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T. Wang et al.
Results
Loss of Nupr1 accelerates the turn-over rates of hematopoietic stem cells under homeostasis
The majority of long-term HSC are quiescent under homeostasis, which is a key mechanism for maintaining the HSC pool for life-long steady hematopoiesis. We hypothesized that genes preferentially expressed in HSC but immediately downregulated in multipotent progeni- tors (MPP) might form an intrinsic regulatory network for maintaining HSC quiescence. To test our hypothesis, we explored candidate factors by RNA-sequencing analysis of sorted HSC (Lin– CD48– Sca1+ c-kit+ CD150+) and MPP (Lin– Sca1+ c-kit+ CD150–). Analysis of differentially expressed genes showed a pattern of transcription factors preferen- tially present in HSC, including Rorc, Hoxb5, Rarb, Gfi1b, Mllt3, and Nupr1. By literature search, we found that most of the candidate genes except Nupr1 were reportedly not involved in regulating HSC homeostasis. Thus, we focused on the Nupr1 gene, the role of which in hematopoiesis has not been reported. The expression of Nupr1 in HSC was significantly higher (>25-fold, P=0.002) than that in MPP (Figure 1A, left). Real-time polymerase chain reaction (PCR) analysis further confirmed the same expression pat- tern (P<0.001), indicating an unknown role for Nupr1 in HSC (Figure 1A, right).
To study whether Nupr1 has any potential impact on the hematopoiesis of HSC, we created Nupr1 conditional knockout mice by introducing two loxp elements flanking exons 1 and 2 of the Nupr1 locus using a C57BL/6 back- ground mESC line (Figure 1B). The resultant Nupr1fl/fl mice were further crossed to Vav-Cre mice to generate Nupr1fl/fl; Vav-Cre compound mice (Nupr1-/- mice). The deletion of Nupr1 was confirmed by PCR in HSC (Online Supplementary Figure S1A-C). Adult Nupr1-/- mice (8-10 weeks old) had a normal percentage of blood lineage cells in peripheral blood, including CD11b+ myeloid, CD19+ B, and CD90.2+ T lineage cells (Online Supplementary Figure S2). We further investigated the potential alterations of HSC homeostasis in the absence of Nupr1. Flow cytometry analysis demonstrated that the Nupr1-/- HSC pool was comparable to the wild-type counterpart in terms of ratios and absolute numbers (Online Supplementary Figure S3). Subsequently, we examined the cell cycle status of Nupr1-/- HSC using the proliferation marker Ki-67 and DAPI stain- ing and found that the ratio of Nupr1-/- HSC in G0-status was reduced significantly (P<0.001). Compared with WT HSC (median value: Nupr1-/- HSC =73.67%, WT HSC = 87.15%), more Nupr1-/- HSC entered G1-S-S2 and M phas- es (Figure 1C, D). To further confirm this novel phenotype, we performed a BrdU incorporation assay, which is con- ventionally used to assess the turn-over rates of blood cells in vivo.35 The 8-week-old Nupr1-/- mice and littermates were injected intraperitoneally with 1 mg BrdU on day 0, fol- lowed by continuous administration of BrdU via water (0.8 mg/mL) for up to 5 days (Figure 1E). After 3 days of BrdU labeling, ~50% of Nupr1-/- HSC became BrdU+ compared with ~35% of WT HSC. The BrdU incorporation rates in HSC differed between the two mouse models (WT and Nupr1-/-, P<0.001), and the dynamics changed along with time elapsed (P=0.012, two-way analysis of variance [ANOVA]). Kinetic analysis of BrdU incorporation from day 3 to day 5 revealed that Nupr1-/- HSC contained a 1.5- fold larger BrdU+ population over WT HSC (Figure 1F, G). Collectively, these data indicate that the Nupr1-deletion
drives HSC to enter the cell cycle and accelerates their turn- over rates in homeostasis.
Nupr1-/- hematopoietic stem cells show repopulating advantage without their multilineage differentiation potential being compromised
To confirm whether Nupr1-/- HSC have a repopulating advantage or disadvantage in vivo, we performed a typical HSC competitive repopulation assay. BMNC (2.5x105) from Nupr1-/- mice (CD45.2) were transplanted into lethally irra- diated recipients (CD45.1) along with the same number of WT (CD45.1) competitor cells. Bone marrow cells from lit- termates (Vav-Cre+, CD45.2+) were mixed with WT (CD45.1) competitors and transplanted into the recipients as the experiment control (Figure 2A). Sixteen weeks later, 1x106 BMNC from the primary recipients were transplant- ed into lethally irradiated recipients to assess long-term engraftment. We observed that donor Nupr1-/- cells account- ed for ~70% of cells in the primary recipients, while the control cells accounted for 50%-60% in the recipients from the transplantation control assay. Nupr1-/- cells gradually dominated in peripheral blood of recipients over time after transplantation (Figure 2B). In the chimeras, ~70% of myeloid cells and B lymphocytes were Nupr1-/- donor- derived cells, while ~60% of T lymphocytes were from CD45.1 competitive cells (Figure 2C). To further explore whether Nupr1-/- HSC dominated in the HSC pool, we sac- rificed the recipients and analyzed HSC 16 weeks after transplantation. The proportion and absolute number of Nupr1-/- HSC were significantly higher (~1.5-fold) than those of the control HSC in primary recipients (Figure 2D, E). Previous research documented that HSC proliferated rapidly at the expense of their long-term repopulating abil- ity.36-40 Interestingly, consistent with the dominating trend in the primary transplants, Nupr1-/- cells continuously domi- nated in the peripheral blood of secondary recipients (Figure 3A). Nupr1-/- HSC further occupied up to 90% of the total HSC in the bone marrow of secondary recipients. However, the control HSC accounted for less than 10% in the secondary recipients (Figure 3B, C). Collectively, these results indicate that the deletion of Nupr1 promotes the repopulating ability of HSC without impairing their long- term engraftment ability.
Nupr1-/- hematopoietic stem cells are highly sensitive to irradiation-stress but re-cover fast
HSC in the cell cycle were reported to be more sensitive to irradiation damage.41,42 To explore whether Nupr1-/- HSC with a fast turn-over rate are more sensitive to irradiation, WT mice and Nupr1-/- mice were exposed to a single dose of total body irradiation (4 Gy dose, 1 Gy/min). Apoptosis and cell cycle status were analyzed 6 h (early stage) and 24 h (later stage) later. As expected, Nupr1-/- HSC showed a sig- nificantly enhanced sensitivity to irradiation: only ~40% of Nupr1-/- HSC lived 6 h after irradiation, whereas ~70% of WT HSC were still alive (Online Supplementary Figure S4A). The proportion of radiation-induced apoptosis (annexin V+) of Nupr1-/- HSC was significantly higher (~2-fold, P=0.02) than that of WT HSC (Online Supplementary Figure S4A). Furthermore, ~60% of the residual Nupr1-/- HSC were in G1-S-G2-M proliferative phases compared with ~50% of the residual WT HSC (P=0.01) (Online Supplementary Figure S4B), indicating an accelerated replenishment rate in response to irradiation damage. At a later stage (24 h) after irradiation, we observed more living Nupr1-/- HSC (WT vs.
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haematologica | 2022; 107(1)