Inhibition of Ptpmt1 maintains long-term HSCs. (A) Lin⁻ cells were cultured for 5 days in StemSpan medium supplemented with Tpo (50 ng/mL), Flt3L (100 ng/mL), and stem cell factor (100 ng/mL) in the presence of AD (200 nM) or vehicle (Con). Cells were collected and transplanted into lethally irradiated isogenic mice (3 × 10⁴ cells per mouse). Survival curves of recipient animals were determined (n = 10 mice/group). (B) Lin⁻ cells (CD45.2⁺) as cultured in panel A or freshly isolated Lin⁻ cells (2 × 10³, 1 × 10⁴, and 5 × 10⁴) were mixed with 1 × 10⁶ fresh BM cells (CD45.1⁺) and transplanted into CD45.1⁺ recipients (4-5 mice per group). CRUs in test cell populations were determined 16 weeks after transplant. Recipient mice that had more than 1% contribution from test donor cells in peripheral blood were considered to have test cell reconstitution. (C-H) Sorted LSK cells were cultured as in panel A. The percentage of LSK cells was analyzed by FACS 7 days later (C), and cultured cells were harvested, washed with phosphate-buffered saline, and assessed by the colony-forming unit (CFU) assay (D); data are presented as mean ± standard error of the mean from 3 independent experiments. (E) Overall reconstituting capabilities of the cultured cells were determined by the competitive repopulation assay as described in “Methods” (n = 10 mice per group); ^*P < .05. CD45.2⁺ donor–derived myeloid (Mac-1⁺/Gr-1⁺), B-lymphoid (B220⁺), and T-lymphoid (CD3⁺) populations in the peripheral blood (F) and CD45.2⁺ donor–derived cells in the BM (G) of recipient animals were assessed by FACS 20 weeks after transplant. (H) BM cells harvested from primary transplants 20 weeks after transplant were transplanted into lethally irradiated secondary recipients (n = 5 mice per group). CD45.2⁺ donor cell reconstitution in peripheral blood was determined by FACS. (I-K) Human apheresis cells were cultured in the presence of AD (250 nM) or vehicle as described in panel A (except that human stem cell factor was used). The percentage (I) and absolute number (J) of Lin⁻CD34⁺CD38⁻ cells were quantified by FACS. (K) CFUs of granulocytes, erythrocytes, macrophages, and megakaryocytes (CFU-GEMM) were determined by the CFU assay. Con, control vehicle; FACS, fluorescence-activated cell sorter.