Figure 2

In vitro and in vivo migration: (A) Migration of HSPC in vitro : Transduced HSPC were seeded onto the upper well of the transwell chamber and allowed to migrate towards SDF1α for 4 hours. The percentage of cells migrated toward a gradient of SDF1α were calculated based on the absolute number of cells recovered in the lower chamber of transwell with respect to the total cells seeded. Values are mean ± SD, n = 4. (B) Short-term homing of RHOA modified HSPC: Transduced HSPCs were expanded in a liquid culture for 7 days containing cytokines. The cells were injected intravenously into NOD/SCIDβ2 microglobulin null mice and bone marrow was collected 24 hours post injection and the percentage homing was analyzed. Values are normalized to the control, Mean ± SEM, n = 3. (C) In vivo migration of RHOA modified HSPC: HSPC were transduced with active RHOA (RHOAV14) and dominant negative RHOA (RHOAN19) after stimulation with the cytokines for 8 hours. 24 hours after transduction, the cells were injected intra-bone into the right femur of the NOD/SCIDβ2 microglobulin null mice. Mice were sacrificed 7 weeks after injection, the left and right femur were collected separately. The percentage of engraftment in the right femur and left femur were analyzed separately by staining for human CD45 and GFP expression and the percentage of migration from right to the left femur were calculated. Values were normalized to the percentage of engraftment, mean ± SEM, n = 4-5. (D) Long-term engraftment of RHOA modified HPC was analyzed in a NOD/SCIDβ2 microglobulin null mice 7 weeks post intra-bone injection. Values are Mean ± SEM, n = 4-5.