MMP-9 deficiency impaired cobblestone area-forming of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. (A) Colony-forming assay of WT or MMP-9–deficient CMPs expressing BCR-ABL and Hes1. Both types of the transduced cells were serially replatable more than 4 times. Bars indicate the number of colonies obtained per 1 × 10³ cells after each round of plating in methylcellulose without cytokines. (B) Typical colonies derived from WT or MMP-9–deficient CMPs expressing BCR-ABL and Hes1. Images were obtained with an IX70 microscope and a DP70 camera (Olympus, Tokyo, Japan); an objective lens, UPlanFl (Olympus); original magnification ×100. (C) Sustained growth of WT or MMP-9–deficient CMPs expressing BCR-ABL and Hes1 in liquid culture without cytokines. The numbers of cells were determined every 4 days by trypan blue staining, and 1 × 10⁵ cells per well were seeded into a 6-well plate. (D) Gelatin zymography for detection of MMP-9. Supernatants of WT or MMP-9–deficient CMPs expressing BCR-ABL and Hes1 were collected after 24 hours of culture, and MMP-9 activity was measured by gelatinolytic zymography. (E) In vivo homing and retention of WT or MMP-9–deficient CMPs expressing BCR-ABL and Hes1. Five days after 2.0 × 10⁶ cells of WT or MMP-9–deficient CMPs expressing BCR-ABL and Hes1 were intravenously injected into nonirradiated mice, GFP⁺ and NGFR⁺ cells migrated into BM were quantified using flow cytometry. (F) Graphic representation of CAFC numbers. Equal numbers of WT or MMP-9–deficient CMPs expressing BCR-ABL and Hes1 were added to each well of a 6-well plate precoated with irradiated C3H10T1/2 cells. After co-culture for 6 days, CAFC numbers per well were counted. (A,C,E-F) All data points correspond to the mean and the SD of 3 independent experiments. Statistically significant differences are shown: *P < .05, **P < .01.