Figure 4. Colony forming assays, hematopoietic stem cell (HSC) and progenitor analysis of mutant Flt3 animals.

(A) BM cells from Flt3^+/+, Flt3^+/ITD, Flt3^ITD/ITD mice were plated on M3434 methylcellulose medium (containing SCF, IL-3, IL-6, EPO) and scored for colony formation 7-10 days later (G=granulocyte, M=macrophage, GM=granulocyte macrophage, BFU-E=burst-forming uniterythroid, GEMM=granulocyte, erythroid, macrophage, megakaryocyte). Results are the average of three independent experiments performed in duplicate (mean +/- S.E.M. are shown). (B) Morphology of increased numbers of CFU-M colonies [left panel, scale bar, 500 μm; middle panel; scale bar, 200 μm, right panel; scale bar, 100 μm (Wright-Giemsa)]. (C) Bone marrow cells harboring the mutant Flt3-ITD allele do not demonstrate serial replating capability and exhibit a dose-dependent decreased replating potential capacity. Results presented are the mean +/- S.E.M. of two independent experiments performed in duplicate. (D-F) Multiparameter flow cytomery demonstrates significantly increased numbers of Lin^-Sca1⁺ckit⁺ cells and a trend towards increased myeloid progenitors in Flt3^ITD/ITD mice over heterozygous and wt animals with a progressive increase in the relative proportions of granulocyte-monocyte progenitors (GMP) in these mice. Plotted in (E) and (F) are the values from two independent experiments (mean +/- S.E.M.; Flt3^+/+, n=5; Flt3^+/ITD, n=6; Flt3^ITD/ITD, n=6). (G) Spleen colony forming unit (CFU-S) ability is diminished in Flt3^ITD/ITD HSC (Lin^-Sca1⁺ckit⁺) cells. Duplicate wt C57BL6 recipient mice were transplanted with 500 Lin^-Sca1⁺ckit⁺ cells sorted from two independent Flt3^+/+ or Flt3^ITD/ITD donor mice and spleens from representative recipient animals are shown. Frequency of CFU-S is calculated as 1/(# cells transplanted/# colonies observed).