In vitro differentiation of cCLP into lineage-positive cells. (A) Representative FACS profiles on differentiation to B cells (upper left), T cells (upper middle), NK cells (upper right), and myeloid cells (lower left and middle) at day 7, and to DC (middle left) at day 14 in different culture conditions of Lin⁻Flk2⁺IL7R⁺CD27⁺Ly6d⁻ cells FACS-purified from cCLP cultured for 2 months. Microscopic images of myeloid cells (lower right) and DC differentiated from cCLP with low (center panel) and high (middle right) magnification. Scale bar, 10 μm. (B) Limiting dilution analyses of 2-month cCLPs for frequencies of proliferating clones under B-cell (left), T-cell (middle), or myeloid cell differentiation conditions (right). For myeloid cell differentiation, cCLPs were cultured on irradiated OP9 (circles) or nonirradiated OP9 (squares). (C) Single-clone analysis of cCLP for the differentiation to B (CD19⁺), T (Thy1⁺), NK (CD122⁺), DC (CD11c⁺MHCII⁺), and myeloid cells (CD11b⁺Gr1⁺). Wells that included >10 lineage-positive cells counted by flow cytometer were indicated as closed circle. For differentiation conditions, see “Materials and methods.” All data are representative of 2 independent experiments.