LSK cells expanded by IL-27 and SCF are multipotent myeloid Progenitors.

(A) Flow cytometry histogram analysis of cell surface markers in the LSK cells expanded by IL-27 and SCF for 4 weeks and primary BM cells using antibodies as indicated (solid line) and their control antibodies (plane line with shading). (B-C) Augmented potential of the LSK cells expanded by IL-27 and SCF to differentiate into mDCs. LSK populations (3 × 10³) purified from the LSK cells expanded by IL-27 and SCF for 2 and 4 weeks and primary BM cells were stimulated with GM-CSF. After the indicated time, these stimulated cells were analyzed for the expression of MHC class II and CD11c (B), and the cell numbers of mDC (MHC class II⁺CD11c⁺) were counted (C). (D-E) Decreased potential of the LSK cells expanded by IL-27 and SCF to differentiate into pDCs (Siglec H⁺PDCA1⁺CD11c⁺) and cDCs (Siglec H⁻PDCA1⁻CD11c⁺). (F-G) Enhanced potential of the LSK cells expanded by IL-27 and SCF for 2 weeks to differentiate into neutrophils. Neutrophil; Ly6G⁺CD11b⁺, macrophage; F4/80⁺CD11b⁺, mast cell; c-Kit⁺FcεR1α⁺CD11b⁻, basophil; CD49b⁺FcεR1α⁺CD11b⁻. (H) Abrogated potential of the LSK cells expanded by IL-27 and SCF for 2 weeks to differentiate into T (day 18) and B (day 15) cells. (I) Enhanced potential of the LSK cells expanded by IL-27 and SCF to differentiate into neutrophils in vivo. LSK populations purified from the LSK cells from CD45.1 congenic mice expanded by IL-27 and SCF for 2 weeks were transferred into sublethally irradiated CD45.2 recipient mice with the same congenic BM cells. Development of Gr-1⁺CD11b⁺ neutrophils in the BM and spleen were analyzed by flow cytometry after 9 days, and the percentage of neutrophils in each CD45.1⁺ and CD45.2⁺ cells was compared. (J) Increased expression of transcription factors critical for the differentiation into myeloid cells in the LSK cells expanded by IL-27 and SCF. RNA was prepared from the LSK population purified from LSK cells expanded for 2 weeks together with other progenitors and subjected to real-time RT-PCR. Data are shown as mean ± SEM (n = 2–4) and are representative of two to three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005.