Figure 1

Normal differentiation in vitro and normal long-term reconstitution ability in vivo of Bmal1 ^−/− HSCs. A, B) Normal in vitro colony formation capacity of Bmal1 ^−/− HSCs. Single HSCs from Bmal1 ^+/+ and Bmal1 ^−/− mice were cultured with cytokines for 11 days. Data shown are the mean numbers ± SDs of colonies of three independent experiments (n = 3). Colony cells were morphologically identified as neutrophils (n), macrophages (m), erythroblasts (E) and megakaryocytes (M). The scale bar in B is 100 μm. C) Comparable proliferation potentials of Bmal1 ^−/− HSCs. CD34⁻KSL HSCs were clonally deposited into 96-well micro-titer plates containing 200 μl of S-Clone SF-03 supplemented with 10% BSA and cultured with the indicated cytokines (50 ng/ml mouse SCF, 50 ng/ml TPO) for 7 days. Cell numbers were counted under a microscope. Data shown are mean numbers ± SEMs of colonies (n = 52). D-F) Comparable long-term reconstitution ability of Bmal1 ^+/+ and Bmal1 ^−/− HSCs during serial transplantation. Lethally irradiated recipient B6-Ly5.1 mice were transplanted with 1 × 10⁶ BM cells (harvested at ZT5) from Bmal1 ^+/+ and Bmal1 ^−/− mice (Ly5.2) and the same number of BM competitor cells from F1 mice in a competitive repopulation assay. Data shown are the mean ratios ± SDs of donor-derived cells in the PB at 12 weeks after the first BMT (D, n = 7), in the BM at 12 weeks after the first transplantation (E, n = 7), and in the PB at 12 weeks after the second BMT (F, n = 5) of three independent experiments.