Homozygous JAK2V617F expression increases the number of erythroid progenitors and fetal liver erythroblast cell proliferation, but does not reduce apoptosis. (A) JAK2^R/R mice show increased total progenitors, mostly from a significant increase in MEP numbers. Frequencies of stem and progenitor cells in WT, JAK2^R/+, and JAK2^R/R mice as determined by flow cytometry. (B) JAK2^R/R mice show increased erythroid colony formation. BM BFU-E (left panel), spleen BFU-E (middle panel), and spleen CFU-E colony assays (right panel), performed with or without EPO. (C) JAK2^R/R mice produce larger spleen BFU-E colonies (left panel), which contain more erythroblasts per colony (right panel). Cell counts per BFU-E were derived by recording colony numbers and total cell number per dish at day 12. (D) Representative flow cytometry plots show Ter119 and CD71 expression before and after 48 hours of in vitro culture of lineage-negative E14.5 fetal liver cells. (E) Increased production of erythroblasts by JAK2^R/R lineage-negative fetal liver cells in culture. Cells were analyzed by flow cytometry after 48 hours in culture. The histograms show the number of cells at individual erythroid differentiation stages (R1-R5). (F) Line graph shows the total cell number in culture. Equal numbers of lineage-negative fetal liver cells were plated on day 0 and cell number was counted on days 1 and 2. (G) JAK2V617F expression does not affect fetal liver erythroblast apoptosis. Erythroblasts were analyzed 48 hours after culture of the lineage-negative fetal liver cells by flow cytometry using Annexin-V antibody and 7-AAD on CD71⁺Ter119⁺. The histograms show percentages of apoptotic cells in the CD71⁺Ter119⁺ erythroblast population. Asterisks indicate significant differences by Student t test (*P < .05; **P < .01; ***P < .001). Data are shown as mean ± SEM.