Accelerated exhaustion of HO-1^+/− HSCs provoked by stress of serial transplantation or repeated myelotoxic injuries in HO-1 deficiency. (A) Experimental design. Five thousand HSCs from luc⁺HO-1^+/− or luc⁺HO-1^+/+ mice were transferred into lethally irradiated primary recipients. Twelve weeks later, one-fifth of the BM recovered from these recipients was transplanted into secondary recipients, and so on, for a total of 4 transplantations. (B) Time course of hematopoietic reconstitution of serially transplanted recipients was measured by BLI. Values are shown as mean (± SD) whole-body BLI intensities (n = 10 for each group). (C,D) Time course of hematopoietic reconstitution from serially transplanted GFP⁺HO-1^+/− or HO-1^+/+ BM cells. Hematopoietic contribution from 2 × 10⁶ BM cells to peripheral myeloid (Mac-1⁺) and lymphoid (CD3⁺ or B220⁺) subsets was analyzed by flow cytometry at days 49 (n = 6, P < .017), 98 (n = 5, P > .22), and 140 (n = 5, P > .35) in the primary recipients (C) and at days 49 (n = 5, P = .028) and 91 (n = 8, P = .0005) in the secondary recipients (D). (E) The adoptive (GFP⁺) HO-1^+/− KTLS HSC compartment assessed at 12 weeks after secondary transplantation. Values displayed are mean (± SD) of 4 samples and P value is one-tailed. (F,G) Repeated myelotoxic injuries and accelerated exhaustion of HO-1^+/− HSCs. HO-1^+/− or HO-1^+/+ mice were treated with doses of 5-FU (150 mg/kg, intraperitoneally) at weeks 1, 2, and 5, respectively. The frequency of KTLS HSC population in nucleated BM cells was analyzed at week 18, and mean (± SD); (n = 4) of KTLS numbers in Lin⁻Thy1.1^lo cells is displayed. P value is 1-tailed (F). In a separate experiment, mice were injected a fourth dose of 5-FU, followed by leukocyte counts in peripheral blood, and their relative WBC counts were compared with that of mice receiving a single dose of 5-FU. Values are mean (± SD) WBC count of HO-1^+/− mice relative to that of HO-1^+/+ after 4th dose (n = 6) of 5-FU versus that of 1st dose (n = 4) (G).