Increased percentages and absolute numbers of LSK cells and LT-HSCs in Rbm15-KO mice without aberrant proliferation or apoptosis. (A) Representative flow analysis of LSK (Lin⁻Sca⁺c-Kit⁺), LT-HSC, and ST-HSC cells in Rbm15-KO mice and littermate control animals. Lin⁻ cells were gated and analyzed for Sca1 and c-Kit, and the LSK cells were then gated to identify LT-HSCs and ST-HSCs based on Flk2, endoglin, or CD150 expression. Increased percentages of LT-HSCs (indicated as LSK/Flk2⁻, endoglin⁺, or CD150⁺ cells) were documented by studies using all 3 of these markers. (B-E) Quantitation of the percentages of LSK cells (B), LT-HSCs (LSK/Flk2⁻; C), and ST-HSCs (LSK/Flk2⁺; E) in total BMNCs, and the absolute number of LT-HSCs (D) in mice BM (n = 18 WT and KO each). (F) In vivo proliferation was assessed by BrdU incorporation. Mice received an intraperitoneal injection of 2 mg of BrdU and were analyzed 12 hours thereafter. BM cells were isolated and stained with a Lin⁺ mixture, c-Kit, and Sca1 antibodies, and analysis of BrdU incorporation in conjunction with 7-aminoactinomycin D was performed using a 5-color flow cytometer. (G) Rbm15-KO HSCs can terminally differentiate in vitro. FACS-sorted LSK/Flk2⁻ cells from WT and Rbm15-KO BM were bulk cultured in stem cell culture medium containing murine SCF and murine IL-3 to allow the cells to differentiate. After 3, 6, and 9 days, the expression of lineage markers Mac1 (macrophages), Gr1 (granulocytes), B220 (B lymphocytes), and CD3 (T lymphocytes) was analyzed. The percentages of these cells as part of the total nucleated cells are shown. (H) The apoptotic responses of freshly isolated HSCs from Rbm15-KO mice and WT littermate controls were assessed by costaining with stem cell markers and annexin V/PI. The percentages of annexin V⁺/PI⁻ apoptotic cells in LSK, LT-, and ST-HSC populations were analyzed. Mean ± SD and P values from 3 independent experiments are shown.