Continued presence of p18-/-HSC progeny without apparent exhaustion during sBMT. BMNCs were collected from p18-/- or p18+/+ male mice at 2 months of age and transplanted into lethally irradiated (10 Gy) female recipients (2 × 106 cells/recipient). After long-term engraftment was established in the primary recipients, the same dose of BMNCs were pooled from the primary recipients and retransplanted into lethally irradiated female recipients. The same procedure was repeated for additional 3 times (A). 0°, I°, II°, III°, and IV° indicate nonrecipient, first, second, third, and fourth sBMTs, respectively. M and F indicate male and female, respectively. Numbers under the arrows indicate the months after each sBMT, and numbers in the parentheses are the cellular age of original transplanted HSCs. In vitro hematopoietic activity was assayed with the CAFC assay and CFC assay as previously described.10 The frequencies of day-35 CAFCs (left) and CFCs (right) normalized to the nonrecipient wild-type control after different sBMTs are shown (B). *P < .05 (Student t test, n = 3). Error bars indicate SD. □ and ▪ indicate p18+/+ and p18-/- groups, respectively. To further assess the exhaustion of hematopoietic regeneration, peripheral blood was collected from the recipients 12 months after IV° sBMT and the PCR-based semiquantitative analysis for mouse Y chromosome–specific sequence (Sry) normalized to the housekeeping gene myogenin (Myo) was applied to calculate the contribution of the original male donor cells (C). The spleen- or blood-nucleated cells from male and female mice were mixed at different ratios to acquire the standardization for the semiquantitative analysis. According to the standardization generated simultaneously (bottom panel), the normalized percentages of donor cells in the blood are indicated under the PCR images (top, middle panels). Numbers above the gel images indicate individual p18-/- or p18+/+ transplant recipients, and engraftment level (%) in each recipient is indicated under the gel image.
