Rbm15-KO HSCs are less competitive in reconstitution of lethally irradiated recipient mice and compromised in serial transplantation due to a LT-HSC differentiation block. (A) Serially diluted donor BM cells (CD45.2⁺) were transplanted into lethally irradiated CD45.1⁺ recipient mice with (groups 2-5) or without (group 1) 2 × 10⁵ competitor WT BM cells (CD45.1⁺). Six weeks after transplantation, PB was analyzed by FACS to examine the donor-derived (CD45.2⁺) monocyte/macrophage (Mac1⁺), granulocyte (Gr1⁺), B-lymphoid (B220⁺), and T-lymphoid (CD3⁺) cells in the recipient animals. The bars represent the averages in each group of mice; error bars show SD. (B) Homing of Rbm15-WT and -KO BM cells. BM cells were labeled with the fluorescent dye CFSE, then transplanted into lethally irradiated (10 Gy) recipient mice. CFSE⁺ cells homing to the BM of the recipient mice were assessed 5 hours after transplantation by flow analysis. (C) Representative BM analysis of competitive transplanted recipient mice (group 5 in A) 5 months after transplantation. The percentages of Lin⁻ cells, LSKs, LT-HSCs, ST-HSCs, and Lin⁻c-Kit⁺Sca1⁻ (LK) progenitors in the total donor-derived cell populations are shown. *P < .05, **P < .01, n = 4. (D) Absolute numbers of different HSC and progenitor subsets derived from CD45.2⁺ donor cells in the BM of the chimeras shown in (C). Note that, whereas Rbm15-KO LT-HSC numbers are increased approximately 3-fold, the numbers of Rbm15-KO ST-HSCs and LK progenitors are reduced markedly compared with the WT counterparts, indicating an inability of LT-HSCs that lack Rbm15 to normally differentiate. *P < .05, **P < .01, n = 2. (E) BMNCs (2 × 10⁶) from either Rbm15-WT or Rbm15-KO mice (CD45.2⁺) were transplanted into lethally irradiated (10 Gy) WT recipient mice (CD45.1⁺) (S1). BMNCs were taken 3 months later from these recipient mice, and the same procedure was applied in secondary (S2) and tertiary (S3) recipients. PB from 3 to 6 mice in each group was analyzed 6 weeks and 3 months after transplantation for CD45.2⁺ donor cell contribution. The data shown were collected from 4 independent experiments using PB samples taken at 6 weeks after transplantation. *P < .05, **P < .01.