The substantial in vitro myeloid potential of DN1 cells is not reproduced in either methylcellulose assays or in vivo. (A) MPP, CLP, or DN1 cells were clone- sorted into 96-well plates containing a confluent layer of a 1:1 mixture of OP9:OP9DL1 stroma in the presence of Flt3L, SCF, IL-3, IL-6, M-CSF, GM-CSF, G-CSF, and IL-7. After 11 days, wells were analyzed by flow cytometry for lineage markers, and those with cells of the phenotype CD45.2⁺ CD25⁻ Mac-1⁺ were scored as myeloid cells. Graphs show the percentage of expanded colonies that contained at least 1% myeloid cells within the hematopoietic readout. Parentheses above columns indicate the number of wells with myeloid readout out of the total number of wells plated (regardless of whether the sorted cells expanded). The clonal readouts were composed almost entirely of either T lineage cells or myeloid lineages. Dendritic cells, as defined by CD11c⁺ Gr1⁻, made up a minor component of the myeloid readout (Figure 2). Results are representative of 2 independent experiments. (B) Expanded wells from the assay in panel A were scored for colonies that contained T cells only, myeloid cells only, or T cells and myeloid cells. T cells were defined as CD45.2⁺ CD25⁺, and myeloid cells were defined as in panel A. The colonies had to contain at least 1% myeloid or T readouts to be scored for the respective lineage. (C) The percentage of myeloid or T cells, as defined earlier in the Figure 1 legend, in all wells with clonal expansion from the assay in panel A. (D) A total of 135 MPP, 1350 CLP, and 1350 DN1 cells were FACS-sorted into methylcellulose containing Flt3L, SCF, IL-3, IL-6, M-CSF, GM-CSF, and G-CSF in 2 separate experiments. After 6 days, colonies were counted, and the number of colonies per 100 cells plated was scored. Parentheses above columns indicate the total number of colonies per number of cells plated in the summation of the 2 experiments. Error bars represent the SD between plates. (E) A total of 5000 MPP, CLP, or DN1 cells were sorted from CD45.1 congenic donors and injected intravenously into sublethally irradiated recipients. After 7 days, splenic chimerism and lineages were assessed by flow cytometry. The graph shows the absolute number of donor-derived myeloid cells (CD45.1⁺Mac-1⁺CD11c⁻) per spleen. Error bars represent the SD between mice. (F) Representative flow cytometric plots of donor splenocytes, showing myeloid readout quantified in panel E. Plots are pregated for CD45.1⁺ (donor) and CD11c⁻ cells.