Functional assessment of long-term repopulating human HSCs. (A) Sublethally irradiated NOG mice were given transplants of CB Lin⁻CD34⁺CD38⁻ cells. Recipient mice were administered with BrdU at the indicated time points (3 recipients in each time point), and Lin⁻CD34⁺CD38⁻ cells were isolated at 2 hours after each administration. The BrdU incorporation rate for the isolated cells was analyzed by flow cytometry (**P < .01 relative to CB and 18 weeks after transplantation). (B) Relative expressions of p16^INK4a, p14^ARF, and p21^CIP1 in Lin⁻CD34⁺CD38⁻ cells were analyzed by quantitative real-time PCR. Each value was normalized to 18S rRNA expression and is presented as a fold induction compared with the levels detected in Lin⁻CD34⁺CD38⁻ cells isolated from CB. Data were collected from 4 independent experiments (**P < .01). (C) Lin⁻CD34⁺CD38⁻ cells and Lin⁻CD34⁺CD38⁺ cells were obtained from CB and primary and secondary recipients' mouse bone marrow at 18 weeks after transplantation (3 recipients at the each time point). At 3 days after cultivation with cytokines, cells were pulsed with BrdU for 30 minutes, and then the BrdU incorporation rate was analyzed by flow cytometry (*P < .05, **P < .01). (D) Whole bone marrow cells obtained from each primary recipient mouse (n = 6) were serially transplanted into secondary (n = 6) and tertiary (n = 5) recipient mice. Human hematopoietic cell engraftment was assessed by the expression of human CD45 by flow cytometry at 18 weeks after each transplantation (**P < .01 relative to primary recipient).