Phenotypic and functional alterations of BM mesenchymal cells in the Sipa1^−/− mice prior to the initiation of MPN. (A) Representative FACS profiles of the analysis of BM stromal cells subsets in 3-month-old Sipa1^+/+ and Sipa1^−/− mice. The CD45⁻TER119⁻CD31⁻PI⁻ cells were first divided into the CD44⁻ and CD44⁺ cells. The SCA1⁺CD51⁺ MSCs, SCA1⁺CD51⁻ MPCs, and SCA1⁻CD51⁻ cells were subsequently gated within the CD44⁻ cells. (B) The frequency of CD31⁺ cells in the Sipa1^+/+ and Sipa1^−/− mouse BM. (C) The percent of the MSCs, MPCs, and the SCA1⁻CD51⁻ cells within total CD45⁻TER119⁻CD31⁻ stromal cells. The data are from 3 independent experiments. (D) CFU-F frequencies in Sipa1^+/+ and Sipa1^−/− mouse BM MNCs and FACS-sorted MSCs. (E) Multilineage differentiation potentials of MSCs from Sipa1^+/+ and Sipa1^−/− BM. Scale bars represent 250 μm (left), 500 μm (middle), and 50 μm (right). n = 3 independent sorting experiments. (F) Representative μCT images of the longitudinal femoral section indicating reduced bone mass of Sipa1^−/− mouse femurs. Scale bars represent 1.0 mm. (G) Femoral bone (left) and marrow (right) volumes of Sipa1^+/+ and Sipa1^−/− mice. n = 3 per group of each genotype. (H) Colony-forming unit in culture (CFU-C) colonies derived from 100 LSK cells cocultured with Sipa1^+/+ BM MSC, MPC, CD51⁻SCA1⁻ mature stromal cells and endothelial cells. Total CFU-C, colonies with GM, G, M, erythrocytes (E), and GME lineages were counted. (I-J) The numbers of CFU-C colonies per 100 LSK cells after coculture with Sipa1^+/+ and Sipa1^−/− MSC (I) and MPC (J). Data were collected from 2 to 3 independent experiments. The statistical difference was determined by unpaired Student t test. See also in supplemental Figure 2.