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Nupr1 regulates the quiescence threshold of HSC
Nupr1-deleted hematopoietic stem cells expand robustly in vitro
We next examined whether the deletion of Nupr1 could enhance HSC expansion in vitro. Fifty HSC sorted from WT and Nupr1-/- mice were cultured in vitro for 10 days following a recently described protocol32 (Figure 4A). After 10 days of culture, the WT input cells yielded more than 2.2×104 cells, while Nupr1-/- HSC produced approximately 5×104 total cells (P<0.001) (Figure 4B). The colonies derived from Nupr1-/- HSC were much larger than those from WT HSC (Figure 4C). Furthermore, we analyzed the phenotypic HSC populations in the expanded cells and found that the absolute number of phenotypic HSC in individual Nupr1-/- colonies was 3 times more than WT HSC (P=0.005) (Figure 4D, E). To determine whether the quantitative expansion of
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phenotypic HSC contained net proliferation of functional HSC, we performed competitive repopulating-unit assays,33 using serial doses of limiting dilutions of the in vitro-expand- ed cells. The WT HSC frequency in the 10-day expanded cells was 1 in 371 cells, which is equivalent to 61 functional HSC, while the Nupr1-/- HSC frequency in the 10-day expanded cells was 1 in 190 cells (Figure 4F),34 which is equivalent to 263 functional HSC (P=0.045). Therefore, the deletion of Nupr1 induced an approximately 4-fold expan- sion of functional HSC numbers over WT HSC. Thus, dele- tion of Nupr1 enhances the expansion ability of HSC in vitro.
Reversion of p53 expression offsets the competitiveness of Nupr1-/- hematopoietic stem cells
To further investigate the underlying molecular mecha-
Figure 3. Nupr1-/- hematopoietic stem cells continuously show a competitive advantage without losing their long-term self-renewing ability in secondary transplantation. (A) Kinetic analysis of donor chimerism (CD45.2+) in peripheral blood (PB) of secondary transplanted recipients. Data were analyzed by two-way analysis of variance and are represented as mean ± standard deviation (SD) (n=5 mice). ***P<0.001. (B) Flow cytometry analysis of donor Nupr1-/- hematopoietic stem cells (HSC) in secondary recipients 16 weeks after trans- plantation. Representative plots from each group of mice are shown. (C) Cell number and percentage of donor-derived HSC in secondary recipients 4 months after competitive transplantation. Data were analyzed using an unpaired Student t-test (two-tailed) and are represented as mean ± SD (n=5 mice). ***P<0.001. BMNC: bone marrow nucleated cells.
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