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M. Ichii et al.
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Figure 1. B-cell reconstitution following transplantation is improved in the absence of STAP-2. (A) The proportion of the indicated subsets (LK; Lineage– Sca1– cKithigh, LSK; Lineage– Sca1+ cKithigh, early lymphoid progenitor (ELP); Flk2high IL7Rα– LSK, common lymphoid progenitor (CLP); Lin– Sca1+ cKitlow Flk2high IL7Rα+ , LT-HSC and mul- tipotent progenitor (MPP)1; Flk2– CD48– CD150+ LSK, MPP2; Flk2– CD48+ CD150+ LSK, MPP3; Flk2– CD48+ CD150- LSK, MPP4; Flk2- CD48- CD150- LSK) in bone marrow (BM) were analyzed with flow cytometry using wild-type (WT) and STAP-2 knockout (KO) mice (WT, n=5; STAP-2 KO, n=4). (B-E) Competitive transplantation experiments were conducted using KO mice. LSK cells (CD45.2+) derived from KO or WT were injected intravenously with competitor BM cells (CD45.1+). (B) Flow cytometry was used to evaluate hematopoietic recovery of Mac1+ or Gr1+ myeloid (M), CD19+ B- and CD3+ T-lineage cells in CD45.2+ donor-derived peripheral blood at the indicated times after transplantation. (C) The lineage distribution in CD45.2+ cells one month after transplantation. (D) BM cells were collected one month after transplantation, and donor chimerism (CD45.2+) was evaluated in Flk2– or Flk2+ LSK cells, ELP and CLP (left panel). The proportion of CD45.2+ cells in the B lineage progenitor subsets (pre-pro-B; B220+ CD43+ CD19– IgM–, pro-B; B220+ CD43+ CD19+ IgM–, pre-B; B220+ CD43- CD19+ IgM-, immature B; B220+ CD43- CD19+ IgM+) was also analyzed (right panel). (E) The same experimental strategy was used to assess the effects of STAP-2 on aged hematopoiesis in 12-22-month old KO mice, using age-matched WT mice as a control. Similar results were obtained in three independent experiments. Six to ten mice were used for each experiment. The results are shown as means±standard deviation. Statistical significances relative to WT controls were determined by unpaired two-tailed Mann-Whitney tests: *P<0.05; **P<0.01.
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haematologica | 2021; 106(2)