Distribution of progenitor cell populations. (A) Highest secondary CFU-GM formation in BM and spleen of Icsbp^−/−Nf1^+/− mice. BM cells from 2- to 3-month-old mice were lineage depleted and stained according to Kondo et al⁴⁴  and Adolfsson et al,⁴⁵  as described in “Fluorescence-activated cell sorter analysis” in “Methods.” Stem cell populations (Figure S3) were identified as follows: HSCs (Lin⁻, Sca1⁺, ckit⁺, Flt3⁻); LMPPs (Lin⁻, Sca1⁺, ckit⁺, Flt3⁺); CMPs (Lin⁻, Sca1⁻, ckit⁺, Flt3⁻, IL7Rα⁻, CD16/32^intermediate, CD34^intermediate); GMPs (Lin⁻, Sca1⁻, ckit⁺, Flt3⁻, IL7Rα ⁻, CD16/32^high, CD34^high); megakaryocyte-erythrocyte progenitors (Lin⁻ Sca1^−, ckit⁺, Flt3⁻, IL7Rα⁻, CD16/32^dim, CD34^dim); and CLPs (Lin⁻, Sca1⁺, ckit⁺, IL7Rα⁺). Lineage-depleted cells were pregated as negative for lineage markers combined with a live gate. Percentages within gates represent the mean ± SD of Lin⁻ cells of n = 3 individual mice of each genotype except for CLP (n = 2). Statistical significance (Mann-Whitney U test) *between Icsbp^−/−Nf1^+/+ and Icsbp^+/+Nf1^+/+ and **between Icsbp^−/−Nf1^+/− and Icsbp^+/+Nf1^+/−. (B,C) Increased secondary CFU-GM from BM and spleen. BM cells were grown in duplicates of 1 mL methylcellulose supplemented with combinations of rmGM-CSF and rrSCF or rrSCF and recombinant murine interleukin-3 (B). In addition, BM and spleen cells were grown in rmGM-CSF (C), as described in “CFU assay” in “Methods.” Cultures were performed in duplicates, incubated at 37°C with 7% CO₂, and cultured for 8 to 10 days (Figure S2). At that time, colonies were counted, and all cells were isolated from methylcellulose and replated again into 1 mL methylcellulose in duplicates using the same cytokines as in the first CFU-GM assay. After 10 to 15 days, number of total colonies was scored again using a stereomicroscope. Dots represent numbers of secondary CFU-GM per total cells plated; and lines, medians. The level of significance (Mann-Whitney U test) comparing Icsbp^−/−Nf1^+/− and Icsbp^−/−Nf1^+/+ mice is shown above dots.