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Y. Hirata et al.
BM cellularity (Figure 1A). CD73 deletion significantly increased the frequencies of cycling cKit+Sca1+Lin- hematopoietic stem and progenitor cells (HSPC) and CD150+CD48-cKit+Sca1+Lin- HSC, as well as numbers of HSPC and HSC (Figure 1A-C, Online Supplementary Figure S1A,B and Online Supplementary Table S1). Consistently, the numbers of colonies formed following in vitro culture of BM cells isolated from CD73 KO mice were significant- ly higher than those from wild-type mice (Online Supplementary Figure S1C). The numbers of other BM cell populations were not significantly altered by CD73 dele- tion (Online Supplementary Figure S1D). To assess the size of the pool of functional HSC and HSPC, we performed competitive BM transplantation assays to evaluate the reconstituting potential of BM cells. BM cells of CD73 KO mice or control wildtype mice (B6 CD45.2; 3 x 105 cells/mouse) were intravenously injected into lethally-irra-
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diated B6 SJL mice (CD45.1), together with competitor SJL BM (CD45.1; 3 x 105 cells/mouse). CD45.2 donor blood chimerism from CD73 KO BM cells remained significant- ly higher than that from control BM cells for 6 months after transplantation, suggesting that CD73 deletion increased functional HSC and HSPC frequencies (Figure 1D). Additionally, no myeloid skewing was observed in donor hematopoietic cells derived from CD73KO BM (Online Supplementary Figure S1E). Taken together, these results indicate that CD73 maintains quiescence and pool size of HSPC and HSC.
CD73 maintains hematopoietic stem cell pool size by preventing oxidative stress
CD73 KO mice showed slight but significant increases in the levels of reactive oxygen species (ROS) in HSPC and HSC but not in Lin+ cells (Figure 2A, Online Supplementary
Figure 1. CD73 deletion increased hematopoietic stem cell pool size. (A) Bone marrow (BM) cellularity of CD73 knockout (KO) mice. We analyzed the numbers of total BM cells isolated from one tibia and one femur by crushing. Data from three independent experiments with nine mice/group were pooled. Data are presented as mean ± SD and analyzed by a two-tailed t-test. (B) Flow cytometric analysis of frequencies of Ki67- cells among hematopoietic stem and progenitor cells (HSPC: cKit+Sca1+Lin-) and hematopoietic stem cells (HSC: CD150+CD48-cKit+Sca1+Lin-) in CD73 KO mice. The results were reproducible in two independent experiments (7 mice/group total). A representative figure from one independent experiment is shown here. Data are presented as mean ± SD and analyzed by a two-tailed t-test. (C) Flow cytometric analysis of HSPC and HSC frequencies and numbers in CD73 KO mice. The results were reproducible in three independent experiments (3 mice/group in each experiment; refer to Online Supplementary Table S1). A representative figure is shown here. Data are presented as mean ± SD and analyzed by a two-tailed t-test. (D) Donor chimerism in the peripheral blood of lethally irradiated mice that received wildtype SJL bone marrow (BM) cells (CD45.1) together with BM cells of CD73 KO or control mice (CD45.2). BM cells from each donor mouse (7 control mice and 9 CD73 KO mice) were transplanted into one recipient. The results were pooled from two independent experiments. Data are presented as mean ± SD and analyzed by a two-tailed t-test. wt: wildtype: M: months.
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