Sustained competitiveness of p18^-/- HSCs after multiple rounds of cBMT. BMNCs from p18^-/- or p18^-/-p21^-/- knockout mice were mixed with an equal number (2 × 10⁶/each) of competitor cells (BMNCs from 2-month-old wild-type mice) and injected into lethally irradiated (10 Gy) recipient mice. Ten months after primary cBMT, 2 × 10⁶ BMNCs from the recipients were rechallenged with an equal amount of the competitor cells and secondarily transplanted into lethally irradiated recipients. This procedure was sequentially repeated twice (donor cells were pooled from 3-5 animals, and there were 10 recipients/each group for each cBMT). The actual interval between each cBMT is shown (A). Some mice were kept longer than the interval. I°, II°, III°, and IV° indicate first, second, third, and fourth cBMTs, respectively. 1:1 indicates that equal amounts of test cells and competitor cells were cotransplanted into the recipients. M and F indicate male and female, respectively. Numbers under the arrows indicate the months after each cBMT, and numbers in the parentheses are the cellular age of original transplanted HSCs. Peripheral blood was collected from the recipients after long-term engraftment during each interval between sequential cBMTs, and PCR-based semiquantitative analysis as described in our previous study¹⁶  was performed to determine the contribution of p18^-/- or p18^-/-p21^-/- to the overall hematopoietic reconstitution. The spleen- or blood-nucleated cells from wild-type and mutant mice were mixed at different ratios to obtain a standardization curve for the semiquantitative analysis. The average levels of engraftment in blood from p18^-/- or p18^-/-p21^-/- origin 12 months after secondary and 6 months after tertiary cBMTs are shown (B). Error bars indicate SD. □ and ▪ indicate p18^-/- and p18^-/-p21^-/- transplantation groups, respectively. Each group includes 8 animals, and the difference between p18^-/- and p18^-/-p21^-/- is significant (P < .05) based on the Student t test. The representative results after tertiary cBMT are shown (C) along with the standardization generated simultaneously (bottom panel). Numbers above the gel images indicate individual animals, and the normalized percentages of test cells of the total in blood are shown below the images. As a control to confirm the accelerated exhaustion of hematopoietic repopulation in the absence of p21 as reported previously,¹⁰  the identical cBMT procedure (A) was used to test the repopulating ability of BMNCs from p21^-/- mice at the age of 12 months (D). Eight to 12 months after the first cBMT, there was virtually no detectable p21^-/- band in blood after 30 cycles of the PCR, and there were 2 blood samples at a barely detectable level (1%-3%) of the engraftment after 35 cycles of the PCR as representatively shown here. This experiment was intended to confirm a previous claim of HSC exhaustion due to p21 absence.¹⁰  It was not performed at the same time as other 2 groups but the competitor cells were from the same type of donor mice as described in “Materials and methods.” Therefore, the results between the p21^-/- group and other groups, though imperfect, are still comparable since all these different test cells were directly assessed with the same type of competitor cells in the irradiated hosts. The singular effect of age would not substantially contribute to the exhaustion based on earlier studies by others showing no disadvantage of aged BMNCs in the competitive repopulation model.^4,19,20