In vitro expanded shJarid1b-HSC retain long-term in vivo multipotency. (A) Analysis of hematopoietic tissues in recipients of day 7 cells (Figure 3B) at 1 year after transplantation. Panel i, spleen weight; panel ii, the total numbers of bone marrow cells pooled from pelvis, femur, and tibia; panel iii, CFC frequency in the GFP⁺ bone marrow cell populations; panel iv, proportions of GFP⁺ multilineage progenitors (CFU-GEMM). Dots in first plot represent the numbers of individual mice for which all the described parameters were analyzed. (B) Contribution of day 7 (Figure 3A) shJarid 1b (GFP⁺) cells to reconstitution of myeloid (Mac1⁺), B-lymphoid (B220⁺), and T-lymphoid lineage (CD4⁺, CD8⁺) at 1 year after transplantation. An example of typical reconstitution observed in all recipients (n > 10) is shown. (C) Jarid1b knockdown promotes the in vitro expansion of LTR-HSCs. Upper panel, experimental outline. Lower panel, CRU numbers in freshly sorted (ie, input) and day 7 shRNA-transduced cell populations (mean ± standard error). shJarid1b CRUs were determined in 2 independent experiments (see supplemental Table 6). (D) Clonal analysis of proviral integrations in DNA isolated from hematopoietic tissues of mouse from shJarid1b cohort introduced in Figure 5C. DNA was digested with EcoRI, which cuts once within the provirus such that each DNA fragment recognized by the ³²P-labeled Gfp probe represents a unique integration event. Mouse ID, the total dose of transplanted cells, and the estimated number of transplanted CRU are shown on top. T, thymus; S, spleen; BM, bone marrow.