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MicroRNA-21 maintains HSC homeostasis
by flow cytometry. There were evident increases in the per- centage and number of LSK, but not lineage-negative cells and myeloid progenitors, in the BM from miR-21D/D mice compared with those in the BM of miR-21fl/fl mice (Figure 2A). Further analysis revealed that the frequency of long- term HSC (LT-HSC) was increased, whereas the proportion of multipotent progenitors was decreased in the LSK com- partments in the absence of miR-21 (Figure 2B). Indeed, the numbers of three LSK subpopulations, especially the LT- HSC, were markedly increased in the BM after miR-21 knockout (Figure 2C). A similar result was obtained by staining LSK with another set of HSC surface markers, CD150 and CD48 (Figure 2D; Online Supplementary Figure S2A). We also observed a dramatic increase in the percent- age of LSK in the spleen, but not in the PB, when miR-21
A
was deleted (Figure 2E; Online Supplementary Figure S2B). These results indicate that miR-21 is responsible for sustain- ing the normal HSC pool.
Loss of miR-21 impairs the quiescence and facilitates the proliferation of hematopoietic stem cells
We then set out to explore the possible reasons for the accumulation of phenotypic HSC after miR-21 knockout. Actually, we found a minor but not significantly different decrease in the apoptosis of HSC upon miR-21 ablation (Online Supplementary Figure S3A). Importantly, cell cycle and in vivo 5-bromodeoxyuridine incorporation analysis revealed that miR-21 deficiency strikingly reduced the qui- escence and increased the proliferation of HSC, but not of myeloid progenitors (Figure 3A, B; Online Supplementary
BC
D
E
Figure 2. Targeted deletion of miR-21 generates an aberrant hematopoietic stem cell pool. (A) Flow cytometric analysis of the percentages and numbers of lineage- negative (Lin-) cells, myeloid progenitors (MP) and Lin-Sca1+c-Kit+ (LSK) cells in miR-21fl/fl and miR-21∆/∆ bone marrow (BM) (n=6 mice per group). (B) Flow cytometric analysis of the proportions of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC) and multipotent progenitors (MPP) in LSK from miR-21fl/fl and miR-21∆/∆ mice (n=6 mice per group). (C) The numbers of LT-HSC, ST-HSC and MPP in miR-21fl/fl and miR-21∆/∆ BM (n=6 mice per group). (D) Flow cytometric analysis of the number of CD150+ CD48- LSK in miR-21fl/fl and miR-21∆/∆ BM (n=6 mice per group). (E) Flow cytometric analysis of the percentage of LSK in the spleen (Sp) of miR-21fl/fl and miR-21∆/∆ mice (n=6 mice per group). All data are shown as means ± standard deviation. *P<0.05, **P<0.01.
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