A CFU-GM assay was performed as described previously [17 (link)]. A colony-forming cell assay for rat cells (MethoCult GFR3774, StemCell Technology) is recommended for the detection and quantification of rat CFU-GM progenitors in bone marrow samples. Briefly, 0.3 mL of cell suspension (2,500 cells/mL for SCE-UCBCs, 1 x 105 cells/mL for UCB-MNCs, and 1 x 106 cells/mL for adult MNCs) was added to 3 mL of MethoCult. Using a 2.5 mL syringe, 1.1 mL of the MethoCult mixture was dispensed into each of two wells of a 6-well plate and cultured. After 10 days, the cultures were photographed using a stereo microscope (M165FC, Leica), and the number of colonies in each of the two wells was counted using a Photoshop counting tool (Adobe) and then averaged. The percentage of colony-forming cells was calculated as the number of colonies divided by the number of seeded cells. The experiments were repeated 3 times (n = 3).
UCB-MNCs were cultured with PBS, Chase ABC (0.2 U/mL, Seikagaku Corporation), CS-A (from whale cartilage, 80% of which consists of ΔDi-4S (A-unit), 100 μg/mL, Seikagaku Corporation), or CS-E (from squid cartilage, 60–65% of which consists of ΔDi-di4, 6S (E-unit), 100 μg/mL, Seikagaku Corporation) (Fig 5). Data are shown as the percentage of the control (PBS). The experiments were repeated 4 times and statistically analyzed.
UCB-MNCs were cultured with PBS, Chase ABC (0.2 U/mL, Seikagaku Corporation), CS-A (from whale cartilage, 80% of which consists of ΔDi-4S (A-unit), 100 μg/mL, Seikagaku Corporation), or CS-E (from squid cartilage, 60–65% of which consists of ΔDi-di4, 6S (E-unit), 100 μg/mL, Seikagaku Corporation) (Fig 5). Data are shown as the percentage of the control (PBS). The experiments were repeated 4 times and statistically analyzed.