35%±5% colony-forming efficiency, 29±7 CellTrace Violet MFI ratio, and 53%±15% cell survival. *p<0.05 and **p<0.01 versus parental control.