Figure 2. Progressive functional decline of miR-146a-deficient HSCs.

(A) Ratio of CD45.2⁺ over CD45.1⁺ cells of various lineages in pretransplanted bone marrow (BM) mixtures consisted of equal numbers of CD45.2⁺ WT and CD45.1⁺ WT total BM cells (blue bar, CD45.2 WT:CD45.1 WT) or equal numbers of CD45.2⁺ miR-146a KO and CD45.1⁺ WT total BM cells (red bar, CD45.2 KO:CD45.1 WT). FACS analysis was performed on the BM mixtures before transplantation to determine the starting ratios of various lineages. (B) Ratio of CD45.2⁺ over CD45.1⁺ cells of various lineages in peripheral blood (PB), spleen (SP) and BM 6 months after transplantation. Blue bar, CD45.2 WT:CD45.1 WT, represents mice received CD45.2 WT:CD45.1 WT BM cells; red bar, CD45.2 KO:CD45.1 WT, represents mice received CD45.2 KO:CD45.1 WT BM cells. All donor mice were 6-week-old female and recipient mice were 2-month-old CD45.1⁺ WT female. Ratio of CD45.2⁺ over CD45.1⁺ of BM LSK cells and HSCs (C) and total white blood (CD45⁺) cells (D) 10-month after transplantation. (E) Number of total HSCs, CD45.2⁺ HSCs and CD45.1^± HSCs in recipient chimera mice. WT:WT, CD45.2 WT:CD45.1 WT; KO:WT, CD45.2 KO:CD45.1 WT. n = 8 for each group. (F)–(K) A repeat of the above experiment with age-and-sex-matched 4-month-old WT and miR-146a KO female mice. (F) Representative FACS plots of BM mixtures before transplantation showing CD45.2/CD45.1 ratio close to 1 for both WT:WT and KO:WT BM mixtures. (G) Ratio of CD45.2⁺ over CD45.1⁺ cells of various lineages before transplantation. (H) Percentage of CD45.2⁺ cells of CD45⁺ peripheral blood nucleated cells at 1, 2, 3 and 6 months. (I) Ratio of CD45.2⁺ over CD45.1⁺ cells of various lineages in PB, SP, and BM 6 months after transplantation. All ratios of CD45.2 WT:CD45.1 WT are normalized to 1. Number of total, CD45.2⁺ and CD45.1⁺ LSK cells (J) and HSCs (K) in recipient chimera mice. LSK, Lin-cKit⁺Sca1⁺; HSC, LSK CD150⁺CD48⁻; L⁻S⁻K⁺, Lin⁻Sca1⁻cKit⁺.

DOI: http://dx.doi.org/10.7554/eLife.00537.005

Figure 2—figure supplement 1. Functional decline of miR-146a-deficient HSCs.

(A) Related to Figure 2C–E. Equal numbers of BM HSCs (LSK CD150⁺CD48⁻) were sorted from 8-week-old CD45.2⁺ WT and CD45.2⁺ miR-146a KO mice. Fifty WT or KO HSCs were mixed with 500,000 CD45.1⁺ WT total BM cells and transplanted into a CD45.1⁺ WT recipient mouse. Percentage of CD45.2⁺ cells in various lineages in peripheral blood (PB), spleen (SP), and bone marrow (BM) was analyzed by FACS 8 months after transplantation. (B)–(E) Related to Figure 2F–K. Quantification of total number of cells (including both CD45.2⁺ and CD45.1⁺ cells) in various lineages in SP and BM of CD45.2 WT:CD45.1 WT and CD45.2 KO:CD45.1 WT recipient mice 6 months after transplantation. (F)–(G) A repeat of the experiment described in Figure 2A with sex- and age-matched 6-month-old WT and miR-146a KO female mice. (F) Ratio of CD45.2⁺ over CD45.1⁺ cells of various lineages in BM mixtures before transplantation. (G) Ratio of CD45.2⁺ over CD45.1⁺ cells of various lineages in PB, SP, and BM 3 months after transplantation. All ratios of CD45.2 WT:CD45.1 WT are normalized to 1. LSK, Lin⁻cKit⁺Sca1⁺; HSC, LSK CD150⁺CD48⁻;L^⁻S⁻K^⁺, Lin-Sca1⁻cKit^⁺.

DOI: http://dx.doi.org/10.7554/eLife.00537.006